Patent Publication Number: US-2023140953-A1

Title: Methods of editing a disease-associated gene using adenosine deaminase base editors, including for the treatment of genetic disease

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is the U.S. national phase application, pursuant to 35 U.S.C. § 371, of PCT International Application No. PCT/US2020/018073, filed Feb. 13, 2020, which claims the benefit of U.S. Provisional Application No. 62/805,271 filed Feb. 13, 2019; U.S. Provisional Application No. 62/850,919, filed May 21, 2019; U.S. Provisional Application No. 62/852,228 filed May 23, 2019; U.S. Provisional Application No. 62/852,224 filed May 23, 2019; U.S. Provisional Application No. 62/873,138 filed Jul. 11, 2019; U.S. Provisional Application No. 62/888,867 filed Aug. 19, 2019; U.S. Provisional Application No. 62/931,722 filed Nov. 6, 2019; U.S. Provisional Application No. 62/941,569 filed Nov. 27, 2019; U.S. Provisional Application No. 62/966,526 filed Jan. 27, 2020, the disclosures of which are hereby incorporated by reference in their entirety. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 29, 2020, is named 52885-802_601_SL.txt and is 1,885,037 bytes in size. 
     INCORPORATION BY REFERENCE 
     All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Absent any indication otherwise, publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entireties. 
     BACKGROUND OF THE DISCLOSURE 
     Targeted editing of nucleic acid sequences, for example, the targeted cleavage or the targeted modification of genomic DNA is a highly promising approach for the study of gene function and also has the potential to provide new therapies for human genetic diseases. Currently available base editors include cytidine base editors (e.g., BE4) that convert target C•G base pairs to T•A and adenine base editors (e.g., ABE7.10) that convert A•T to G•C. There is a need in the art for improved base editors capable of inducing modifications within a target sequence with greater specificity and efficiency. 
     SUMMARY OF THE DISCLOSURE 
     The invention provides compositions comprising novel adenine base editors (e.g., ABE8) that have increased efficiency and methods of using base editors comprising adenosine deaminase variants for editing a target sequence. 
     In some aspects, provided herein, is a method of treating a neurological disorder in a subject, the method comprising: administering to the subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide directs the adenosine base editor to effect an A-to-G nucleobase alteration in a target gene or a regulatory element thereof associated with the neurological disorder in the subject, thereby treating the neurological disorder in the subject. In another embodiment of this aspect, the target gene is an alpha-L-iduronidase (IDUA) gene and the neurological disease is Hurler syndrome. In one embodiment of this aspect, the target gene is a leucine-rich repeat kinase-2 (LRRK2) gene and the neurological disease is Parkinson&#39;s disease. In one embodiment of this aspect, the target gene is a methyl CpG binding protein 2 (MECP2) gene and the neurological disease is Rett syndrome. In another embodiment of this aspect, the target gene is an ATP-binding cassette subfamily member 4 (ABCA4) gene and the neurological disease is Stargardt disease. 
     In some aspects, provided herein, is a method of treating Hurler syndrome in a subject, the method comprising administering to the subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide directs the adenosine base editor to effect an A-to-G nucleobase alteration in an alpha-L-iduronidase (IDUA) gene or a regulatory element thereof in the subject, thereby treating Hurler syndrome in the subject. 
     In some embodiments, the administration ameliorates at least one symptom related to Hurler syndrome. In some embodiments, the administration results in faster amelioration of at least one symptom related to Hurler syndrome as compared to treatment with a base editor without the amino acid substitution in the adenosine deaminase. 
     In some embodiments, the IDUA gene or regulatory element thereof comprises a SNP associated with Hurler syndrome. In some embodiments, the A-to-G nucleobase alteration is at the SNP associated with Hurler syndrome. In some embodiments, the SNP associated with Hurler syndrome results in a W402X or a W401X amino acid mutation in an IDUA polypeptide as numbered in SEQ ID NO: 4, or a variant thereof, encoded by the IDUA gene, wherein X is a stop codon. In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Hurler syndrome to a wild type nucleobase. In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Hurler syndrome to a non-wild type nucleobase that results in one or more ameliorated symptoms of Hurler syndrome. In some embodiments, the A-to-G alteration at the SNP associated with Hurler Syndrome changes a stop codon to a tryptophan in an IDUA polypeptide encoded by the IDUA gene. 
     In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to the IDUA gene or regulatory element thereof comprising the SNP associated with Hurler syndrome. In some embodiments, the adenosine base editor is in complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to the IDUA gene or regulatory element thereof comprising the SNP associated with Hurler syndrome. In some embodiments, the sgRNA comprises a nucleic acid sequence selected from the group consisting of: 5′-GACUCUAGGCAGAGGUCUCAA-3′ (SEQ ID NO: 7), 5′-ACUCUAGGCAGAGGUCUCAA-3′ (SEQ ID NO: 8), 5′-CUCUAGGCCGAAGUGUCGC-3′ (SEQ ID NO: 9), and 5′-GCUCUAGGCCGAAGUGUCGC-3′ (SEQ ID NO: 10). 
     In some aspects, provided herein, is a method of treating Parkinson&#39;s disease in a subject, the method comprising: administering to the subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide directs the adenosine base editor to effect an A-to-G nucleobase alteration a leucine-rich repeat kinase-2 (LRRK2) gene or a regulatory element thereof in the subject, thereby treating Parkinson&#39;s disease in the subject. 
     In some embodiments, the administration ameliorates at least one symptom related to Parkinson&#39;s disease. In some embodiments, the administration results in faster amelioration of at least one symptom related to Parkinson&#39;s disease as compared to treatment with a base editor without the amino acid substitution in the adenosine deaminase. 
     In some embodiments, the LRRK2 gene or regulatory element thereof comprises a SNP associated with Parkinson&#39;s disease. In some embodiments, the A-to-G nucleobase alteration is at the SNP associated with Parkinson&#39;s disease. In some embodiments, the SNP associated with Parkinson Disease results in a A419V, a R1441C, a R1441H, or a G2019S amino acid mutation in a LRRK2 polypeptide as numbered in SEQ ID NO: 3, or a variant thereof, encoded by the LRRK2 gene. 
     In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Parkinson&#39;s disease to a wild type nucleobase. In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Parkinson&#39;s disease to a non-wild type nucleobase that results in one or more ameliorated symptoms of Parkinson&#39;s disease. In some embodiments, the A-to-G nucleobase alteration changes a Cysteine or Histidine to an Arginine in a LRRK2 polypeptide encoded by the LRRK2 gene. In some embodiments, the A-to-G alteration changes a Serine to a Glycine in a LRRK2 polypeptide encoded by the LRRK2 gene. In some embodiments, the A-to-G alteration replaces the Cysteine (C) or Histidine (H) with an Arginine (R) at position 144 or replaces the Serine with a Glycine (G) at position 2019 of a LRRK2 polypeptide as numbered in SEQ ID NO: 3, or a variant thereof, encoded by the LRRK2 gene. 
     In some aspects, provided herein, is a method of treating Parkinson&#39;s disease in a subject, the method comprising: administering to the subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, and wherein the guide polynucleotide directs the adenosine base editor to effect an A-to-G nucleobase alteration at a SNP in a LRRK2 gene associated with Parkinson&#39;s disease, wherein the SNP does not encode a G2019S mutation in a LRRK2 polypeptide as numbered in SEQ ID NO: 3, or a variant thereof. 
     In some embodiments, the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2 or a corresponding position thereof. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to the LRRK2 gene or regulatory element thereof comprising the SNP associated with Parkinson&#39;s Disease. In some embodiments, the adenosine base editor is in complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to the LRRK2 gene or regulatory element thereof comprising the SNP associated with Parkinson Disease. In some embodiments, the sgRNA comprises a nucleic acid sequence: 5′-AAGCGCAAGCCUGGAGGGAA-3′ (SEQ ID NO: 11); or 5′-ACUACAGCAUUGCUCAGUAC-3′ (SEQ ID NO: 12). 
     In some aspects, provided herein, is a method of treating Rett syndrome in a subject, the method comprising administering to the subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide directs the adenosine base editor to effect an A-to-G nucleobase alteration in a methyl CpG binding protein 2 (MECP2) gene or a regulatory element thereof in the subject, thereby treating Rett syndrome in the subject. 
     In some embodiments, the administration ameliorates at least one symptom related to Rett syndrome. In some embodiments, the administration results in faster amelioration of at least one symptom related to Rett syndrome as compared to treatment with a base editor without the amino acid substitution in the adenosine deaminase. In some embodiments, the MECP2 gene or regulatory element thereof comprises a SNP associated with Rett syndrome. In some embodiments, the A-to-G nucleobase alteration is at the SNP associated with Rett Syndrome. In some embodiments, the SNP associated with Rett syndrome results in a R106W or a T158M amino acid mutation in a MECP2 polypeptide as numbered in SEQ ID NO: 5, or a variant thereof, encoded by the MECP2 gene. In some embodiments, the SNP associated with Rett syndrome results in a R255X or a R270X amino acid mutation in a MECP2 polypeptide encoded by the MECP2 gene, wherein X is a stop codon. 
     In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Rett syndrome to a wild type nucleobase. In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Rett syndrome to a non-wild type nucleobase that results in ameliorated Rett syndrome symptoms. In some embodiments, the A-to-G nucleobase alteration at the SNP associated with Rett Syndrome changes a stop codon to tryptophan in MECP2 polypeptide. 
     In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to the MECP2 gene or regulatory element thereof comprising the SNP associated with Rett syndrome. In some embodiments, the adenosine base editor is in complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to the MECP2 gene or regulatory element thereof comprising the SNP associated with Rett syndrome. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of: 5′-CUUUUCACUUCCUGCCGGGG-3′ (SEQ ID NO: 13), 5′-AGCUUCCAUGUCCAGCCUUC-3′ (SEQ ID NO: 14), 5′-ACCAUGAAGUCAAAAUCAUU-3′ (SEQ ID NO: 15), and 5′-GCUUUCAGCCCCGUUUCUUG-3′ (SEQ ID NO: 16). 
     In some aspects, provided herein, is a method of treating Stargardt disease in a subject, the method comprising administering to the subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide directs the adenosine base editor to effect an A-to-G nucleobase alteration in an ATP-binding cassette subfamily member 4 (ABCA4) gene or a regulatory element thereof in the subject, thereby treating Stargardt disease in the subject. 
     In some embodiments, the administration ameliorates at least one symptom related to Stargardt disease. In some embodiments, the administration results in faster amelioration of at least one symptom related to Stargardt disease as compared to treatment with a base editor without the amino acid substitution in the adenosine deaminase. 
     In some embodiments, the ABCA4 gene comprises a SNP associated with Stargardt disease. In some embodiments, the A-to-G nucleobase alteration is at the SNP associated with Stargardt disease. In some embodiments, the SNP associated with Stargardt disease results in a A1038V or a G1961E amino acid mutation in an ABCA4 polypeptide as numbered in SEQ ID NO: 6, or a variant thereof, encoded by the ABCA4 gene. In some embodiments, the SNP associated with Stargardt disease results in a G1961E amino acid mutation in the ABCA4 polypeptide as numbered in SEQ ID NO: 6, or a variant thereof. 
     In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Stargardt disease to a wild type nucleobase. In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Stargardt disease to a non-wild type nucleobase that results in one or more ameliorated symptoms of Stargardt disease. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to the ABCA4 gene or regulatory element thereof comprising the SNP associated with Stargardt disease. 
     In some embodiments, the adenosine base editor is in complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to the ABCA4 gene or regulatory element thereof comprising the SNP associated with Stargardt Disease. In some embodiments, the sgRNA comprises the sequence 5′-CUCCAGGGCGAACUUCGACACACAGC-3′ (SEQ ID NO: 17). 
     In various aspects, the treatment described herein results in ameliorated symptoms of the neurological disorder compared to treatment with a base editor comprising an adenosine deaminase domain without the amino acid substitutions. 
     In some aspects, provided herein, is a method of editing a target gene or regulatory element thereof associated with a neurological disorder, the method comprising contacting the target gene or regulatory element thereof with (i) an adenosine base editor and (ii) a guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2 or a corresponding position thereof, wherein the guide polynucleotide directs the adenosine base editor to effect an A-to-G nucleobase alteration in a target gene or a regulatory element thereof associated with the neurological disorder. In one embodiment of this aspect, the target gene is a leucine-rich repeat kinase-2 (LRRK2) gene and the neurological disease is Parkinson&#39;s disease. In another embodiment of this aspect, the target gene is an alpha-L-iduronidase (IDUA) gene and the neurological disease is Hurler syndrome. In one embodiment of this aspect, the target gene is a methyl CpG binding protein 2 (MECP2) gene and the neurological disease is Rett syndrome. In another embodiment of this aspect, the target gene is an ATP-binding cassette subfamily member 4 (ABCA4) gene and the neurological disease is Stargardt disease. 
     In some aspects, provided herein, is a method of editing a leucine-rich repeat kinase-2 (LRRK2) gene or a regulatory element thereof, the method comprising contacting the LRRK2 gene or regulatory element thereof with (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide directs the adenosine base editor to effect an A-to-G nucleobase alteration in the LRRK2 gene a regulatory element thereof. 
     In some embodiments, the A-to-G nucleobase alteration is at the SNP associated with Parkinson&#39;s disease. In some embodiments, the SNP associated with Parkinson Disease results in a A419V, a R1441C, a R1441H, or a G2019S amino acid mutation in a LRRK2 polypeptide as numbered in SEQ ID NO: 3, or a variant thereof, encoded by the LRRK2 gene. In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Parkinson&#39;s disease to a wild type nucleobase. In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Parkinson&#39;s disease to a non-wild type nucleobase that results in one or more ameliorated symptoms of Parkinson&#39;s disease. 
     In some embodiments, the A-to-G nucleobase alteration changes a Cysteine or Histidine to an Arginine in a LRRK2 polypeptide encoded by the LRRK2 gene. In some embodiments, the A-to-G alteration changes a Serine to a Glycine in a LRRK2 polypeptide encoded by the LRRK2 gene. In some embodiments, the A-to-G alteration replaces the Cysteine (C) or Histidine (H) with an Arginine (R) at position 144 or replaces the Serine with a Glycine (G) at position 2019 of a LRRK2 polypeptide as numbered in SEQ ID NO: 3, or a variant thereof, encoded by the LRRK2 gene. 
     In some aspects, provided herein, is a method of editing a leucine-rich repeat kinase-2 (LRRK2) gene or a regulatory element thereof, the method comprising contacting the LRRK2 gene or regulatory element thereof with (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, and wherein the guide polynucleotide directs the adenosine base editor to effect an A-to-G nucleobase alteration at a SNP in a LRRK2 gene, wherein the SNP does not encode a G2019S mutation in a LRRK2 polypeptide as numbered in SEQ ID NO: 3, or a variant thereof. 
     In some embodiments, the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2 or a corresponding position thereof. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to the LRRK2 gene or regulatory element thereof comprising the SNP associated with Parkinson&#39;s Disease. 
     In some embodiments, the adenosine base editor is in complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to the LRRK2 gene or regulatory element thereof comprising the SNP associated with Parkinson Disease. In some embodiments, the sgRNA comprises a nucleic acid sequence: 5′-AAGCGCAAGCCUGGAGGGAA-3′ (SEQ ID NO: 11); or 5′-ACUACAGCAUUGCUCAGUAC-3′ (SEQ ID NO: 12). 
     In some aspects, provided herein, is a method of editing an alpha-L-iduronidase (IDUA) gene or a regulatory element thereof, the method comprising contacting the IDUA gene or regulatory element thereof with (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide directs the adenosine base editor to effect an A-to-G nucleobase alteration in the IDUA gene or a regulatory element thereof. 
     In some embodiments, the IDUA gene or regulatory element thereof comprises a SNP associated with Hurler syndrome. In some embodiments, the A-to-G nucleobase alteration is at the SNP associated with Hurler syndrome. In some embodiments, the SNP associated with Hurler syndrome results in a W402X or a W401X amino acid mutation in an IDUA polypeptide as numbered in SEQ ID NO: 4, or a variant thereof, encoded by the IDUA gene, wherein X is a stop codon. 
     In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Hurler syndrome to a wild type nucleobase. In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Hurler syndrome to a non-wild type nucleobase that results in one or more ameliorated symptoms of Hurler syndrome. In some embodiments, the A-to-G alteration at the SNP associated with Hurler Syndrome changes a stop codon to a tryptophan in an IDUA polypeptide encoded by the IDUA gene. 
     In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to the IDUA gene or regulatory element thereof comprising the SNP associated with Hurler syndrome. In some embodiments, the adenosine base editor is in complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to the IDUA gene or regulatory element thereof comprising the SNP associated with Hurler syndrome. In some embodiments, the sgRNA comprises a nucleic acid sequence selected from the group consisting of: 5′-GACUCUAGGCAGAGGUCUCAA-3′ (SEQ ID NO: 7), 5′-ACUCUAGGCAGAGGUCUCAA-3′ (SEQ ID NO: 8), 5′-CUCUAGGCCGAAGUGUCGC-3′ (SEQ ID NO: 9), and 5′-GCUCUAGGCCGAAGUGUCGC-3′ (SEQ ID NO: 10). 
     In some aspects, provided herein, is a method of editing a methyl CpG binding protein 2 (MECP2) gene or regulatory element thereof, the method comprising administering to the subject (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide directs the adenosine base editor to effect an A-to-G nucleobase alteration in the MECP2 gene or a regulatory element thereof. 
     In some embodiments, the MECP2 gene or regulatory element thereof comprises a SNP associated with Rett syndrome. In some embodiments, the A-to-G nucleobase alteration is at the SNP associated with Rett Syndrome. In some embodiments, the SNP associated with Rett syndrome results in a R106W or a T158M amino acid mutation in a MECP2 polypeptide as numbered in SEQ ID NO: 5, or a variant thereof, encoded by the MECP2 gene. In some embodiments, the SNP associated with Rett syndrome results in a R255X or a R270X amino acid mutation in a MECP2 polypeptide encoded by the MECP2 gene, wherein X is a stop codon. 
     In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Rett syndrome to a wild type nucleobase. In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Rett syndrome to a non-wild type nucleobase that results in one or more ameliorated symptoms of Rett syndrome. In some embodiments, the A-to-G nucleobase alteration at the SNP associated with Rett Syndrome changes a stop codon to tryptophan in MECP2 polypeptide. 
     In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to the MECP2 gene or regulatory element thereof comprising the SNP associated with Rett syndrome. In some embodiments, the adenosine base editor is in complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to the MECP2 gene or regulatory element thereof comprising the SNP associated with Rett syndrome. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of: 5′-CUUUUCACUUCCUGCCGGGG-3′ (SEQ ID NO: 13), 5′-AGCUUCCAUGUCCAGCCUUC-3′ (SEQ ID NO: 14), 5′-ACCAUGAAGUCAAAAUCAUU-3′ (SEQ ID NO: 15), and 5′-GCUUUCAGCCCCGUUUCUUG-3′ (SEQ ID NO: 16). 
     In some aspects, provided herein, is a method of editing an ATP binding cassette subfamily member 4 (ABCA4) gene or regulatory element thereof, the method comprising contacting the ABCA4 gene or regulatory element thereof with (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide directs the adenosine base editor to effect an A-to-G nucleobase alteration in the ABCA4 gene or a regulatory element thereof. 
     In some embodiments, the administration ameliorates at least one symptom related to Stargardt disease. In some embodiments, the administration results in faster amelioration of at least one symptom related to Stargardt disease as compared to treatment with a base editor without the amino acid substitution in the adenosine deaminase. 
     In some embodiments, the ABCA4 gene comprises a SNP associated with Stargardt disease. In some embodiments, the A-to-G nucleobase alteration is at the SNP associated with Stargardt disease. In some embodiments, the SNP associated with Stargardt disease results in a A1038V, or a G1961E amino acid mutation in an ABCA4 polypeptide as numbered in SEQ ID NO: 6, or a variant thereof, encoded by the ABCA4 gene. In some embodiments, the SNP associated with Stargardt disease results in a G1961E amino acid mutation in the ABCA4 polypeptide as numbered in SEQ ID NO: 6, or a variant thereof. 
     In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Stargardt disease to a wild type nucleobase. In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Stargardt disease to a non-wild type nucleobase that results in one or more ameliorated symptoms of Stargardt disease. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to the ABCA4 gene or regulatory element thereof comprising the SNP associated with Stargardt Disease. 
     In some embodiments, the adenosine base editor is in complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to the ABCA4 gene or regulatory element thereof comprising the SNP associated with Stargardt Disease. In some embodiments, the sgRNA comprises the sequence 5′-CUCCAGGGCGAACUUCGACACACAGC-3′ (SEQ ID NO: 17). 
     In various embodiments of the above aspects, the contacting is in a cell. In some embodiments, the contacting results in less than 10% indels in a genome in the cell, wherein indel rate is measured by mismatch frequency between sequences flanking the single nucleotide modification and an unmodified sequence. In some embodiments, the contacting results in less than 5% indels in a genome in the cell, wherein indel rate is measured by mismatch frequency between sequences flanking the single nucleotide modification and an unmodified sequence. In some embodiments, the contacting results in less than 1% indels in a genome in the cell, wherein indel rate is measured by mismatch frequency between sequences flanking the single nucleotide modification and an unmodified sequence. 
     In various embodiments of the above aspects, the cell is a neuron. In some embodiments, the contacting is in a population of cells. In some embodiments, the contacting results in the A-to-G nucleobase alteration in at least 40% of the population of cells after the contacting step. In some embodiments, the contacting results in the A-to-G nucleobase alteration in at least 50% of the population of cells after the contacting step. In some embodiments, the contacting results in the A-to-G nucleobase alteration in at least 70% of the population of cells after the contacting step. In some embodiments, at least 90% of the cells are viable after the contacting step. In some embodiments, the population of cells was not enriched after the contacting step. In some embodiments, the population of cells are neurons. In some embodiments, the contacting is in vivo or ex vivo. 
     In various aspects and embodiments above, the polynucleotide programmable DNA binding domain is a Cas9. In some embodiments, the Cas9 is a SpCas9, a SaCas9, or a variant thereof. In some embodiments, the polynucleotide programmable DNA binding domain comprises a modified SpCas9 having an altered protospacer-adjacent motif (PAM) specificity. In some embodiments, the Cas9 has specificity for a PAM sequence selected from the group consisting of NGG, NGA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, and NGC; wherein N is A, G, C, or T; and wherein R is A or G. In some embodiments, the polynucleotide programmable DNA binding domain is a nuclease inactive variant. In some embodiments, the polynucleotide programmable DNA binding domain is a nickase variant. In some embodiments, the nickase variant comprises an amino acid substitution D10A or a corresponding amino acid substitution thereof. In various aspects and embodiments provided herein, the adenosine deaminase domain comprises a TadA domain. In some embodiments, the adenosine deaminase comprises a TadA deaminase comprising a V82S alteration and/or a T166R alteration. 
     In various aspects and embodiments above, the adenosine deaminase further comprises one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, Q154R, or a combination thereof. In various aspects and embodiments provided herein, the adenosine deaminase comprises a combination of alterations selected from the group consisting of: Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y147T+Q154R; Y147T+Q154S; and Y123H+Y147R+Q154R+I76Y. In various aspects and embodiments provided herein, the adenosine base editor domain comprises an adenosine deaminase monomer. In various aspects and embodiments provided herein, the adenosine base editor comprises an adenosine deaminase dimer. In some embodiments, the TadA deaminase is a TadA*8 variant. In some embodiments, the TadA*8 variant is selected from the group consisting of: TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, and TadA*8.13. In some embodiments, the adenosine base editor is an ABE8 base editor selected from the group consisting of: ABE8.1, ABE8.2, ABE8.3, ABE8.4, ABE8.5, ABE8.6, ABE8.7, ABE8.8, ABE8.9, ABE8.10, ABE8.11, ABE8.12, and ABE8.13. 
     In some aspects, provided herein, is a cell produced by the method described in various aspects and embodiments disclosed herein. In some aspects, provided herein, is a population of cells produced by the method described in various aspects and embodiments disclosed herein. 
     In some aspects, provided herein, is a base editor system comprising (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide directs the adenosine base editor to effect an A-to-G nucleobase alteration in a target gene or a regulatory element thereof associated with the neurological disorder. In one embodiment of the above aspect, the target gene is a leucine-rich repeat kinase-2 (LRRK2) gene and the neurological disease is Parkinson&#39;s disease. In another embodiment of this aspect, the target gene is an alpha-L-iduronidase (IDUA) gene and the neurological disease is Hurler syndrome. In one embodiment of this aspect, the target gene is a methyl CpG binding protein 2 (MECP2) gene and the neurological disease is Rett syndrome. In another embodiment of this aspect, the target gene is an ATP-binding cassette subfamily member 4 (ABCA4) gene and the neurological disease is Stargardt disease. 
     In some aspects, provided herein, is a base editor system comprising (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide directs the adenosine base editor to effect an A-to-G nucleobase alteration in a LRRK2 gene a regulatory element thereof. 
     In some embodiments, the A-to-G nucleobase alteration is at a SNP associated with Parkinson&#39;s disease in the LRRK2 gene or regulatory element thereof. In some embodiments, the SNP associated with Parkinson Disease results in a A419V, a R1441C, a R1441H, or a G2019S amino acid mutation in a LRRK2 polypeptide as numbered in SEQ ID NO: 3, or a variant thereof, encoded by the LRRK2 gene. 
     In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Parkinson&#39;s disease to a wild type nucleobase. In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Parkinson&#39;s disease to a non-wild type nucleobase that results in ameliorated Parkinson&#39;s symptoms. In some embodiments, the A-to-G nucleobase alteration changes a Cysteine or Histidine to an Arginine in a LRRK2 polypeptide encoded by the LRRK2 gene. In some embodiments, the A-to-G alteration changes a Serine to a Glycine in a LRRK2 polypeptide encoded by the LRRK2 gene. In some embodiments, the A-to-G alteration replaces the Cysteine (C) or Histidine (H) with an Arginine (R) at position 144 or replaces the Serine with a Glycine (G) at position 2019 of a LRRK2 polypeptide as numbered in SEQ ID NO: 3, or a variant thereof, encoded by the LRRK2 gene. In some embodiments, the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2 or a corresponding position thereof. 
     In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to the LRRK2 gene or regulatory element thereof comprising the SNP associated with Parkinson&#39;s Disease. In some embodiments, the adenosine base editor is in complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to the LRRK2 gene or regulatory element thereof comprising the SNP associated with Parkinson Disease. In some embodiments, the sgRNA comprises a nucleic acid sequence: 5′-AAGCGCAAGCCUGGAGGGAA-3′ (SEQ ID NO: 11); or 5′-ACUACAGCAUUGCUCAGUAC-3′ (SEQ ID NO: 12). 
     In some aspects, provided herein, is a base editor system comprising (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide directs the adenosine base editor to effect an A-to-G nucleobase alteration in an alpha-L-iduronidase (IDUA) gene or a regulatory element thereof. 
     In some embodiments, the IDUA gene or regulatory element thereof comprises a SNP associated with Hurler syndrome. In some embodiments, the A-to-G nucleobase alteration is at the SNP associated with Hurler syndrome. In some embodiments, the SNP associated with Hurler syndrome results in a W402X or a W401X amino acid mutation in an IDUA polypeptide as numbered in SEQ ID NO: 4, or a variant thereof, encoded by the IDUA gene, wherein X is a stop codon. 
     In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Hurler syndrome to a wild type nucleobase. In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Hurler syndrome to a non-wild type nucleobase that results in one or more ameliorated symptoms of Hurler syndrome. In some embodiments, the A-to-G alteration at the SNP associated with Hurler Syndrome changes a stop codon to a tryptophan in an IDUA polypeptide encoded by the IDUA gene. 
     In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to the IDUA gene or regulatory element thereof comprising the SNP associated with Hurler syndrome. In some embodiments, the adenosine base editor is in complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to the IDUA gene or regulatory element thereof comprising the SNP associated with Hurler syndrome. In some embodiments, the sgRNA comprises a nucleic acid sequence selected from the group consisting of: 5′-GACUCUAGGCAGAGGUCUCAA-3′ (SEQ ID NO: 7), 5′-ACUCUAGGCAGAGGUCUCAA-3′ (SEQ ID NO: 8), 5′-CUCUAGGCCGAAGUGUCGC-3′ (SEQ ID NO: 9), and 5′-GCUCUAGGCCGAAGUGUCGC-3′ (SEQ ID NO: 10). 
     In some aspects, provided herein, is a base editor system comprising (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide directs the adenosine base editor to effect an A-to-G nucleobase alteration in a methyl CpG binding protein 2 (MECP2) gene or regulatory element thereof. 
     In some embodiments, the MECP2 gene or regulatory element thereof comprises a SNP associated with Rett syndrome. In some embodiments, the A-to-G nucleobase alteration is at the SNP associated with Rett Syndrome. In some embodiments, the SNP associated with Rett syndrome results in a R106W or a T158M amino acid mutation in a MECP2 polypeptide as numbered in SEQ ID NO: 5, or a variant thereof, encoded by the MECP2 gene. In some embodiments, the SNP associated with Rett syndrome results in a R255X or a R270X amino acid mutation in a MECP2 polypeptide encoded by the MECP2 gene, wherein X is a stop codon. 
     In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Rett syndrome to a wild type nucleobase. In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Rett syndrome to a non-wild type nucleobase that results in one or more ameliorated symptoms of Rett syndrome. In some embodiments, the A-to-G nucleobase alteration at the SNP associated with Rett Syndrome changes a stop codon to tryptophan in MECP2 polypeptide. 
     In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to the MECP2 gene or regulatory element thereof comprising the SNP associated with Rett syndrome. In some embodiments, the adenosine base editor is in complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to the MECP2 gene or regulatory element thereof comprising the SNP associated with Rett syndrome. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence selected from the group consisting of: 5′-CUUUUCACUUCCUGCCGGGG-3′ (SEQ ID NO: 13), 5′-AGCUUCCAUGUCCAGCCUUC-3′ (SEQ ID NO: 14), 5′-ACCAUGAAGUCAAAAUCAUU-3′ (SEQ ID NO: 15), and 5′-GCUUUCAGCCCCGUUUCUUG-3′ (SEQ ID NO: 16). 
     In some aspects, provided herein, is a base editor system comprising contacting (i) an adenosine base editor or a nucleic acid sequence encoding the adenosine base editor and (ii) a guide polynucleotide or a nucleic acid sequence encoding the guide polynucleotide, wherein the adenosine base editor comprises a programmable DNA binding domain and an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid substitution at amino acid position 82 or 166 as numbered in SEQ ID NO: 2 or a corresponding position thereof, and wherein the guide polynucleotide directs the adenosine base editor to effect an A-to-G nucleobase alteration in an ATP binding cassette subfamily member 4 (ABCA4) gene or regulatory element thereof. 
     In some embodiments, the administration ameliorates at least one symptom related to Stargardt disease. In some embodiments, the administration results in faster amelioration of at least one symptom related to Stargardt disease as compared to treatment with a base editor without the amino acid substitution in the adenosine deaminase. In some embodiments, the ABCA4 gene comprises a SNP associated with Stargardt disease. In some embodiments, the A-to-G nucleobase alteration is at the SNP associated with Stargardt disease. In some embodiments, the SNP associated with Stargardt disease results in a A1038V, or a G1961E amino acid mutation in an ABCA4 polypeptide as numbered in SEQ ID NO: 6, or a variant thereof, encoded by the ABCA4 gene. In some embodiments, the SNP associated with Stargardt disease results in a G1961E amino acid mutation in the ABCA4 polypeptide as numbered in SEQ ID NO: 6, or a variant thereof. 
     In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Stargardt disease to a wild type nucleobase. In some embodiments, the A-to-G nucleobase alteration changes the SNP associated with Stargardt disease to a non-wild type nucleobase that results in ameliorated Stargardt Disease symptoms. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence complementary to the ABCA4 gene or regulatory element thereof comprising the SNP associated with Stargardt Disease. 
     In some embodiments, the adenosine base editor is in complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to the ABCA4 gene or regulatory element thereof comprising the SNP associated with Stargardt Disease. In some embodiments, the sgRNA comprises the sequence 5′-CUCCAGGGCGAACUUCGACACACAGC-3′ (SEQ ID NO: 17). 
     In various aspects and embodiments provided herein, the polynucleotide programmable DNA binding domain is a Cas9. In some embodiments, the Cas9 is a SpCas9, a SaCas9, or a variant thereof. In some embodiments, the polynucleotide programmable DNA binding domain comprises a modified SpCas9 having an altered protospacer-adjacent motif (PAM) specificity. In some embodiments, the Cas9 has specificity for a PAM sequence selected from the group consisting of NGG, NGA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, and NGC, wherein N is A, G, C, or T and wherein R is A or G. In some embodiments, the polynucleotide programmable DNA binding domain is a nuclease inactive variant. In some embodiments, the polynucleotide programmable DNA binding domain is a nickase variant. In some embodiments, the nickase variant comprises an amino acid substitution D10A or a corresponding amino acid substitution thereof. 
     In various aspects and embodiments provided herein, the adenosine deaminase domain comprises a TadA domain. In some embodiments, the adenosine deaminase comprises a TadA deaminase comprising a V82S alteration and/or a T166R alteration. 
     In various aspects and embodiments provided herein, the adenosine deaminase further comprises one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, Q154R, or a combination thereof. In various aspects and embodiments provided herein, the adenosine deaminase comprises a combination of alterations selected from the group consisting of: Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y147T+Q154R; Y147T+Q154S; and Y123H+Y147R+Q154R+I76Y. In some embodiments, the adenosine base editor domain comprises an adenosine deaminase monomer. In some embodiments, the adenosine base editor comprises an adenosine deaminase dimer. 
     In various aspects and embodiments provided herein, the TadA deaminase is a TadA*8 variant. In some embodiments, the TadA*8 variant is selected from the group consisting of: TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, and TadA*8.13. In some embodiments, the adenosine base editor is an ABE8 base editor selected from the group consisting of: ABE8.1, ABE8.2, ABE8.3, ABE8.4, ABE8.5, ABE8.6, ABE8.7, ABE8.8, ABE8.9, ABE8.10, ABE8.11, ABE8.12, and ABE8.13. 
     In some aspects, provided herein, is a vector comprising the nucleic acid sequence encoding the adenosine base editor described herein. In some aspects, provided herein, is a vector comprising the nucleic acid sequence encoding the adenosine base editor and the guide polynucleotide described herein. In some embodiments, the vector is a viral vector, a lentiviral vector, or an AAV vector. 
     In some aspects, provided herein, is a cell comprising the base editor system or the vector described herein. In some embodiments, the cell is a central nervous system cell. In some embodiments, the cell is a neuron. In some embodiments, the cell is a photoreceptor. In some embodiments, the cell is in vitro, in vivo, or ex vivo. 
     In some aspects, provided herein, is a pharmaceutical composition comprising the base editor, the vector, or the cell described herein and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition described herein further comprises a lipid. In another embodiment, the pharmaceutical composition described herein further comprises a virus. 
     In some aspects, provided herein, is a kit comprising the base editor or the vector described herein. 
     In various embodiments of the methods described herein, at least one nucleotide of the guide polynucleotide comprises a non-naturally occurring modification. In various embodiments of the methods described herein, at least one nucleotide of the nucleic acid sequence comprises a non-naturally occurring modification. In various embodiments, at least one nucleotide of the nucleic acid sequence of the base editor system comprises a non-naturally occurring modification. In some embodiments, the non-naturally occurring modification is a chemical modification. In some embodiments, the chemical modification is a 2′-O-methylation. In some embodiments, the nucleic acid sequence comprises a phosphorothioate. 
     The description and examples herein illustrate embodiments of the present disclosure in detail. It is to be understood that this disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this disclosure, which are encompassed within its scope. 
     The practice of some embodiments disclosed herein employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R. I. Freshney, ed. (2010)). 
     The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. 
     Although various features of the present disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. 
     The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and in view of the accompanying drawings as described hereinbelow. 
     Definitions 
     The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. 
     Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale &amp; Marham, The Harper Collins Dictionary of Biology (1991). 
     In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or,” unless stated otherwise, and is understood to be inclusive. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting. 
     As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure. 
     The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, such as within 5-fold or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed. 
     Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. 
     Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures. 
     By “abasic base editor” is meant an agent capable of excising a nucleobase and inserting a DNA nucleobase (A, T, C, or G). Abasic base editors comprise a nucleic acid glycosylase polypeptide or fragment thereof. In one embodiment, the nucleic acid glycosylase is a mutant human uracil DNA glycosylase comprising an Asp at amino acid 204 (e.g., replacing an Asn at amino acid 204) in the following sequence, or corresponding position in a uracil DNA glycosylase, and having cytosine-DNA glycosylase activity, or active fragment thereof. In one embodiment, the nucleic acid glycosylase is a mutant human uracil DNA glycosylase comprising an Ala, Gly, Cys, or Ser at amino acid 147 (e.g., replacing a Tyr at amino acid 147) in the following sequence, or corresponding position in a uracil DNA glycosylase, and having thymine-DNA glycosylase activity, or an active fragment thereof. The sequence of exemplary human uracil-DNA glycosylase, isoform 1, follows: 
     
       
         
           
               
               
               
            
               
                   
                   
                 (SEQ ID NO: 18) 
               
               
                   
                 1 
                 mgvfclgpwg igrklrtpgk gplqllsrlc 
               
               
                   
               
               
                   
                   
                 gdhlqaipak kapagqeepg tppssplsae 
               
               
                   
               
               
                   
                 61 
                 qldriqrnka aallrlaarn vpvgfgeswk 
               
               
                   
               
               
                   
                   
                 khlsgefgkp yfikimgfva eerkhytvyp 
               
               
                   
               
               
                   
                 121 
                 pphqvftwtq mcdikdvkvv ilgqdp   y   hgp 
               
               
                   
               
               
                   
                   
                 nqahglcfsv qrpvppppsl eniykelstd 
               
               
                   
               
               
                   
                 181 
                 iedfvhpghg dlsgwakqgv lll   n   avltvr 
               
               
                   
               
               
                   
                   
                 ahqanshker gweqftdavv swlnqnsngl 
               
               
                   
               
               
                   
                 241 
                 vfllwgsyaq kkgsaidrkr hhvlqtahps 
               
               
                   
               
               
                   
                   
                 p   l   svy   r   gffg crhfsktnel iqksgkkpid 
               
               
                   
               
               
                   
                 301 
                 wkel 
               
            
           
         
       
     
     The sequence of human uracil-DNA glycosylase, isoform 2, follows: 
     
       
         
           
               
               
               
            
               
                   
                   
                 (SEQ ID NO: 19) 
               
               
                   
                 1 
                 migqktlysf fspsparkrh apspepavqg 
               
               
                   
               
               
                   
                   
                 tgvagvpees gdaaaipakk apagqeepgt 
               
               
                   
               
               
                   
                 61 
                 ppssplsaeq idriqrnkaa allrlaarnv 
               
               
                   
               
               
                   
                   
                 pvgfgeswkk hlsgefgkpy fikimgfvae 
               
               
                   
               
               
                   
                 121 
                 erkhytvypp phqvftwtqm cdikdvkvvi 
               
               
                   
               
               
                   
                   
                 lgqdp   y   hgpn qahglcfsvq rpvppppsle 
               
               
                   
               
               
                   
                 181 
                 niykelstdi edfvhpghgd isgwakqgvl 
               
               
                   
               
               
                   
                   
                 ll   n   avltvra hqanshkerg weqftdavvs 
               
               
                   
               
               
                   
                 241 
                 wlnqnsnglv fllwgsyaqk kgsaidrkrh 
               
               
                   
               
               
                   
                   
                 hvlqtahpsp    l   svy   r   gffgc rhfsktnell 
               
               
                   
               
               
                   
                 301 
                 qksgkkpidw kel 
               
            
           
         
       
     
     In other embodiments, the abasic editor is any one of the abasic editors described in PCT/JP2015/080958 and US20170321210, which are incorporated herein by reference. In particular embodiments, the abasic editor comprises a mutation at a position shown in the sequence above in bold with underlining or at a corresponding amino acid in any other abasic editor or uracil deglycosylase known in the art. In one embodiment, the abasic editor comprises a mutation at Y147, N204, L272, and/or 8276, or corresponding position. In another embodiment, the abasic editor comprises a Y147A or Y147G mutation, or corresponding mutation. In another embodiment, the abasic editor comprises a N204D mutation, or corresponding mutation. In another embodiment, the abasic editor comprises a L272A mutation, or corresponding mutation. In another embodiment, the abasic editor comprises a R276E or R276C mutation, or corresponding mutation. 
     By “adenosine deaminase” is meant a polypeptide or fragment thereof capable of catalyzing the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase catalyzing the hydrolytic deamination of adenosine to inosine or deoxy adenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases (e.g., engineered adenosine deaminases, evolved adenosine deaminases) provided herein may be from any organism, such as a bacterium. 
     In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is TadA variant. In some embodiments, the TadA variant is a TadA*8. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to a naturally occurring deaminase. For example, deaminase domains are described in International PCT Application Nos. PCT/2017/045381 (WO 2018/027078) and PCT/US2016/058344 (WO 2017/070632), each of which is incorporated herein by reference for its entirety. Also, see Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3: eaao4774 (2017)), and Rees, H. A., et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 December; 19(12):770-788. doi: 10.1038/s41576-018-0059-1, the entire contents of which are hereby incorporated by reference. 
     A wild type TadA(wt) adenosine deaminase has the following sequence (also termed TadA reference sequence): 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 2) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVG 
               
               
                   
               
               
                   
                 AVLVHNNRVIGEGWNRPIGRHDPTAHAEIMA 
               
               
                   
               
               
                   
                 LRQGGLVMQNYRLIDATLYVTLEPCVMCAGA 
               
               
                   
               
               
                   
                 MIHSRIGRVVFGARDAKTGAAGSLMDVLHHP 
               
               
                   
               
               
                   
                 GMNHRVEITEGILADECAALLSDFFRMRRQE 
               
               
                   
               
               
                   
                 IKAQKKAQSSTD. 
               
            
           
         
       
     
     In some embodiments, the adenosine deaminase comprises an alteration in the following sequence: 
                            (SEQ ID NO: 20)           MSEVEESHEY WMRHALTLAK RARDEREVPV                   GAVLVLNNRV IGEGWNRAIG LHDPTAHAEI                   MALRQGGLVM QNYRLIDATL YVTFEPCVMC                   AGAMIHSRIG RVVFGVRNAK TGAAGSLMDV                   LHYPGMNHRV EITEGILADE CAALLCYFFR                   MPRQVFNAQK KAQSSTD            
(also termed TadA*7.10).
 
     In some embodiments, TadA*7.10 comprises at least one alteration. In some embodiments, TadA*7.10 comprises an alteration at amino acid 82 and/or 166. In particular embodiments, a variant of the above-referenced sequence comprises one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and/or Q154R. The alteration Y123H is also referred to herein as H123H (the alteration H123Y in TadA*7.10 reverted back to Y123H (wt)). In other embodiments, a variant of the TadA*7.10 sequence comprises a combination of alterations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. 
     In other embodiments, the invention provides adenosine deaminase variants that include deletions, e.g., TadA*8, comprising a deletion of the C terminus beginning at residue 149, 150, 151, 152, 153, 154, 155, 156, or 157. In other embodiments, the adenosine deaminase variant is a TadA (e.g., TadA*8) monomer comprising one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and/or Q154R. In other embodiments, the adenosine deaminase variant is TadA (e.g., TadA*8) a monomer comprising a combination of alterations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. 
     In still other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8) each having one or more of the following alterations Y147T, Y147R, Q154S, Y123H, V82S, T166R, and/or Q154R. In other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8) each having a combination of alterations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. 
     In other embodiments, the adenosine deaminase variant is a heterodimer comprising a wild-type TadA adenosine deaminase domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising one or more of the following alterations Y147T, Y147R, Q154S, Y123H, V82S, T166R, and/or Q154R. In other embodiments, the adenosine deaminase variant is a heterodimer comprising a wild-type TadA adenosine deaminase domain and an adenosine deaminase variant domain (e.g. TadA*8) comprising a combination of alterations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. 
     In other embodiments, the adenosine deaminase variant is a heterodimer comprising a TadA*7.10 domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising one or more of the following alterations Y147T, Y147R, Q154S, Y123H, V82S, T166R, and/or Q154R. In other embodiments, the adenosine deaminase variant is a heterodimer comprising a TadA*7.10 domain and an adenosine deaminase variant domain (e.g. TadA*8) comprising a combination of the following alterations: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; or I76Y+V82S+Y123H+Y147R+Q154R. 
     In one embodiment, the adenosine deaminase is a TadA*8 that comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: 
     
       
         
           
               
               
            
               
                   
                 SEQ ID NO: 21) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRARDEREVP 
               
               
                   
               
               
                   
                 VGAVLVLNNRVIGEGWNRAIGLHDPTAHA 
               
               
                   
               
               
                   
                 EIMALRQGGLVMQNYRLIDATLYVTFEPC 
               
               
                   
               
               
                   
                 VMCAGAMIHSRIGRVVFGVRNAKTGAAGS 
               
               
                   
               
               
                   
                 LMDVLHYPGMNHRVEITEGILADECAALL 
               
               
                   
               
               
                   
                 CTFFRMPRQVFNAQKKAQSSTD. 
               
            
           
         
       
     
     In some embodiments, the TadA*8 is truncated. In some embodiments, the truncated TadA*8 is missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to the full length TadA*8. In some embodiments, the truncated TadA*8 is missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to the full length TadA*8. In some embodiments the adenosine deaminase variant is a full-length TadA*8. 
     In particular embodiments, an adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from one of the following: 
     In particular embodiments, an adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from one of the following: 
     
       
         
           
               
               
            
               
                   
                   Escherichia   coli  TadA: 
               
               
                   
                 (SEQ ID NO: 22) 
               
               
                   
                 MRRAFITGVFFLSEVEFSHEYWMRHALTLAKRAWD 
               
               
                   
               
               
                   
                 EREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAE 
               
               
                   
               
               
                   
                 IMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAM 
               
               
                   
               
               
                   
                 IHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHR 
               
               
                   
               
               
                   
                 VEITEGILADECAALLSDFFRMRRQEIKAQKKAQS 
               
               
                   
               
               
                   
                 STD 
               
               
                   
               
               
                   
                   E.   coli  TadA (N-terminal truncated): 
               
               
                   
                 (SEQ ID NO: 2) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLV 
               
               
                   
               
               
                   
                 HNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVM 
               
               
                   
               
               
                   
                 ONYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFG 
               
               
                   
               
               
                   
                 ARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADE 
               
               
                   
               
               
                   
                 CAALLSDFFRMRRQEIKAQKKAQSSTD 
               
               
                   
               
               
                   
                 
                   Staphylococcus aureus 
                 
               
               
                   
                 ( S. aureus ) TadA: 
               
               
                   
                 (SEQ ID NO: 23) 
               
               
                   
                 MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIIT 
               
               
                   
               
               
                   
                 KDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVL 
               
               
                   
               
               
                   
                 GSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYG 
               
               
                   
               
               
                   
                 ADDPKGGCSGSLMNLLQOSNFNHRAIVDKGVLKEA 
               
               
                   
               
               
                   
                 CSTLLTTFFKNLRANKKSTN 
               
               
                   
               
               
                   
                 
                   Bacillus subtilis 
                 
               
               
                   
                 ( B. subtilis ) TadA: 
               
               
                   
                 (SEQ ID NO: 24) 
               
               
                   
                 MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGE 
               
               
                   
               
               
                   
                 IIARAHNLRETEQRSIAHAEMLVIDEACKALGTWR 
               
               
                   
               
               
                   
                 LEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDP 
               
               
                   
               
               
                   
                 KGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGM 
               
               
                   
               
               
                   
                 LSAFFRELRKKKKAARKNLSE 
               
               
                   
               
               
                   
                 Salmonella typhimurium 
               
               
                   
                 (S. typhimurium) TadA: 
               
               
                   
                 (SEQ ID NO: 25) 
               
               
                   
                 MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWD 
               
               
                   
               
               
                   
                 EREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAHAE 
               
               
                   
               
               
                   
                 IMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAM 
               
               
                   
               
               
                   
                 VHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHR 
               
               
                   
               
               
                   
                 VEIIEGVLRDECATLLSDFFRMRRQEIKALKKADR 
               
               
                   
               
               
                   
                 AEGAGPAV 
               
               
                   
               
               
                   
                 
                   Shewanella putrefaciens 
                 
               
               
                   
                 ( S.   putrefaciens ) TadA: 
               
               
                   
                 (SEQ ID NO: 26) 
               
               
                   
                 MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQI 
               
               
                   
               
               
                   
                 ATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLL 
               
               
                   
               
               
                   
                 DATLYITLEPCAMCAGAMVHSRIARWYGARDEKTG 
               
               
                   
               
               
                   
                 AAGTWNLLQHPAFNHQVEVTSGVLAEACSAQLSRF 
               
               
                   
               
               
                   
                 FKRRRDEKKALKLAQRAQQGIE 
               
               
                   
               
               
                   
                   Haemophilus influenzae  F3031 
               
               
                   
                 ( H. influenzae ) TadA: 
               
               
                   
                 (SEQ ID NO: 27) 
               
               
                   
                 MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGA 
               
               
                   
               
               
                   
                 VLVDDARNIIGEGWNLSIVQSDPTAHAEIIALRNG 
               
               
                   
               
               
                   
                 AKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKR 
               
               
                   
               
               
                   
                 LVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGV 
               
               
                   
               
               
                   
                 LAEECSQKLSTFFQKRREEKKIEKALLKSLSDK 
               
               
                   
               
               
                   
                 
                   Caulobacter crescentus 
                 
               
               
                   
                 ( C.   crescentus ) TadA: 
               
               
                   
                 (SEQ ID NO: 28) 
               
               
                   
                 MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAV 
               
               
                   
               
               
                   
                 ILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAA 
               
               
                   
               
               
                   
                 AAKLGNYRLTDLTLWTLEPCAMCAGAISHARIGRW 
               
               
                   
               
               
                   
                 FGADDPKGGAWHGPKFFAQPTCHWRPEVTGGVLAD 
               
               
                   
               
               
                   
                 ESADLLRGFFRARRKAKI 
               
               
                   
               
               
                   
                 
                   Geobacter sulfurreducens 
                 
               
               
                   
                 ( G.   sulfurreducens ) TadA: 
               
               
                   
                 (SEQ ID NO: 29) 
               
               
                   
                 MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIG 
               
               
                   
               
               
                   
                 AVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQA 
               
               
                   
               
               
                   
                 ARRSANWRLTGATLYVTLEPCLMCMGAIILARLER 
               
               
                   
               
               
                   
                 VVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGV 
               
               
                   
               
               
                   
                 CQEECGTMLSDFFRDLRRRKKAKATPALFIDERKV 
               
               
                   
               
               
                   
                 PPEP 
               
               
                   
               
               
                   
                 TadA*7.10 
               
               
                   
                 (SEQ ID NO: 20) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLV 
               
               
                   
               
               
                   
                 LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVM 
               
               
                   
               
               
                   
                 QNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFG 
               
               
                   
               
               
                   
                 VRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADE 
               
               
                   
               
               
                   
                 CAALLCYFFRMPRQVFNAQKKAQSSTD 
               
            
           
         
       
     
     Additional TadA7.10 or TadA7.10 variants contemplated as a component of a heterodimer with a TadA*8 include: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 30) 
               
               
                   
                 GSSGSETPGTSESATPESSGSEVEFSHEYWMRHAL 
               
               
                   
               
               
                   
                 TLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGL 
               
               
                   
               
               
                   
                 HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEP 
               
               
                   
               
               
                   
                 CVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVL 
               
               
                   
               
               
                   
                 HYPGMNHRVEITEGILADECAALLCYFFRMPRQVF 
               
               
                   
               
               
                   
                 NAQKKAQSSTD 
               
               
                   
               
               
                   
                 TadA7.10 CP65 
               
               
                   
                 (SEQ ID NO: 31) 
               
               
                   
                 TAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVM 
               
               
                   
               
               
                   
                 CAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP 
               
               
                   
               
               
                   
                 GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQ 
               
               
                   
               
               
                   
                 KKAQSSTDGSSGSETPGTSESATPESSGSEVEFSH 
               
               
                   
               
               
                   
                 EYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGE 
               
               
                   
               
               
                   
                 GWNRAIGLHDP 
               
               
                   
               
               
                   
                 TadA7.10 CP83 
               
               
                   
                 (SEQ ID NO: 32) 
               
               
                   
                 YRL1DATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKT 
               
               
                   
               
               
                   
                 GAAGSLMDVLHYPGMNHRVEITEGILADECAALLC 
               
               
                   
               
               
                   
                 YFFRMPRQVFNAQKKAQSSTDGSSGSETPGTSESA 
               
               
                   
               
               
                   
                 TPESSGSEVEFSHEYWMRHALTLAKRARDEREVPV 
               
               
                   
               
               
                   
                 GAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQ 
               
               
                   
               
               
                   
                 GGLVMQN 
               
               
                   
               
               
                   
                 TadA7.10 CP 136 
               
               
                   
                 (SEQ ID NO: 33) 
               
               
                   
                 MNHRVEITEGILADECAALLCYFFRMPRQVFNAQK 
               
               
                   
               
               
                   
                 KAQSSTDGSSGSETPGTSESATPESSGSEVEFSHE 
               
               
                   
               
               
                   
                 YWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEG 
               
               
                   
               
               
                   
                 WNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDAT 
               
               
                   
               
               
                   
                 LYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAA 
               
               
                   
               
               
                   
                 GSLMDVLHYPG 
               
               
                   
               
               
                   
                 TadA7.10 C-truncate 
               
               
                   
                 (SEQ ID NO: 34) 
               
               
                   
                 GSSGSETPGTSESATPESSGSEVEFSHEYWMRHAL 
               
               
                   
               
               
                   
                 TLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGL 
               
               
                   
               
               
                   
                 HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEP 
               
               
                   
               
               
                   
                 CVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVL 
               
               
                   
               
               
                   
                 HYPGMNHRVEITEGILADECAALLCYFFRMPRQVF 
               
               
                   
               
               
                   
                 N 
               
               
                   
               
               
                   
                 TadA7.10 C-truncate 2 
               
               
                   
                 (SEQ ID NO: 35) 
               
               
                   
                 GSSGSETPGTSESATPESSGSEVEFSHEYWMRHAL 
               
               
                   
               
               
                   
                 TLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGL 
               
               
                   
               
               
                   
                 HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEP 
               
               
                   
               
               
                   
                 CVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVL 
               
               
                   
               
               
                   
                 HYPGMNHRVEITEGILADECAALLCYFFRMPRQ 
               
               
                   
               
               
                   
                 TadA7.10 delta59-66+C-truncate 
               
               
                   
                 (SEQ ID NO: 36) 
               
               
                   
                 GSSGSETPGTSESATPESSGSEVEFSHEYWMRHAL 
               
               
                   
               
               
                   
                 TLAKRARDEREVPVGAVLVLNNRVIGEGWNRAHAE 
               
               
                   
               
               
                   
                 IMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAM 
               
               
                   
               
               
                   
                 IHSRIGRWFGVRNAKTGAAGSLMDVLHYPGMNHRV 
               
               
                   
               
               
                   
                 EITEGILADECAALLCYFFRMPRQVFN 
               
               
                   
               
               
                   
                 TadA7.10 delta 59-66 
               
               
                   
                 (SEQ ID NO: 37) 
               
               
                   
                 GSSGSETPGTSESATPESSGSEVEFSHEYWMRHAL 
               
               
                   
               
               
                   
                 TLAKRARDEREVPVGAVLVLNNRVIGEGWNRAHAE 
               
               
                   
               
               
                   
                 IMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAM 
               
               
                   
               
               
                   
                 IHSRIGRWFGVRNAKTGAAGSLMDVLHYPGMNHRV 
               
               
                   
               
               
                   
                 EITEGILADECAALLCYFFRMPRQVFNAQKKAQSS 
               
               
                   
               
               
                   
                 TD. 
               
            
           
         
       
     
     In some embodiments, the adenosine deaminase variant comprises an alteration in TadA7.10. In some embodiments, TadA7.10 comprises an alteration at amino acid 82 or 166. In particular embodiments, a variant in the above-referenced sequence comprises one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R. In other embodiments, the adenosine deaminase variant comprises a combination of alterations selected from the group consisting of Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y147T+Q154R; Y147T+Q154S; and Y123H+Y147R+Q154R+I76Y. 
     In other embodiments, the invention provides adenosine deaminase variants that include deletions, e.g., TadA7.10 comprising a deletion of the C terminus beginning at residue 149, 150, 151, 152, 153, 154, 155, 156, or 157. In other embodiments, the adenosine deaminase variant is a TadA monomer comprising one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, Q154R. In other embodiments, the adenosine deaminase variant is a monomer comprising the following alterations: Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y147T+Q154R; Y147T+Q154S; and Y123H+Y147 R+Q154R+I76Y. In still other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains each having one or more of the following alterations Y147T, Y147R, Q154S, Y123H, V82S, T166R, Q154R. In other embodiments, the adenosine deaminase variant is a heterodimer comprising a wild-type adenosine deaminase domain or a TadA7.10 domain and an adenosine deaminase variant domain comprising one or more of the following alterations Y147T, Y147R, Q154S, Y123H, V82S, T166R, Q154R. In other embodiments, the adenosine deaminase variant is a heterodimer comprising a TadA7.10 domain and an adenosine deaminase variant of TadA7.10 comprising the following alterations: Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y147T+Q154R; Y147T+Q154S; and Y123H+Y147R+Q154R+I76Y. 
     “Administering” is referred to herein as providing one or more compositions described herein to a patient or a subject. By way of example and without limitation, composition administration, e.g., injection, can be performed by intravenous (i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes can be employed. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. In some embodiments, parenteral administration includes infusing or injecting intravascularly, intravenously, intramuscularly, intraarterially, intrathecally, intratumorally, intradermally, intraperitoneally, transtracheally, subcutaneously, subcuticularly, intraarticularly, subcapsularly, subarachnoidly and intrasternally. Alternatively, or concurrently, administration can be by the oral route. 
     By “agent” is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof. 
     By “alteration” is meant a change (e.g. increase or decrease) in the structure, expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a change in a polynucleotide or polypeptide sequence or a change in expression levels, such as a 10% change, a 25% change, a 40% change, a 50% change, or greater. 
     By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. 
     By “analog” is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polynucleotide or polypeptide analog retains the biological activity of a corresponding naturally-occurring polynucleotide or polypeptide, while having certain modifications that enhance the analog&#39;s function relative to a naturally occurring polynucleotide or polypeptide. Such modifications could increase the analog&#39;s affinity for DNA, efficiency, specificity, protease or nuclease resistance, membrane permeability, and/or half-life, without altering, for example, ligand binding. An analog may include an unnatural nucleotide or amino acid. 
     By “base editor (BE)” or “nucleobase editor (NBE)” is meant an agent that binds a polynucleotide and has nucleobase modifying activity. In various embodiment, the base editor comprises a nucleobase modifying polypeptide (e.g., a deaminase) and a nucleic acid programmable nucleotide binding domain in conjunction with a guide polynucleotide (e.g., guide RNA). In various embodiments, the agent is a biomolecular complex comprising a protein domain having base editing activity, i.e., a domain capable of modifying a base (e.g., A, T, C, G, or U) within a nucleic acid molecule (e.g., DNA). In some embodiments, the polynucleotide programmable DNA binding domain is fused or linked to a deaminase domain. In one embodiment, the agent is a fusion protein comprising a domain having base editing activity. In another embodiment, the protein domain having base editing activity is linked to the guide RNA (e.g., via an RNA binding motif on the guide RNA and an RNA binding domain fused to the deaminase). In some embodiments, the domain having base editing activity is capable of deaminating a base within a nucleic acid molecule. In some embodiments, the base editor is capable of deaminating one or more bases within a DNA molecule. In some embodiments, the base editor is capable of deaminating an adenosine (A) within DNA. In some embodiments, the base editor is an adenosine base editor (ABE). 
     By “cytidine deaminase” is meant a polypeptide or fragment thereof capable of catalyzing a deamination reaction that converts an amino group to a carbonyl group. In some embodiments the cytidine deaminase has at least about 85% identity to APOBEC or AID. In one embodiment, the cytidine deaminase converts cytosine to uracil or 5-methylcytosine to thymine. PmCDA1, which is derived from  Petromyzon marinus  ( Petromyzon marinus  cytosine deaminase 1, “PmCDA1”), AID (Activation-induced cytidine deaminase; AICDA), which is derived from a mammal (e.g., human, swine, bovine, horse, monkey etc.), and APOBEC are exemplary cytidine deaminases. 
     In some embodiments, the base editor is a reprogrammable base editor fused to a deaminase (e.g., an adenosine deaminase or cytidine deaminase). In some embodiments, the base editor is a Cas9 fused to a deaminase (e.g., an adenosine deaminase or cytidine deaminase). In some embodiments, the base editor is a nuclease-inactive Cas9 (dCas9) fused to a deaminase (e.g., an adenosine deaminase or cytidine deaminase). In some embodiments, the Cas9 is a circular permutant Cas9 (e.g., spCas9 or saCas9). Circular permutant Cas9s are known in the art and described, for example, in Oakes et al., Cell 176, 254-267, 2019. In some embodiments, the base editor is fused to an inhibitor of base excision repair, for example, a UGI domain, or a dISN domain. In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase and an inhibitor of base excision repair, such as a UGI or dISN domain. In other embodiments, the base editor is an abasic base editor. 
     In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, an adenosine deaminase is evolved from TadA. In some embodiments, the base editors of the present invention comprise a napDNAbp domain with an internally fused catalytic (e.g., deaminase) domain. In some embodiments, the napDNAbp is a Cas12a (Cpf1) with an internally fused deaminase domain. In some embodiments, the napDNAbp is a Cas12b (c2c1) with an internally fused deaminase domain. In some embodiments, the napDNAbp is a Cas12c (c2c3) with an internally fused deaminase domain. In some embodiments, the napDNAbp is a Cas12d (CasX) with an internally fused deaminase domain. In some embodiments, the napDNAbp is a Cas12e (CasY) with an internally fused deaminase domain. In some embodiments, the napDNAbp is a Cas12g with an internally fused deaminase domain. In some embodiments, the napDNAbp is a Cas12 h with an internally fused deaminase domain. In some embodiments, napDNAbp is a Cas12i with an internally fused deaminase domain. In some embodiments, the base editor is a catalytically dead Cas12 (dCas12) fused to a deaminase domain. In some embodiments, the base editor is a Cas12 nickase (nCas12) fused to a deaminase domain. 
     In some embodiments, base editors are generated (e.g., ABE8) by cloning an adenosine deaminase variant (e.g., TadA*8) into a scaffold that includes a circular permutant Cas9 (e.g., spCAS9 or saCAS9) and a bipartite nuclear localization sequence. Circular permutant Cas9s are known in the art and described, for example, in Oakes et al., Cell 176, 254-267, 2019. Exemplary circular permutants follow where the bold sequence indicates sequence derived from Cas9, the italics sequence denotes a linker sequence, and the underlined sequence denotes a bipartite nuclear localization sequence. 
     CP5 (with MSP “NGC=Pam Variant with mutations Regular Cas9 likes NGG” PID=Protein Interacting Domain and “D10A” nickase): 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 38) 
               
               
                   
                 
                   EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPL 
                 
               
               
                   
                   
               
               
                   
                 
                   IETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKK 
                 
               
               
                   
                   
               
               
                   
                 
                   TEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYG 
                 
               
               
                   
                   
               
               
                   
                 
                   GFMQPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT 
                 
               
               
                   
                   
               
               
                   
                 
                   IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS 
                 
               
               
                   
                   
               
               
                   
                 
                   LFELENGRKRMLASAKFLQKGNELALPSKYVNFLY 
                 
               
               
                   
                   
               
               
                   
                 
                   LASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQ 
                 
               
               
                   
                   
               
               
                   
                 
                   ISEFSKRVILADANLDKVLSAYNKHRDKPIREQAE 
                 
               
               
                   
                   
               
               
                   
                 
                   NIIHLFTLTNLGAPRAFKYFDTTIARKEYRSTKEV 
                 
               
               
                   
                   
               
               
                   
                 
                   LDATLIHQSITGLYETRIDLSQLGGD 
                   GGSGGSGGS 
                 
               
               
                   
                   
               
               
                   
                 
                   GGSGGSGGSGGM 
                   DKKYSIGLAIGTNSVGWAVITDE 
                 
               
               
                   
                   
               
               
                   
                 
                   YKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA 
                 
               
               
                   
                   
               
               
                   
                 
                   EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVD 
                 
               
               
                   
                   
               
               
                   
                 
                   DSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYH 
                 
               
               
                   
                   
               
               
                   
                 
                   EKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF 
                 
               
               
                   
                   
               
               
                   
                 
                   RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEN 
                 
               
               
                   
                   
               
               
                   
                 
                   PINASGVDAKAILSARLSKSRRLENLIAQLPGEKK 
                 
               
               
                   
                   
               
               
                   
                 
                   NGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKD 
                 
               
               
                   
                   
               
               
                   
                 
                   TYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD 
                 
               
               
                   
                   
               
               
                   
                 
                   ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALV 
                 
               
               
                   
                   
               
               
                   
                 
                   RQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYK 
                 
               
               
                   
                   
               
               
                   
                 
                   FIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS 
                 
               
               
                   
                   
               
               
                   
                 
                   IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKIL 
                 
               
               
                   
                   
               
               
                   
                 
                   TFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE 
                 
               
               
                   
                   
               
               
                   
                 
                   EVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLL 
                 
               
               
                   
                   
               
               
                   
                 
                   YEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV 
                 
               
               
                   
                   
               
               
                   
                 
                   DLLFKTNRKVTVKQLKEDYEKKIECFDSVEISGVE 
                 
               
               
                   
                   
               
               
                   
                 
                   DRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDI 
                 
               
               
                   
                   
               
               
                   
                 
                   VLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR 
                 
               
               
                   
                   
               
               
                   
                 
                   RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFA 
                 
               
               
                   
                   
               
               
                   
                 
                   NRNEMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHI 
                 
               
               
                   
                   
               
               
                   
                 
                   ANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI 
                 
               
               
                   
                   
               
               
                   
                 
                   VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQ 
                 
               
               
                   
                   
               
               
                   
                 
                   ILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD 
                 
               
               
                   
                   
               
               
                   
                 
                   INRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNR 
                 
               
               
                   
                   
               
               
                   
                 
                   GKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN 
                 
               
               
                   
                   
               
               
                   
                 
                   LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQI 
                 
               
               
                   
                   
               
               
                   
                 
                   LDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD 
                 
               
               
                   
                   
               
               
                   
                 
                   FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL 
                 
               
               
                   
                   
               
               
                   
                 
                   ESEFVYGDYKVYDVRKMIAKSEQ 
                   EGADKRTADGSE 
                 
               
               
                   
                   
               
               
                   
                 
                   FESPKKKRKV* 
                 
               
            
           
         
       
     
     In some embodiments, the ABE8 is selected from a base editor from Table 6-9, 13, or 14 infra. In some embodiments, ABE8 contains an adenosine deaminase variant evolved from TadA. In some embodiments, the adenosine deaminase variant of ABE8 is a TadA*8 variant as described in Table 7, 9, 13 or 14 infra. In some embodiments, the adenosine deaminase variant is TadA*7.10 variant (e.g. TadA*8) comprising one or more of an alteration selected from the group of Y147T, Y147R, Q154S, Y123H, V82S, T166R, and/or Q154R. In various embodiments, ABE8 comprises TadA*7.10 variant (e.g. TadA*8) with a combination of alterations selected from the group consisting of Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. In some embodiments ABE8 is a monomeric construct. In some embodiments, ABE8 is a heterodimeric construct. In some embodiments, the ABE8 base editor comprises the sequence: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 21) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVG 
               
               
                   
                   
               
               
                   
                 AVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA 
               
               
                   
                   
               
               
                   
                 LRQGGLVMONYRLIDATLYVTFEPCVMCAGA 
               
               
                   
                   
               
               
                   
                 MIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP 
               
               
                   
                   
               
               
                   
                 GMNHRVEITEGILADECAALLCTFFRMPRQV 
               
               
                   
                   
               
               
                   
                 FNAQKKAQSSTD. 
               
            
           
         
       
     
     In some embodiments, the polynucleotide programmable DNA binding domain is a CRISPR associated (e.g., Cas or Cpf1) enzyme. In some embodiments, the base editor is a catalytically dead Cas9 (dCas9) fused to a deaminase domain. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to a deaminase domain. In some embodiments, the base editor is fused to an inhibitor of base excision repair (BER). In some embodiments, the inhibitor of base excision repair is a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the inhibitor of base excision repair is an inosine base excision repair inhibitor. 
     Details of base editors are described in International PCT Application Nos. PCT/2017/045381 (WO 2018/027078) and PCT/US2016/058344 (WO 2017/070632), each of which is incorporated herein by reference for its entirety. Also see Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), and Rees, H. A., et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 December; 19(12):770-788. doi: 10.1038/s41576-018-0059-1, the entire contents of which are hereby incorporated by reference. 
     By way of example, a cytidine base editor as used in the base editing compositions, systems and methods described herein has the following nucleic acid sequence (8877 base pairs), (Addgene, Watertown, Mass.; Komor A C, et al., 2017, Sci Adv., 30; 3(8):eaao4774. doi: 10.1126/sciadv.aao4774) as provided below. Polynucleotide sequences having at least 95% or greater identity to the BE4 nucleic acid sequence are also encompassed. 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 39) 
               
               
                   
                    1 atatgccaag tacgccccct attgacgtca 
               
               
                   
                   
               
               
                   
                      atgacggtaa atggcccgcc tggcattatg 
               
               
                   
                   
               
               
                   
                   61 cccagtacat gaccttatgg gactttccta 
               
               
                   
                   
               
               
                   
                      cttggcagta catctacgta ttagtcatcg 
               
               
                   
                   
               
               
                   
                  121 ctattaccat ggtgatgcgg ttttggcagt 
               
               
                   
                   
               
               
                   
                      acatcaatgg gcgtggatag cggtttgact 
               
               
                   
                   
               
               
                   
                  181 cacggggatt tccaagtctc caccccattg 
               
               
                   
                   
               
               
                   
                      acgtcaatgg gagtttgttt tggcaccaaa 
               
               
                   
                   
               
               
                   
                  241 atcaacggga ctttccaaaa tgtcgtaaca 
               
               
                   
                   
               
               
                   
                      actccgcccc attgacgcaa atgggcggta 
               
               
                   
                   
               
               
                   
                  301 ggcgtgtacg gtgggaggtc tatataagca 
               
               
                   
                   
               
               
                   
                      gagctggttt agtgaaccgt cagatccgct 
               
               
                   
                   
               
               
                   
                  361 agagatccgc ggccgctaat acgactcact 
               
               
                   
                   
               
               
                   
                      atagggagag ccgccaccat gagctcagag 
               
               
                   
                   
               
               
                   
                  421 actggcccag tggctgtgga ccccacattg 
               
               
                   
                   
               
               
                   
                      agacggcgga tcgagcccca tgagtttgag 
               
               
                   
                   
               
               
                   
                  481 gtattcttcg atccgagaga gctccgcaag 
               
               
                   
                   
               
               
                   
                      gagacctgcc tgctttacga aattaattgg 
               
               
                   
                   
               
               
                   
                  541 gggggccggc actccatttg gcgacataca 
               
               
                   
                   
               
               
                   
                      tcacagaaca ctaacaagca cgtcgaagtc 
               
               
                   
                   
               
               
                   
                  601 aacttcatcg agaagttcac gacagaaaga 
               
               
                   
                   
               
               
                   
                      tatttctgtc cgaacacaag gtgcagcatt 
               
               
                   
                   
               
               
                   
                  661 acctggtttc tcagctggag cccatgcggc 
               
               
                   
                   
               
               
                   
                      gaatgtagta gggccatcac tgaattcctg 
               
               
                   
                   
               
               
                   
                  721 tcaaggtatc cccacgtcac tctgtttatt 
               
               
                   
                   
               
               
                   
                      tacatcgcaa ggctgtacca ccacgctgac 
               
               
                   
                   
               
               
                   
                  781 ccccgcaatc gacaaggcct gcgggatttg 
               
               
                   
                   
               
               
                   
                      atctcttcag gtgtgactat ccaaattatg 
               
               
                   
                   
               
               
                   
                  841 actgagcagg agtcaggata ctgctggaga 
               
               
                   
                   
               
               
                   
                      aactttgtga attatagccc gagtaatgaa 
               
               
                   
                   
               
               
                   
                  901 gcccactggc ctaggtatcc ccatctgtgg 
               
               
                   
                   
               
               
                   
                      gtacgactgt acgttcttga actgtactgc 
               
               
                   
                   
               
               
                   
                  961 atcatactgg gcctgcctcc ttgtctcaac 
               
               
                   
                   
               
               
                   
                      attctgagaa ggaagcagcc acagctgaca 
               
               
                   
                   
               
               
                   
                 1021 ttctttacca tcgctcttca gtcttgtcat 
               
               
                   
                   
               
               
                   
                      taccagcgac tgcccccaca cattctctgg 
               
               
                   
                   
               
               
                   
                 1081 gccaccgggt tgaaatctgg tggttcttct 
               
               
                   
                   
               
               
                   
                      ggtggttcta gcggcagcga gactcccggg 
               
               
                   
                   
               
               
                   
                 1141 acctcagagt ccgccacacc cgaaagttct 
               
               
                   
                   
               
               
                   
                      ggtggttctt ctggtggttc tgataaaaag 
               
               
                   
                   
               
               
                   
                 1201 tattctattg gtttagccat cggcactaat 
               
               
                   
                   
               
               
                   
                      tccgttggat gggctgtcat aaccgatgaa 
               
               
                   
                   
               
               
                   
                 1261 tacaaagtac cttcaaagaa atttaaggtg 
               
               
                   
                   
               
               
                   
                      ttggggaaca cagaccgtca ttcgattaaa 
               
               
                   
                   
               
               
                   
                 1321 aagaatctta tcggtgccct cctattcgat 
               
               
                   
                   
               
               
                   
                      agtggcgaaa cggcagaggc gactcgcctg 
               
               
                   
                   
               
               
                   
                 1381 aaacgaaccg ctcggagaag gtatacacgt 
               
               
                   
                   
               
               
                   
                      cgcaagaacc gaatatgtta cttacaagaa 
               
               
                   
                   
               
               
                   
                 1441 atttttagca atgagatggc caaagttgac 
               
               
                   
                   
               
               
                   
                      gattctttct ttcaccgttt ggaagagtcc 
               
               
                   
                   
               
               
                   
                 1501 ttccttgtcg aagaggacaa gaaacatgaa 
               
               
                   
                   
               
               
                   
                      cggcacccca tctttggaaa catagtagat 
               
               
                   
                   
               
               
                   
                 1561 gaggtggcat atcatgaaaa gtacccaacg 
               
               
                   
                   
               
               
                   
                      atttatcacc tcagaaaaaa gctagttgac 
               
               
                   
                   
               
               
                   
                 1621 tcaactgata aagcggacct gaggttaatc 
               
               
                   
                   
               
               
                   
                      tacttggctc ttgcccatat gataaagttc 
               
               
                   
                   
               
               
                   
                 1681 cgtgggcact ttctcattga gggtgatcta 
               
               
                   
                   
               
               
                   
                      aatccggaca actcggatgt cgacaaactg 
               
               
                   
                   
               
               
                   
                 1741 ttcatccagt tagtacaaac ctataatcag 
               
               
                   
                   
               
               
                   
                      ttgtttgaag agaaccctat aaatgcaagt 
               
               
                   
                   
               
               
                   
                 1801 ggcgtggatg cgaaggctat tcttagcgcc 
               
               
                   
                   
               
               
                   
                      cgcctctcta aatcccgacg gctagaaaac 
               
               
                   
                   
               
               
                   
                 1861 ctgatcgcac aattacccgg agagaagaaa 
               
               
                   
                   
               
               
                   
                      aatgggttgt tcggtaacct tatagcgctc 
               
               
                   
                   
               
               
                   
                 1921 tcactaggcc tgacaccaaa ttttaagtcg 
               
               
                   
                   
               
               
                   
                      aacttcgact tagctgaaga tgccaaattg 
               
               
                   
                   
               
               
                   
                 1981 cagcttagta aggacacgta cgatgacgat 
               
               
                   
                   
               
               
                   
                      ctcgacaatc tactggcaca aattggagat 
               
               
                   
                   
               
               
                   
                 2041 cagtatgcgg acttattttt ggctgccaaa 
               
               
                   
                   
               
               
                   
                      aaccttagcg atgcaatcct cctatctgac 
               
               
                   
                   
               
               
                   
                 2101 atactgagag ttaatactga gattaccaag 
               
               
                   
                   
               
               
                   
                      gcgccgttat ccgcttcaat gatcaaaagg 
               
               
                   
                   
               
               
                   
                 2161 tacgatgaac atcaccaaga cttgacactt 
               
               
                   
                   
               
               
                   
                      ctcaaggccc tagtccgtca gcaactgcct 
               
               
                   
                   
               
               
                   
                 2221 gagaaatata aggaaatatt ctttgatcag 
               
               
                   
                   
               
               
                   
                      tcgaaaaacg ggtacgcagg ttatattgac 
               
               
                   
                   
               
               
                   
                 2281 ggcggagcga gtcaagagga attctacaag 
               
               
                   
                   
               
               
                   
                      tttatcaaac ccatattaga gaagatggat 
               
               
                   
                   
               
               
                   
                 2341 gggacggaag agttgcttgt aaaactcaat 
               
               
                   
                   
               
               
                   
                      cgcgaagatc tactgcgaaa gcagcggact 
               
               
                   
                   
               
               
                   
                 2401 ttcgacaacg gtagcattcc acatcaaatc 
               
               
                   
                   
               
               
                   
                      cacttaggcg aattgcatgc tatacttaga 
               
               
                   
                   
               
               
                   
                 2461 aggcaggagg atttttatcc gttcctcaaa 
               
               
                   
                   
               
               
                   
                      gacaatcgtg aaaagattga gaaaatccta 
               
               
                   
                   
               
               
                   
                 2521 acctttcgca taccttacta tgtgggaccc 
               
               
                   
                   
               
               
                   
                      ctggcccgag ggaactctcg gttcgcatgg 
               
               
                   
                   
               
               
                   
                 2581 atgacaagaa agtccgaaga aacgattact 
               
               
                   
                   
               
               
                   
                      ccatggaatt ttgaggaagt tgtcgataaa 
               
               
                   
                   
               
               
                   
                 2641 ggtgcgtcag ctcaatcgtt catcgagagg 
               
               
                   
                   
               
               
                   
                      atgaccaact ttgacaagaa tttaccgaac 
               
               
                   
                   
               
               
                   
                 2701 gaaaaagtat tgcctaagca cagtttactt 
               
               
                   
                   
               
               
                   
                      tacgagtatt tcacagtgta caatgaactc 
               
               
                   
                   
               
               
                   
                 2761 acgaaagtta agtatgtcac tgagggcatg 
               
               
                   
                   
               
               
                   
                      cgtaaacccg cctttctaag cggagaacag 
               
               
                   
                   
               
               
                   
                 2821 aagaaagcaa tagtagatct gttattcaag 
               
               
                   
                   
               
               
                   
                      accaaccgca aagtgacagt taagcaattg 
               
               
                   
                   
               
               
                   
                 2881 aaagaggact actttaagaa aattgaatgc 
               
               
                   
                   
               
               
                   
                      ttcgattctg tcgagatctc cggggtagaa 
               
               
                   
                   
               
               
                   
                 2941 gatcgattta atgcgtcact tggtacgtat 
               
               
                   
                   
               
               
                   
                      catgacctcc taaagataat taaagataag 
               
               
                   
                   
               
               
                   
                 3001 gacttcctgg ataacgaaga gaatgaagat 
               
               
                   
                   
               
               
                   
                      atcttagaag atatagtgtt gactcttacc 
               
               
                   
                   
               
               
                   
                 3061 ctctttgaag atcgggaaat gattgaggaa 
               
               
                   
                   
               
               
                   
                      agactaaaaa catacgctca cctgttcgac 
               
               
                   
                   
               
               
                   
                 3121 gataaggtta tgaaacagtt aaagaggcgt 
               
               
                   
                   
               
               
                   
                      cgctatacgg gctggggacg attgtcgcgg 
               
               
                   
                   
               
               
                   
                 3181 aaacttatca acgggataag agacaagcaa 
               
               
                   
                   
               
               
                   
                      agtggtaaaa ctattctcga ttttctaaag 
               
               
                   
                   
               
               
                   
                 3241 agcgacggct tcgccaatag gaactttatg 
               
               
                   
                   
               
               
                   
                      cagctgatcc atgatgactc tttaaccttc 
               
               
                   
                   
               
               
                   
                 3301 aaagaggata tacaaaaggc acaggtttcc 
               
               
                   
                   
               
               
                   
                      ggacaagggg actcattgca cgaacatatt 
               
               
                   
                   
               
               
                   
                 3361 gcgaatcttg ctggttcgcc agccatcaaa 
               
               
                   
                   
               
               
                   
                      aagggcatac tccagacagt caaagtagtg 
               
               
                   
                   
               
               
                   
                 3421 gatgagctag ttaaggtcat gggacgtcac 
               
               
                   
                   
               
               
                   
                      aaaccggaaa acattgtaat cgagatggca 
               
               
                   
                   
               
               
                   
                 3481 cgcgaaaatc aaacgactca gaaggggcaa 
               
               
                   
                   
               
               
                   
                      aaaaacagtc gagagcggat gaagagaata 
               
               
                   
                   
               
               
                   
                 3541 gaagagggta ttaaagaact gggcagccag 
               
               
                   
                   
               
               
                   
                      atcttaaagg agcatcctgt ggaaaatacc 
               
               
                   
                   
               
               
                   
                 3601 caattgcaga acgagaaact ttacctctat 
               
               
                   
                   
               
               
                   
                      tacctacaaa atggaaggga catgtatgtt 
               
               
                   
                   
               
               
                   
                 3661 gatcaggaac tggacataaa ccgtttatct 
               
               
                   
                   
               
               
                   
                      gattacgacg tcgatcacat tgtaccccaa 
               
               
                   
                   
               
               
                   
                 3721 tcctttttga aggacgattc aatcgacaat 
               
               
                   
                   
               
               
                   
                      aaagtgctta cacgctcgga taagaaccga 
               
               
                   
                   
               
               
                   
                 3781 gggaaaagtg acaatgttcc aagcgaggaa 
               
               
                   
                   
               
               
                   
                      gtcgtaaaga aaatgaagaa ctattggcgg 
               
               
                   
                   
               
               
                   
                 3841 cagctcctaa atgcgaaact gataacgcaa 
               
               
                   
                   
               
               
                   
                      agaaagttcg ataacttaac taaagctgag 
               
               
                   
                   
               
               
                   
                 3901 aggggtggct tgtctgaact tgacaaggcc 
               
               
                   
                   
               
               
                   
                      ggatttatta aacgtcagct cgtggaaacc 
               
               
                   
                   
               
               
                   
                 3961 cgccaaatca caaagcatgt tgcacagata 
               
               
                   
                   
               
               
                   
                      ctagattccc gaatgaatac gaaatacgac 
               
               
                   
                   
               
               
                   
                 4021 gagaacgata agctgattcg ggaagtcaaa 
               
               
                   
                   
               
               
                   
                      gtaatcactt taaagtcaaa attggtgtcg 
               
               
                   
                   
               
               
                   
                 4081 gacttcagaa aggattttca attctataaa 
               
               
                   
                   
               
               
                   
                      gttagggaga taaataacta ccaccatgcg 
               
               
                   
                   
               
               
                   
                 4141 cacgacgctt atcttaatgc cgtcgtaggg 
               
               
                   
                   
               
               
                   
                      accgcactca ttaagaaata cccgaagcta 
               
               
                   
                   
               
               
                   
                 4201 gaaagtgagt ttgtgtatgg tgattacaaa 
               
               
                   
                   
               
               
                   
                      gtttatgacg tccgtaagat gatcgcgaaa 
               
               
                   
                   
               
               
                   
                 4261 agcgaacagg agataggcaa ggctacagcc 
               
               
                   
                   
               
               
                   
                      aaatacttct tttattctaa cattatgaat 
               
               
                   
                   
               
               
                   
                 4321 ttctttaaga cggaaatcac tctggcaaac 
               
               
                   
                   
               
               
                   
                      ggagagatac gcaaacgacc tttaattgaa 
               
               
                   
                   
               
               
                   
                 4381 accaatgggg agacaggtga aatcgtatgg 
               
               
                   
                   
               
               
                   
                      gataagggcc gggacttcgc gacggtgaga 
               
               
                   
                   
               
               
                   
                 4441 aaagttttgt ccatgcccca agtcaacata 
               
               
                   
                   
               
               
                   
                      gtaaagaaaa ctgaggtgca gaccggaggg 
               
               
                   
                   
               
               
                   
                 4501 ttttcaaagg aatcgattct tccaaaaagg 
               
               
                   
                   
               
               
                   
                      aatagtgata agctcatcgc tcgtaaaaag 
               
               
                   
                   
               
               
                   
                 4561 gactgggacc cgaaaaagta cggtggcttc 
               
               
                   
                   
               
               
                   
                      gatagcccta cagttgccta ttctgtccta 
               
               
                   
                   
               
               
                   
                 4621 gtagtggcaa aagttgagaa gggaaaatcc 
               
               
                   
                   
               
               
                   
                      aagaaactga agtcagtcaa agaattattg 
               
               
                   
                   
               
               
                   
                 4681 gggataacga ttatggagcg ctcgtctttt 
               
               
                   
                   
               
               
                   
                      gaaaagaacc ccatcgactt ccttgaggcg 
               
               
                   
                   
               
               
                   
                 4741 aaaggttaca aggaagtaaa aaaggatctc 
               
               
                   
                   
               
               
                   
                      ataattaaac taccaaagta tagtctgttt 
               
               
                   
                   
               
               
                   
                 4801 gagttagaaa atggccgaaa acggatgttg 
               
               
                   
                   
               
               
                   
                      gctagcgccg gagagcttca aaaggggaac 
               
               
                   
                   
               
               
                   
                 4861 gaactcgcac taccgtctaa atacgtgaat 
               
               
                   
                   
               
               
                   
                      ttcctgtatt tagcgtccca ttacgagaag 
               
               
                   
                   
               
               
                   
                 4921 ttgaaaggtt cacctgaaga taacgaacag 
               
               
                   
                   
               
               
                   
                      aagcaacttt ttgttgagca gcacaaacat 
               
               
                   
                   
               
               
                   
                 4981 tatctcgacg aaatcataga gcaaatttcg 
               
               
                   
                   
               
               
                   
                      gaattcagta agagagtcat cctagctgat 
               
               
                   
                   
               
               
                   
                 5041 gccaatctgg acaaagtatt aagcgcatac 
               
               
                   
                   
               
               
                   
                      aacaagcaca gggataaacc catacgtgag 
               
               
                   
                   
               
               
                   
                 5101 caggcggaaa atattatcca tttgtttact 
               
               
                   
                   
               
               
                   
                      cttaccaacc tcggcgctcc agccgcattc 
               
               
                   
                   
               
               
                   
                 5161 aagtattttg acacaacgat agatcgcaaa 
               
               
                   
                   
               
               
                   
                      cgatacactt ctaccaagga ggtgctagac 
               
               
                   
                   
               
               
                   
                 5221 gcgacactga ttcaccaatc catcacggga 
               
               
                   
                   
               
               
                   
                      ttatatgaaa ctcggataga tttgtcacag 
               
               
                   
                   
               
               
                   
                 5281 cttgggggtg actctggtgg ttctggagga 
               
               
                   
                   
               
               
                   
                      tctggtggtt ctactaatct gtcagatatt 
               
               
                   
                   
               
               
                   
                 5341 attgaaaagg agaccggtaa gcaactggtt 
               
               
                   
                   
               
               
                   
                      atccaggaat ccatcctcat gctcccagag 
               
               
                   
                   
               
               
                   
                 5401 gaggtggaag aagtcattgg gaacaagccg 
               
               
                   
                   
               
               
                   
                      gaaagcgata tactcgtgca caccgcctac 
               
               
                   
                   
               
               
                   
                 5461 gacgagagca ccgacgagaa tgtcatgctt 
               
               
                   
                   
               
               
                   
                      ctgactagcg acgcccctga atacaagcct 
               
               
                   
                   
               
               
                   
                 5521 tgggctctgg tcatacagga tagcaacggt 
               
               
                   
                   
               
               
                   
                      gagaacaaga ttaagatgct ctctggtggt 
               
               
                   
                   
               
               
                   
                 5581 tctggaggat ctggtggttc tactaatctg 
               
               
                   
                   
               
               
                   
                      tcagatatta ttgaaaagga gaccggtaag 
               
               
                   
                   
               
               
                   
                 5641 caactggtta tccaggaatc catcctcatg 
               
               
                   
                   
               
               
                   
                      ctcccagagg aggtggaaga agtcattggg 
               
               
                   
                   
               
               
                   
                 5701 aacaagccgg aaagcgatat actcgtgcac 
               
               
                   
                   
               
               
                   
                      accgcctacg acgagagcac cgacgagaat 
               
               
                   
                   
               
               
                   
                 5761 gtcatgcttc tgactagcga cgcccctgaa 
               
               
                   
                   
               
               
                   
                      tacaagcctt gggctctggt catacaggat 
               
               
                   
                   
               
               
                   
                 5821 agcaacggtg agaacaagat taagatgctc 
               
               
                   
                   
               
               
                   
                      tctggtggtt ctcccaagaa gaagaggaaa 
               
               
                   
                   
               
               
                   
                 5881 gtctaaccgg tcatcatcac catcaccatt 
               
               
                   
                   
               
               
                   
                      gagtttaaac ccgctgatca gcctcgactg 
               
               
                   
                   
               
               
                   
                 5941 tgccttctag ttgccagcca tctgttgttt 
               
               
                   
                   
               
               
                   
                      gcccctcccc cgtgccttcc ttgaccctgg 
               
               
                   
                   
               
               
                   
                 6001 aaggtgccac tcccactgtc ctttcctaat 
               
               
                   
                   
               
               
                   
                      aaaatgagga aattgcatcg cattgtctga 
               
               
                   
                   
               
               
                   
                 6061 gtaggtgtca ttctattctg gggggtgggg 
               
               
                   
                   
               
               
                   
                      tggggcagga cagcaagggg gaggattggg 
               
               
                   
                   
               
               
                   
                 6121 aagacaatag caggcatgct ggggatgcgg 
               
               
                   
                   
               
               
                   
                      tgggctctat ggcttctgag gcggaaagaa 
               
               
                   
                   
               
               
                   
                 6181 ccagctgggg ctcgataccg tcgacctcta 
               
               
                   
                   
               
               
                   
                      gctagagctt ggcgtaatca tggtcatagc 
               
               
                   
                   
               
               
                   
                 6241 tgtttcctgt gtgaaattgt tatccgctca 
               
               
                   
                   
               
               
                   
                      caattccaca caacatacga gccggaagca 
               
               
                   
                   
               
               
                   
                 6301 taaagtgtaa agcctagggt gcctaatgag 
               
               
                   
                   
               
               
                   
                      tgagctaact cacattaatt gcgttgcgct 
               
               
                   
                   
               
               
                   
                 6361 cactgcccgc tttccagtcg ggaaacctgt 
               
               
                   
                   
               
               
                   
                      cgtgccagct gcattaatga atcggccaac 
               
               
                   
                   
               
               
                   
                 6421 gcgcggggag aggcggtttg cgtattgggc 
               
               
                   
                   
               
               
                   
                      gctcttccgc ttcctcgctc actgactcgc 
               
               
                   
                   
               
               
                   
                 6481 tgcgctcggt cgttcggctg cggcgagcgg 
               
               
                   
                   
               
               
                   
                      tatcagctca ctcaaaggcg gtaatacggt 
               
               
                   
                   
               
               
                   
                 6541 tatccacaga atcaggggat aacgcaggaa 
               
               
                   
                   
               
               
                   
                      agaacatgtg agcaaaaggc cagcaaaagg 
               
               
                   
                   
               
               
                   
                 6601 ccaggaaccg taaaaaggcc gcgttgctgg 
               
               
                   
                   
               
               
                   
                      cgtttttcca taggctccgc ccccctgacg 
               
               
                   
                   
               
               
                   
                 6661 agcatcacaa aaatcgacgc tcaagtcaga 
               
               
                   
                   
               
               
                   
                      ggtggcgaaa cccgacagga ctataaagat 
               
               
                   
                   
               
               
                   
                 6721 accaggcgtt tccccctgga agctccctcg 
               
               
                   
                   
               
               
                   
                      tgcgctctcc tgttccgacc ctgccgctta 
               
               
                   
                   
               
               
                   
                 6781 ccggatacct gtccgccttt ctcccttcgg 
               
               
                   
                   
               
               
                   
                      gaagcgtggc gctttctcat agctcacgct 
               
               
                   
                   
               
               
                   
                 6841 gtaggtatct cagttcggtg taggtcgttc 
               
               
                   
                   
               
               
                   
                      gctccaagct gggctgtgtg cacgaacccc 
               
               
                   
                   
               
               
                   
                 6901 ccgttcagcc cgaccgctgc gccttatccg 
               
               
                   
                   
               
               
                   
                      gtaactatcg tcttgagtcc aacccggtaa 
               
               
                   
                   
               
               
                   
                 6961 gacacgactt atcgccactg gcagcagcca 
               
               
                   
                   
               
               
                   
                      ctggtaacag gattagcaga gcgaggtatg 
               
               
                   
                   
               
               
                   
                 7021 taggcggtgc tacagagttc ttgaagtggt 
               
               
                   
                   
               
               
                   
                      ggcctaacta cggctacact agaagaacag 
               
               
                   
                   
               
               
                   
                 7081 tatttggtat ctgcgctctg ctgaagccag 
               
               
                   
                   
               
               
                   
                      ttaccttcgg aaaaagagtt ggtagctctt 
               
               
                   
                   
               
               
                   
                 7141 gatccggcaa acaaaccacc gctggtagcg 
               
               
                   
                   
               
               
                   
                      gtggtttttt tgtttgcaag cagcagatta 
               
               
                   
                   
               
               
                   
                 7201 cgcgcagaaa aaaaggatct caagaagatc 
               
               
                   
                   
               
               
                   
                      ctttgatctt ttctacgggg tctgacgctc 
               
               
                   
                   
               
               
                   
                 7261 agtggaacga aaactcacgt taagggattt 
               
               
                   
                   
               
               
                   
                      tggtcatgag attatcaaaa aggatcttca 
               
               
                   
                   
               
               
                   
                 7321 cctagatcct tttaaattaa aaatgaagtt 
               
               
                   
                   
               
               
                   
                      ttaaatcaat ctaaagtata tatgagtaaa 
               
               
                   
                   
               
               
                   
                 7381 cttggtctga cagttaccaa tgcttaatca 
               
               
                   
                   
               
               
                   
                      gtgaggcacc tatctcagcg atctgtctat 
               
               
                   
                   
               
               
                   
                 7441 ttcgttcatc catagttgcc tgactccccg 
               
               
                   
                   
               
               
                   
                      tcgtgtagat aactacgata cgggagggct 
               
               
                   
                   
               
               
                   
                 7501 taccatctgg ccccagtgct gcaatgatac 
               
               
                   
                   
               
               
                   
                      cgcgagaccc acgctcaccg gctccagatt 
               
               
                   
                   
               
               
                   
                 7561 tatcagcaat aaaccagcca gccggaaggg 
               
               
                   
                   
               
               
                   
                      ccgagcgcag aagtggtcct gcaactttat 
               
               
                   
                   
               
               
                   
                 7621 ccgcctccat ccagtctatt aattgttgcc 
               
               
                   
                   
               
               
                   
                      gggaagctag agtaagtagt tcgccagtta 
               
               
                   
                   
               
               
                   
                 7681 atagtttgcg caacgttgtt gccattgcta 
               
               
                   
                   
               
               
                   
                      caggcatcgt ggtgtcacgc tcgtcgtttg 
               
               
                   
                   
               
               
                   
                 7741 gtatggcttc attcagctcc ggttcccaac 
               
               
                   
                   
               
               
                   
                      gatcaaggcg agttacatga tcccccatgt 
               
               
                   
                   
               
               
                   
                 7801 tgtgcaaaaa agcggttagc tccttcggtc 
               
               
                   
                   
               
               
                   
                      ctccgatcgt tgtcagaagt aagttggccg 
               
               
                   
                   
               
               
                   
                 7861 cagtgttatc actcatggtt atggcagcac 
               
               
                   
                   
               
               
                   
                      tgcataattc tcttactgtc atgccatccg 
               
               
                   
                   
               
               
                   
                 7921 taagatgctt ttctgtgact ggtgagtact 
               
               
                   
                   
               
               
                   
                      caaccaagtc attctgagaa tagtgtatgc 
               
               
                   
                   
               
               
                   
                 7981 ggcgaccgag ttgctcttgc ccggcgtcaa 
               
               
                   
                   
               
               
                   
                      tacgggataa taccgcgcca catagcagaa 
               
               
                   
                   
               
               
                   
                 8041 ctttaaaagt gctcatcatt ggaaaacgtt 
               
               
                   
                   
               
               
                   
                      cttcggggcg aaaactctca aggatcttac 
               
               
                   
                   
               
               
                   
                 8101 cgctgttgag atccagttcg atgtaaccca 
               
               
                   
                   
               
               
                   
                      ctcgtgcacc caactgatct tcagcatctt 
               
               
                   
                   
               
               
                   
                 8161 ttactttcac cagcgtttct gggtgagcaa 
               
               
                   
                   
               
               
                   
                      aaacaggaag gcaaaatgcc gcaaaaaagg 
               
               
                   
                   
               
               
                   
                 8221 gaataagggc gacacggaaa tgttgaatac 
               
               
                   
                   
               
               
                   
                      tcatactctt cctttttcaa tattattgaa 
               
               
                   
                   
               
               
                   
                 8281 gcatttatca gggttattgt ctcatgagcg 
               
               
                   
                   
               
               
                   
                      gatacatatt tgaatgtatt tagaaaaata 
               
               
                   
                   
               
               
                   
                 8341 aacaaatagg ggttccgcgc acatttcccc 
               
               
                   
                   
               
               
                   
                      gaaaagtgcc acctgacgtc gacggatcgg 
               
               
                   
                   
               
               
                   
                 8401 gagatcgatc tcccgatccc ctagggtcga 
               
               
                   
                   
               
               
                   
                      ctctcagtac aatctgctct gatgccgcat 
               
               
                   
                   
               
               
                   
                 8461 agttaagcca gtatctgctc cctgcttgtg 
               
               
                   
                   
               
               
                   
                      tgttggaggt cgctgagtag tgcgcgagca 
               
               
                   
                   
               
               
                   
                 8521 aaatttaagc tacaacaagg caaggcttga 
               
               
                   
                   
               
               
                   
                      ccgacaattg catgaagaat ctgcttaggg 
               
               
                   
                   
               
               
                   
                 8581 ttaggcgttt tgcgctgctt cgcgatgtac 
               
               
                   
                   
               
               
                   
                      gggccagata tacgcgttga cattgattat 
               
               
                   
                   
               
               
                   
                 8641 tgactagtta ttaatagtaa tcaattacgg 
               
               
                   
                   
               
               
                   
                      ggtcattagt tcatagccca tatatggagt 
               
               
                   
                   
               
               
                   
                 8701 tccgcgttac ataacttacg gtaaatggcc 
               
               
                   
                   
               
               
                   
                      cgcctggctg accgcccaac gacccccgcc 
               
               
                   
                   
               
               
                   
                 8761 cattgacgtc aataatgacg tatgttccca 
               
               
                   
                   
               
               
                   
                      tagtaacgcc aatagggact ttccattgac 
               
               
                   
                   
               
               
                   
                 8821 gtcaatgggt ggagtattta cggtaaactg 
               
               
                   
                   
               
               
                   
                      cccacttggc agtacatcaa gtgtatc 
               
               
                   
                   
               
               
                   
                 BE4 amino acid sequence: 
               
               
                   
                 (SEQ ID NO: 40) 
               
               
                   
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELR 
               
               
                   
                   
               
               
                   
                 KETCLLYEINWGGRHSIWRHTSQNTNKHVEVNF 
               
               
                   
                   
               
               
                   
                 IEKFTTERYFCPNTRCSITWFLSWSPCGECSRA 
               
               
                   
                   
               
               
                   
                 ITEFLSRYPHVTLFIYIARLYHHADPRNRQGLR 
               
               
                   
                   
               
               
                   
                 DLISSGVTIQIMTEQESGYCWRNFVNYSPSNEA 
               
               
                   
                   
               
               
                   
                 HWPRYPHLWVRLYVLELYCIILGLPPCLNILRR 
               
               
                   
                   
               
               
                   
                 KQPQLTFFTIALQSCHYQRLPPHILWATGLKSG 
               
               
                   
                   
               
               
                   
                 GSSGGSSGSETPGTSESATPESSGGSSGGSDKK 
               
               
                   
                   
               
               
                   
                 YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGN 
               
               
                   
                   
               
               
                   
                 TDRHSIKKNLIGALLFDSGETAEATRLKRTARR 
               
               
                   
                   
               
               
                   
                 RYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEE 
               
               
                   
                   
               
               
                   
                 SFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY 
               
               
                   
                   
               
               
                   
                 HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFL 
               
               
                   
                   
               
               
                   
                 IEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN 
               
               
                   
                   
               
               
                   
                 ASGVDAKAILSARLSKSRRLENLIAQLPGEKKN 
               
               
                   
                   
               
               
                   
                 GLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK 
               
               
                   
                   
               
               
                   
                 DTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL 
               
               
                   
                   
               
               
                   
                 LSDILRVNTEITKAPLSASMIKRYDEHHQDLTL 
               
               
                   
                   
               
               
                   
                 LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGA 
               
               
                   
                   
               
               
                   
                 SQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
                   
               
               
                   
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFL 
               
               
                   
                   
               
               
                   
                 KDNREKIEKILTFRIPYYVGPLARGNSRFAWMT 
               
               
                   
                   
               
               
                   
                 RKSEETITPWNFEEVVDKGASAQSFIERMTNFD 
               
               
                   
                   
               
               
                   
                 KNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE 
               
               
                   
                   
               
               
                   
                 GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLK 
               
               
                   
                   
               
               
                   
                 EDYFKKIECFDSVEISGVEDRFNASLGTYHDLL 
               
               
                   
                   
               
               
                   
                 KIIKDKDFLDNEENEDILEDIVLTLTLFEDREM 
               
               
                   
                   
               
               
                   
                 IEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSR 
               
               
                   
                   
               
               
                   
                 KLINGIRDKQSGKTILDFLKSDGFANRNFMQLI 
               
               
                   
                   
               
               
                   
                 HDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGS 
               
               
                   
                   
               
               
                   
                 PAIKKGILQTVKVVDELVKVMGRHKPENIVIEM 
               
               
                   
                   
               
               
                   
                 ARENQTTQKGQKNSRERMKRIEEGIKELGSQIL 
               
               
                   
                   
               
               
                   
                 KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD 
               
               
                   
                   
               
               
                   
                 INRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK 
               
               
                   
                   
               
               
                   
                 NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQR 
               
               
                   
                   
               
               
                   
                 KFDNLTKAERGGLSELDKAGFIKRQLVETRQIT 
               
               
                   
                   
               
               
                   
                 KHVAQILDSRMNTKYDENDKLIREVKVITLKSK 
               
               
                   
                   
               
               
                   
                 LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVG 
               
               
                   
                   
               
               
                   
                 TALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ 
               
               
                   
                   
               
               
                   
                 EIGKATAKYFFYSNIMNFFKTEITLANGEIRKR 
               
               
                   
                   
               
               
                   
                 PLIETNGETGEIVWDKGRDFATVRKVLSMPQVN 
               
               
                   
                   
               
               
                   
                 IVKKTEVOTGGFSKESILPKRNSDKLIARKKDW 
               
               
                   
                   
               
               
                   
                 DPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKS 
               
               
                   
                   
               
               
                   
                 VKELLGITIMERSSFEKNPIDFLEAKGYKEVKK 
               
               
                   
                   
               
               
                   
                 DLIIKLPKYSLFELENGRKRMLASAGELQKGNE 
               
               
                   
                   
               
               
                   
                 LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLF 
               
               
                   
                   
               
               
                   
                 VEQHKHYLDEIIEQISEFSKRVILADANLDKVL 
               
               
                   
                   
               
               
                   
                 SAYNKHRDKPIREQAENIIHLFTLTNLGAPAAF 
               
               
                   
                   
               
               
                   
                 KYFDTTIDRKRYTSTKEVLDATLIHQSITGLYE 
               
               
                   
                   
               
               
                   
                 TRIDLSQLGGDSGGSGGSGGSTNLSDIIEKETG 
               
               
                   
                   
               
               
                   
                 KQLVIQESILMLPEEVEEVIGNKPESDILVHTA 
               
               
                   
                   
               
               
                   
                 YDESTDENVMLLTSDAPEYKPWALVIQDSNGEN 
               
               
                   
                   
               
               
                   
                 KIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQ 
               
               
                   
                   
               
               
                   
                 ESILMLPEEVEEVIGNKPESDILVHTAYDESTD 
               
               
                   
                   
               
               
                   
                 ENVMLLTSDAPEYKPWALVIQDSNGENKIKMLS 
               
               
                   
                   
               
               
                   
                 GGSPKKKRK 
               
            
           
         
       
     
     By way of example, the adenine base editor (ABE) as used in the base editing compositions, systems and methods described herein has the nucleic acid sequence (8877 base pairs), (Addgene, Watertown, Mass.; Gaudelli N M, et al., Nature. 2017 Nov. 23; 551(7681):464-471. doi: 10.1038/nature24644; Koblan L W, et al., Nat Biotechnol. 2018 October; 36(9):843-846. doi: 10.1038/nbt.4172.) as provided below. Polynucleotide sequences having at least 95% or greater identity to the ABE nucleic acid sequence are also encompassed. 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 41) 
               
               
                   
                 ATATGCCAAGTACGCCCCCTATTGACGTCAATGACG 
               
               
                   
                   
               
               
                   
                 GTAAATGGCCCGCCTGGCATTATGCCCAGTACATG 
               
               
                   
                   
               
               
                   
                 ACCTTATGGGACTTTCCTACTTGGCAGTACATCTA 
               
               
                   
                   
               
               
                   
                 CGTATTAGTCATCGCTATTACCATGGTGATGCGGT 
               
               
                   
                   
               
               
                   
                 TTTGGCAGTACATCAATGGGCGTGGATAGCGGTTT 
               
               
                   
                   
               
               
                   
                 GACTCACGGGGATTTCCAAGTCTCCACCCCATTGA 
               
               
                   
                   
               
               
                   
                 CGTCAATGGGAGTTTGTTTTGGCACCAAAATCAAC 
               
               
                   
                   
               
               
                   
                 GGGACTTTCCAAAATGTCGTAACAACTCCGCCCCA 
               
               
                   
                   
               
               
                   
                 TTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGA 
               
               
                   
                   
               
               
                   
                 GGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTC 
               
               
                   
                   
               
               
                   
                 AGATCCGCTAGAGATCCGCGGCCGCTAATACGACT 
               
               
                   
                   
               
               
                   
                 CACTATAGGGAGAGCCGCCACCATGAAACGGACAG 
               
               
                   
                   
               
               
                   
                 CCGACGGAAGCGAGTTCGAGTCACCAAAGAAGAAG 
               
               
                   
                   
               
               
                   
                 CGGAAAGTCTCTGAAGTCGAGTTTAGCCACGAGTA 
               
               
                   
                   
               
               
                   
                 TTGGATGAGGCACGCACTGACCCTGGCAAAGCGAG 
               
               
                   
                   
               
               
                   
                 CATGGGATGAAAGAGAAGTCCCCGTGGGCGCCGTG 
               
               
                   
                   
               
               
                   
                 CTGGTGCACAACAATAGAGTGATCGGAGAGGGATG 
               
               
                   
                   
               
               
                   
                 GAACAGGCCAATCGGCCGCCACGACCCTACCGGAC 
               
               
                   
                   
               
               
                   
                 ACGCAGAGATCATGGCACTGAGGCAGGGAGGCCTG 
               
               
                   
                   
               
               
                   
                 GTCATGCAGAATTACCGCCTGATCGATGCCACCCT 
               
               
                   
                   
               
               
                   
                 GTATGTGACACTGGAGCCATGCGTGATGTGCGCAG 
               
               
                   
                   
               
               
                   
                 GAGCAATGATCCACAGCAGGATCGGAAGAGTGGTG 
               
               
                   
                   
               
               
                   
                 TTCGGAGCACGGGACGCCAAGACCGGCGCAGCAGG 
               
               
                   
                   
               
               
                   
                 CTCCCTGATGGATGTGCTGCACCACCCCGGCATGA 
               
               
                   
                   
               
               
                   
                 ACCACCGGGTGGAGATCACAGAGGGAATCCTGGCA 
               
               
                   
                   
               
               
                   
                 GACGAGTGCGCCGCCCTGCTGAGCGATTTCTTTAG 
               
               
                   
                   
               
               
                   
                 AATGCGGAGACAGGAGATCAAGGCCCAGAAGAAGG 
               
               
                   
                   
               
               
                   
                 CACAGAGCTCCACCGACTCTGGAGGATCTAGCGGA 
               
               
                   
                   
               
               
                   
                 GGATCCTCTGGAAGCGAGACACCAGGCACAAGCGA 
               
               
                   
                   
               
               
                   
                 GTCCGCCACACCAGAGAGCTCCGGCGGCTCCTCCG 
               
               
                   
                   
               
               
                   
                 GAGGATCCTCTGAGGTGGAGTTTTCCCACGAGTAC 
               
               
                   
                   
               
               
                   
                 TGGATGAGACATGCCCTGACCCTGGCCAAGAGGGC 
               
               
                   
                   
               
               
                   
                 ACGCGATGAGAGGGAGGTGCCTGTGGGAGCCGTGC 
               
               
                   
                   
               
               
                   
                 TGGTGCTGAACAATAGAGTGATCGGCGAGGGCTGG 
               
               
                   
                   
               
               
                   
                 AACAGAGCCATCGGCCTGCACGACCCAACAGCCCA 
               
               
                   
                   
               
               
                   
                 TGCCGAAATTATGGCCCTGAGACAGGGCGGCCTGG 
               
               
                   
                   
               
               
                   
                 TCATGCAGAACTACAGACTGATTGACGCCACCCTG 
               
               
                   
                   
               
               
                   
                 TACGTGACATTCGAGCCTTGCGTGATGTGCGCCGG 
               
               
                   
                   
               
               
                   
                 CGCCATGATCCACTCTAGGATCGGCCGCGTGGTGT 
               
               
                   
                   
               
               
                   
                 TTGGCGTGAGGAACGCAAAAACCGGCGCCGCAGGC 
               
               
                   
                   
               
               
                   
                 TCCCTGATGGACGTGCTGCACTACCCCGGCATGAA 
               
               
                   
                   
               
               
                   
                 TCACCGCGTCGAAATTACCGAGGGAATCCTGGCAG 
               
               
                   
                   
               
               
                   
                 ATGAATGTGCCGCCCTGCTGTGCTATTTCTTTCGG 
               
               
                   
                   
               
               
                   
                 ATGCCTAGACAGGTGTTCAATGCTCAGAAGAAGGC 
               
               
                   
                   
               
               
                   
                 CCAGAGCTCCACCGACTCCGGAGGATCTAGCGGAG 
               
               
                   
                   
               
               
                   
                 GCTCCTCTGGCTCTGAGACACCTGGCACAAGCGAG 
               
               
                   
                   
               
               
                   
                 AGCGCAACACCTGAAAGCAGCGGGGGCAGCAGCGG 
               
               
                   
                   
               
               
                   
                 GGGGTCAGACAAGAAGTACAGCATCGGCCTGGCCA 
               
               
                   
                   
               
               
                   
                 TCGGCACCAACTCTGTGGGCTGGGCCGTGATCACC 
               
               
                   
                   
               
               
                   
                 GACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGT 
               
               
                   
                   
               
               
                   
                 GCTGGGCAACACCGACCGGCACAGCATCAAGAAGA 
               
               
                   
                   
               
               
                   
                 ACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAA 
               
               
                   
                   
               
               
                   
                 ACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAG 
               
               
                   
                   
               
               
                   
                 AAGAAGATACACCAGACGGAAGAACCGGATCTGCT 
               
               
                   
                   
               
               
                   
                 ATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAG 
               
               
                   
                   
               
               
                   
                 GTGGACGACAGCTTCTTCCACAGACTGGAAGAGTC 
               
               
                   
                   
               
               
                   
                 CTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGC 
               
               
                   
                   
               
               
                   
                 ACCCCATCTTCGGCAACATCGTGGACGAGGTGGCC 
               
               
                   
                   
               
               
                   
                 TACCACGAGAAGTACCCCACCATCTACCACCTGAG 
               
               
                   
                   
               
               
                   
                 AAAGAAACTGGTGGACAGCACCGACAAGGCCGACC 
               
               
                   
                   
               
               
                   
                 TGCGGCTGATCTATCTGGCCCTGGCCCACATGATC 
               
               
                   
                   
               
               
                   
                 AAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCT 
               
               
                   
                   
               
               
                   
                 GAACCCCGACAACAGCGACGTGGACAAGCTGTTCA 
               
               
                   
                   
               
               
                   
                 TCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAG 
               
               
                   
                   
               
               
                   
                 GAAAACCCCATCAACGCCAGCGGCGTGGACGCCAA 
               
               
                   
                   
               
               
                   
                 GGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGAC 
               
               
                   
                   
               
               
                   
                 GGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAG 
               
               
                   
                   
               
               
                   
                 AAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCT 
               
               
                   
                   
               
               
                   
                 GAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACT 
               
               
                   
                   
               
               
                   
                 TCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGC 
               
               
                   
                   
               
               
                   
                 AAGGACACCTACGACGACGACCTGGACAACCTGCT 
               
               
                   
                   
               
               
                   
                 GGCCCAGATCGGCGACCAGTACGCCGACCTGTTTC 
               
               
                   
                   
               
               
                   
                 TGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTG 
               
               
                   
                   
               
               
                   
                 AGCGACATCCTGAGAGTGAACACCGAGATCACCAA 
               
               
                   
                   
               
               
                   
                 GGCCCCCCTGAGCGCCTCTATGATCAAGAGATACG 
               
               
                   
                   
               
               
                   
                 ACGAGCACCACCAGGACCTGACCCTGCTGAAAGCT 
               
               
                   
                   
               
               
                   
                 CTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGA 
               
               
                   
                   
               
               
                   
                 GATTTTCTTCGACCAGAGCAAGAACGGCTACGCCG 
               
               
                   
                   
               
               
                   
                 GCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTC 
               
               
                   
                   
               
               
                   
                 TACAAGTTCATCAAGCCCATCCTGGAAAAGATGGA 
               
               
                   
                   
               
               
                   
                 CGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAG 
               
               
                   
                   
               
               
                   
                 AGGACCTGCTGCGGAAGCAGCGGACCTTCGACAAC 
               
               
                   
                   
               
               
                   
                 GGCAGCATCCCCCACCAGATCCACCTGGGAGAGCT 
               
               
                   
                   
               
               
                   
                 GCACGCCATTCTGCGGCGGCAGGAAGATTTTTACC 
               
               
                   
                   
               
               
                   
                 CATTCCTGAAGGACAACCGGGAAAAGATCGAGAAG 
               
               
                   
                   
               
               
                   
                 ATCCTGACCTTCCGCATCCCCTACTACGTGGGCCC 
               
               
                   
                   
               
               
                   
                 TCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGA 
               
               
                   
                   
               
               
                   
                 CCAGAAAGAGCGAGGAAACCATCACCCCCTGGAAC 
               
               
                   
                   
               
               
                   
                 TTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCA 
               
               
                   
                   
               
               
                   
                 GAGCTTCATCGAGCGGATGACCAACTTCGATAAGA 
               
               
                   
                   
               
               
                   
                 ACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGC 
               
               
                   
                   
               
               
                   
                 CTGCTGTACGAGTACTTCACCGTGTATAACGAGCT 
               
               
                   
                   
               
               
                   
                 GACCAAAGTGAAATACGTGACCGAGGGAATGAGAA 
               
               
                   
                   
               
               
                   
                 AGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCC 
               
               
                   
                   
               
               
                   
                 ATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGT 
               
               
                   
                   
               
               
                   
                 GACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGA 
               
               
                   
                   
               
               
                   
                 AAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGC 
               
               
                   
                   
               
               
                   
                 GTGGAAGATCGGTTCAACGCCTCCCTGGGCACATA 
               
               
                   
                   
               
               
                   
                 CCACGATCTGCTGAAAATTATCAAGGACAAGGACT 
               
               
                   
                   
               
               
                   
                 TCCTGGACAATGAGGAAAACGAGGACATTCTGGAA 
               
               
                   
                   
               
               
                   
                 GATATCGTGCTGACCCTGACACTGTTTGAGGACAG 
               
               
                   
                   
               
               
                   
                 AGAGATGATCGAGGAACGGCTGAAAACCTATGCCC 
               
               
                   
                   
               
               
                   
                 ACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAG 
               
               
                   
                   
               
               
                   
                 CGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCG 
               
               
                   
                   
               
               
                   
                 GAAGCTGATCAACGGCATCCGGGACAAGCAGTCCG 
               
               
                   
                   
               
               
                   
                 GCAAGACAATCCTGGATTTCCTGAAGTCCGACGGC 
               
               
                   
                   
               
               
                   
                 TTCGCCAACAGAAACTTCATGCAGCTGATCCACGA 
               
               
                   
                   
               
               
                   
                 CGACAGCCTGACCTTTAAAGAGGACATCCAGAAAG 
               
               
                   
                   
               
               
                   
                 CCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAG 
               
               
                   
                   
               
               
                   
                 CACATTGCCAATCTGGCCGGCAGCCCCGCCATTAA 
               
               
                   
                   
               
               
                   
                 GAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACG 
               
               
                   
                   
               
               
                   
                 AGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAG 
               
               
                   
                   
               
               
                   
                 AACATCGTGATCGAAATGGCCAGAGAGAACCAGAC 
               
               
                   
                   
               
               
                   
                 CACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAA 
               
               
                   
                   
               
               
                   
                 TGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGC 
               
               
                   
                   
               
               
                   
                 AGCCAGATCCTGAAAGAACACCCCGTGGAAAACAC 
               
               
                   
                   
               
               
                   
                 CCAGCTGCAGAACGAGAAGCTGTACCTGTACTACC 
               
               
                   
                   
               
               
                   
                 TGCAGAATGGGCGGGATATGTACGTGGACCAGGAA 
               
               
                   
                   
               
               
                   
                 CTGGACATCAACCGGCTGTCCGACTACGATGTGGA 
               
               
                   
                   
               
               
                   
                 CCATATCGTGCCTCAGAGCTTTCTGAAGGACGACT 
               
               
                   
                   
               
               
                   
                 CCATCGACAACAAGGTGCTGACCAGAAGCGACAAG 
               
               
                   
                   
               
               
                   
                 AACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGA 
               
               
                   
                   
               
               
                   
                 GGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGC 
               
               
                   
                   
               
               
                   
                 TGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTC 
               
               
                   
                   
               
               
                   
                 GACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAG 
               
               
                   
                   
               
               
                   
                 CGAACTGGATAAGGCCGGCTTCATCAAGAGACAGC 
               
               
                   
                   
               
               
                   
                 TGGTGGAAACCCGGCAGATCACAAAGCACGTGGCA 
               
               
                   
                   
               
               
                   
                 CAGATCCTGGACTCCCGGATGAACACTAAGTACGA 
               
               
                   
                   
               
               
                   
                 CGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGA 
               
               
                   
                   
               
               
                   
                 TCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGG 
               
               
                   
                   
               
               
                   
                 AAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAA 
               
               
                   
                   
               
               
                   
                 CAACTACCACCACGCCCACGACGCCTACCTGAACG 
               
               
                   
                   
               
               
                   
                 CCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCT 
               
               
                   
                   
               
               
                   
                 AAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAA 
               
               
                   
                   
               
               
                   
                 GGTGTACGACGTGCGGAAGATGATCGCCAAGAGCG 
               
               
                   
                   
               
               
                   
                 AGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTC 
               
               
                   
                   
               
               
                   
                 TTCTACAGCAACATCATGAACTTTTTCAAGACCGA 
               
               
                   
                   
               
               
                   
                 GATTACCCTGGCCAACGGCGAGATCCGGAAGCGGC 
               
               
                   
                   
               
               
                   
                 CTCTGATCGAGACAAACGGCGAAACCGGGGAGATC 
               
               
                   
                   
               
               
                   
                 GTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCG 
               
               
                   
                   
               
               
                   
                 GAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGA 
               
               
                   
                   
               
               
                   
                 AAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAA 
               
               
                   
                   
               
               
                   
                 GAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCT 
               
               
                   
                   
               
               
                   
                 GATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGT 
               
               
                   
                   
               
               
                   
                 ACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCT 
               
               
                   
                   
               
               
                   
                 GTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTC 
               
               
                   
                   
               
               
                   
                 CAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGA 
               
               
                   
                   
               
               
                   
                 TCACCATCATGGAAAGAAGCAGCTTCGAGAAGAAT 
               
               
                   
                   
               
               
                   
                 CCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGA 
               
               
                   
                   
               
               
                   
                 AGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGT 
               
               
                   
                   
               
               
                   
                 ACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGA 
               
               
                   
                   
               
               
                   
                 ATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAA 
               
               
                   
                   
               
               
                   
                 CGAACTGGCCCTGCCCTCCAAATATGTGAACTTCC 
               
               
                   
                   
               
               
                   
                 TGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGC 
               
               
                   
                   
               
               
                   
                 TCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGT 
               
               
                   
                   
               
               
                   
                 GGAACAGCACAAGCACTACCTGGACGAGATCATCG 
               
               
                   
                   
               
               
                   
                 AGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTG 
               
               
                   
                   
               
               
                   
                 GCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTA 
               
               
                   
                   
               
               
                   
                 CAACAAGCACCGGGATAAGCCCATCAGAGAGCAGG 
               
               
                   
                   
               
               
                   
                 CCGAGAATATCATCCACCTGTTTACCCTGACCAAT 
               
               
                   
                   
               
               
                   
                 CTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACAC 
               
               
                   
                   
               
               
                   
                 CACCATCGACCGGAAGAGGTACACCAGCACCAAAG 
               
               
                   
                   
               
               
                   
                 AGGTGCTGGACGCCACCCTGATCCACCAGAGCATC 
               
               
                   
                   
               
               
                   
                 ACCGGCCTGTACGAGACACGGATCGACCTGTCTCA 
               
               
                   
                   
               
               
                   
                 GCTGGGAGGTGACTCTGGCGGCTCAAAAAGAACCG 
               
               
                   
                   
               
               
                   
                 CCGACGGCAGCGAATTCGAGCCCAAGAAGAAGAGG 
               
               
                   
                   
               
               
                   
                 AAAGTCTAACCGGTCATCATCACCATCACCATTGA 
               
               
                   
                   
               
               
                   
                 GTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTC 
               
               
                   
                   
               
               
                   
                 TAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCG 
               
               
                   
                   
               
               
                   
                 TGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACT 
               
               
                   
                   
               
               
                   
                 GTCCTTTCCTAATAAAATGAGGAAATTGCATCGCA 
               
               
                   
                   
               
               
                   
                 TTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG 
               
               
                   
                   
               
               
                   
                 GGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAA 
               
               
                   
                   
               
               
                   
                 GACAATAGCAGGCATGCTGGGGATGCGGTGGGCTC 
               
               
                   
                   
               
               
                   
                 TATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCT 
               
               
                   
                   
               
               
                   
                 CGATACCGTCGACCTCTAGCTAGAGCTTGGCGTAA 
               
               
                   
                   
               
               
                   
                 TCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTA 
               
               
                   
                   
               
               
                   
                 TCCGCTCACAATTCCACACAACATACGAGCCGGAA 
               
               
                   
                   
               
               
                   
                 GCATAAAGTGTAAAGCCTAGGGTGCCTAATGAGTG 
               
               
                   
                   
               
               
                   
                 AGCTAACTCACATTAATTGCGTTGCGCTCACTGCC 
               
               
                   
                   
               
               
                   
                 CGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGC 
               
               
                   
                   
               
               
                   
                 ATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGT 
               
               
                   
                   
               
               
                   
                 TTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCAC 
               
               
                   
                   
               
               
                   
                 TGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAG 
               
               
                   
                   
               
               
                   
                 CGGTATCAGCTCACTCAAAGGCGGTAATACGGTTA 
               
               
                   
                   
               
               
                   
                 TCCACAGAATCAGGGGATAACGCAGGAAAGAACAT 
               
               
                   
                   
               
               
                   
                 GTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTA 
               
               
                   
                   
               
               
                   
                 AAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTC 
               
               
                   
                   
               
               
                   
                 CGCCCCCCTGACGAGCATCACAAAAATCGACGCTC 
               
               
                   
                   
               
               
                   
                 AAGTCAGAGGTGGCGAAACCCGACAGGACTATAAA 
               
               
                   
                   
               
               
                   
                 GATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTG 
               
               
                   
                   
               
               
                   
                 CGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATA 
               
               
                   
                   
               
               
                   
                 CCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGC 
               
               
                   
                   
               
               
                   
                 TTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCG 
               
               
                   
                   
               
               
                   
                 GTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCA 
               
               
                   
                   
               
               
                   
                 CGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTAT 
               
               
                   
                   
               
               
                   
                 CCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGA 
               
               
                   
                   
               
               
                   
                 CACGACTTATCGCCACTGGCAGCAGCCACTGGTAA 
               
               
                   
                   
               
               
                   
                 CAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTA 
               
               
                   
                   
               
               
                   
                 CAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTAC 
               
               
                   
                   
               
               
                   
                 ACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCT 
               
               
                   
                   
               
               
                   
                 GAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCT 
               
               
                   
                   
               
               
                   
                 CTTGATCCGGCAAACAAACCACCGCTGGTAGCGGT 
               
               
                   
                   
               
               
                   
                 GGTTTTTTTGTTTGCAAGCAGCAGATTACGCGGAG 
               
               
                   
                   
               
               
                   
                 AAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTT 
               
               
                   
                   
               
               
                   
                 CTACGGGGTCTGACACTCAGTGGAACGAAAACTCA 
               
               
                   
                   
               
               
                   
                 CGTTAAGGGATTTTGGTCATGAGATTATCAAAAAG 
               
               
                   
                   
               
               
                   
                 GATCTTCACCTAGATCCTTTTAAATTAAAAATGAA 
               
               
                   
                   
               
               
                   
                 GTTTTAAATCAATCTAAAGTATATATGAGTAAACT 
               
               
                   
                   
               
               
                   
                 TGGTCTGACAGTTACCAATGCTTAATCAGTGAGGC 
               
               
                   
                   
               
               
                   
                 ACCTATCTCAGCGATCTGTCTATTTCGTTCATCCA 
               
               
                   
                   
               
               
                   
                 TAGTTGCCTGACTCCCCGTCGTGTAGATAACTACG 
               
               
                   
                   
               
               
                   
                 ATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGC 
               
               
                   
                   
               
               
                   
                 AATGATACCGCGAGACCCACGCTCACCGGCTCCAG 
               
               
                   
                   
               
               
                   
                 ATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCC 
               
               
                   
                   
               
               
                   
                 GAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTC 
               
               
                   
                   
               
               
                   
                 CATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAG 
               
               
                   
                   
               
               
                   
                 TAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTT 
               
               
                   
                   
               
               
                   
                 GTTGCCATTGCTACAGGCATCGTGGTGTCACGCTC 
               
               
                   
                   
               
               
                   
                 GTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCC 
               
               
                   
                   
               
               
                   
                 AACGATCAAGGCGAGTTACATGATCCCCCATGTTG 
               
               
                   
                   
               
               
                   
                 TGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGAT 
               
               
                   
                   
               
               
                   
                 CGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCAC 
               
               
                   
                   
               
               
                   
                 TCATGGTTATGGCAGCACTGCATAATTCTCTTACT 
               
               
                   
                   
               
               
                   
                 GTCATGCCATCCGTAAGATGCTTTTCTGTGACTGG 
               
               
                   
                   
               
               
                   
                 TGAGTACTCAACCAAGTCATTCTGAGAATAGTGTA 
               
               
                   
                   
               
               
                   
                 TGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATA 
               
               
                   
                   
               
               
                   
                 CGGGATAATACCGCGCCACATAGCAGAACTTTAAA 
               
               
                   
                   
               
               
                   
                 AGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAA 
               
               
                   
                   
               
               
                   
                 AACTCTCAAGGATCTTACCGCTGTTGAGATCCAGT 
               
               
                   
                   
               
               
                   
                 TCGATGTAACCCACTCGTGCACCCAACTGATCTTC 
               
               
                   
                   
               
               
                   
                 AGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAG 
               
               
                   
                   
               
               
                   
                 CAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGA 
               
               
                   
                   
               
               
                   
                 ATAAGGGCGACACGGAAATGTTGAATACTCATACT 
               
               
                   
                   
               
               
                   
                 CTTCCTTTTTCAATATTATTGAAGCATTTATCAGG 
               
               
                   
                   
               
               
                   
                 GTTATTGTCTCATGAGCGGATACATATTTGAATGT 
               
               
                   
                   
               
               
                   
                 ATTTAGAAAAATAAACAAATAGGGGTTCCGCGCAC 
               
               
                   
                   
               
               
                   
                 ATTTCCCCGAAAAGTGCCACCTGACGTCGACGGAT 
               
               
                   
                   
               
               
                   
                 CGGGAGATCGATCTCCCGATCCCCTAGGGTCGACT 
               
               
                   
                   
               
               
                   
                 CTCAGTACAATCTGCTCTGATGCCGCATAGTTAAG 
               
               
                   
                   
               
               
                   
                 CCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCG 
               
               
                   
                   
               
               
                   
                 CTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACA 
               
               
                   
                   
               
               
                   
                 AGGCAAGGCTTGACCGACAATTGCATGAAGAATCT 
               
               
                   
                   
               
               
                   
                 GCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATG 
               
               
                   
                   
               
               
                   
                 TACGGGCCAGATATACGCGTTGACATTGATTATTG 
               
               
                   
                   
               
               
                   
                 ACTAGTTATTAATAGTAATCAATTACGGGGTCATT 
               
               
                   
                   
               
               
                   
                 AGTTCATAGCCCATATATGGAGTTCCGCGTTACAT 
               
               
                   
                   
               
               
                   
                 AACTTACGGTAAATGGCCCGCCTGGCTGACCGCCC 
               
               
                   
                   
               
               
                   
                 AACGACCCCCGCCCATTGACGTCAATAATGACGTA 
               
               
                   
                   
               
               
                   
                 TGTTCCCATAGTAACGCCAATAGGGACTTTCCATT 
               
               
                   
                   
               
               
                   
                 GACGTCAATGGGTGGAGTATTTACGGTAAACTGCC 
               
               
                   
                   
               
               
                   
                 CACTTGGCAGTACATCAAGTGTATC 
               
            
           
         
       
     
     By “base editing activity” is meant acting to chemically alter a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, e.g., converting target C•G to T•A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, e.g., converting A•T to G•C. In another embodiment, the base editing activity is cytidine deaminase activity, e.g., converting target C•G to T•A and adenosine or adenine deaminase activity, e.g., converting A•T to G•C. In some embodiments, base editing activity is assessed by efficiency of editing. Base editing efficiency may be measured by any suitable means, for example, by sanger sequencing or next generation sequencing. In some embodiments, base editing efficiency is measured by percentage of total sequencing reads with nucleobase conversion effected by the base editor, for example, percentage of total sequencing reads with target A•T base pair converted to a G•C base pair. In some embodiments, base editing efficiency is measured by percentage of total cells with nucleobase conversion effected by the abse editor, when base editing is performed in a population of cells. 
     The term “base editor system” refers to a system for editing a nucleobase of a target nucleotide sequence. In various embodiments, the base editor system comprises (1) a polynucleotide programmable nucleotide binding domain (e.g., Cas9); (2) a deaminase domain (e.g., an adenosine deaminase or a cytidine deaminase) for deaminating said nucleobase; and (3) one or more guide polynucleotide (e.g., guide RNA). In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor system is ABE8. 
     In some embodiments, a base editor system may comprise more than one base editing component. For example, a base editor system may include more than one deaminase. In some embodiments, a base editor system may include one or more adenosine deaminases. In some embodiments, a single guide polynucleotide may be utilized to target different deaminases to a target nucleic acid sequence. In some embodiments, a single pair of guide polynucleotides may be utilized to target different deaminases to a target nucleic acid sequence. 
     The deaminase domain and the polynucleotide programmable nucleotide binding component of a base editor system may be associated with each other covalently or non-covalently, or any combination of associations and interactions thereof. For example, in some embodiments, a deaminase domain can be targeted to a target nucleotide sequence by a polynucleotide programmable nucleotide binding domain. In some embodiments, a polynucleotide programmable nucleotide binding domain can be fused or linked to a deaminase domain. In some embodiments, a polynucleotide programmable nucleotide binding domain can target a deaminase domain to a target nucleotide sequence by non-covalently interacting with or associating with the deaminase domain. For example, in some embodiments, the deaminase domain can comprise an additional heterologous portion or domain that is capable of interacting with, associating with, or capable of forming a complex with an additional heterologous portion or domain that is part of a polynucleotide programmable nucleotide binding domain. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous portion may be a protein domain. In some embodiments, the additional heterologous portion may be a K Homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a steril alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif. 
     A base editor system may further comprise a guide polynucleotide component. It should be appreciated that components of the base editor system may be associated with each other via covalent bonds, noncovalent interactions, or any combination of associations and interactions thereof. In some embodiments, a deaminase domain can be targeted to a target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the deaminase domain can comprise an additional heterologous portion or domain (e.g., polynucleotide binding domain such as an RNA or DNA binding protein) that is capable of interacting with, associating with, or capable of forming a complex with a portion or segment (e.g., a polynucleotide motif) of a guide polynucleotide. In some embodiments, the additional heterologous portion or domain (e.g., polynucleotide binding domain such as an RNA or DNA binding protein) can be fused or linked to the deaminase domain. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous portion may be a protein domain. In some embodiments, the additional heterologous portion may be a K Homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif. 
     In some embodiments, a base editor system can further comprise an inhibitor of base excision repair (BER) component. It should be appreciated that components of the base editor system may be associated with each other via covalent bonds, noncovalent interactions, or any combination of associations and interactions thereof. The inhibitor of BER component may comprise a BER inhibitor. In some embodiments, the inhibitor of BER can be a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the inhibitor of BER can be an inosine BER inhibitor. In some embodiments, the inhibitor of BER can be targeted to the target nucleotide sequence by the polynucleotide programmable nucleotide binding domain. In some embodiments, a polynucleotide programmable nucleotide binding domain can be fused or linked to an inhibitor of BER. In some embodiments, a polynucleotide programmable nucleotide binding domain can be fused or linked to a deaminase domain and an inhibitor of BER. In some embodiments, a polynucleotide programmable nucleotide binding domain can target an inhibitor of BER to a target nucleotide sequence by non-covalently interacting with or associating with the inhibitor of BER. For example, in some embodiments, the inhibitor of BER component can comprise an additional heterologous portion or domain that is capable of interacting with, associating with, or capable of forming a complex with an additional heterologous portion or domain that is part of a polynucleotide programmable nucleotide binding domain. 
     In some embodiments, the inhibitor of BER can be targeted to the target nucleotide sequence by the guide polynucleotide. For example, in some embodiments, the inhibitor of BER can comprise an additional heterologous portion or domain (e.g., polynucleotide binding domain such as an RNA or DNA binding protein) that is capable of interacting with, associating with, or capable of forming a complex with a portion or segment (e.g., a polynucleotide motif) of a guide polynucleotide. In some embodiments, the additional heterologous portion or domain of the guide polynucleotide (e.g., polynucleotide binding domain such as an RNA or DNA binding protein) can be fused or linked to the inhibitor of BER. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous portion may be a protein domain. In some embodiments, the additional heterologous portion may be a K Homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif. 
     The term “Cas9” or “Cas9 domain” refers to an RNA guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9). A Cas9 nuclease is also referred to sometimes as a Casnl nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat) associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gNRA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., et al.,  Science  337:816-821(2012), the entire contents of which is hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an M1 strain of  Streptococcus pyogenes .” Ferretti et al., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., et al., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., et al.,  Science  337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to,  S. pyogenes  and  S. thermophilus . Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. 
     An exemplary Cas9, is  Streptococcus pyogenes  Cas9 (spCas9), the amino acid sequence of which is provided below: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 42) 
               
               
                   
                 MDKK YSIGLDIGTNSVGWAVITDDYKVPSKKFKVL   
               
               
                   
                   
               
               
                   
                   GNTDRHSIKKNLIGALLFGSGET AEATRLKRTARR 
               
               
                   
                   
               
               
                   
                 RYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF 
               
               
                   
                   
               
               
                   
                 LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRK 
               
               
                   
                   
               
               
                   
                 KLADSTDKADLRLIYLALAHMIKFRGHFLIEGDLN 
               
               
                   
                   
               
               
                   
                 PDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKA 
               
               
                   
                   
               
               
                   
                 ILSARLSKSRRLENLIAQLPGEKRNGLFGNLIALS 
               
               
                   
                   
               
               
                   
                 LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA 
               
               
                   
                   
               
               
                   
                 QIGDQYADLFLAAKNLSDAILLSDILRVNSEITKA 
               
               
                   
                   
               
               
                   
                 PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
                   
               
               
                   
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG 
               
               
                   
                   
               
               
                   
                 TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELH 
               
               
                   
                   
               
               
                   
                 AILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL 
               
               
                   
                   
               
               
                   
                 ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQS 
               
               
                   
                   
               
               
                   
                 FIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELT 
               
               
                   
                   
               
               
                   
                 KVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT 
               
               
                   
                   
               
               
                   
                 VKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYH 
               
               
                   
                   
               
               
                   
                 DLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRG 
               
               
                   
                   
               
               
                   
                 MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRK 
               
               
                   
                   
               
               
                   
                 LINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
                   
               
               
                   
                 SLTFKEDIQKAQVSGQG HSLHEQIANLAGSPAIKK   
               
               
                   
                   
               
               
                   
                   GILQTVKIVDELVKVMGHKPENIVIEMAR ENQTTQ 
               
               
                   
                   
               
               
                   
                 K GQKNSRERMKRIEEGIKELGSQILKEHPVENTQL   
               
               
                   
                   
               
               
                   
                 
                   QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHI 
                 
               
               
                   
                   
               
               
                   
                 
                   VPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVV 
                 
               
               
                   
                   
               
               
                   
                   KKMKNYWRQLLNAKLITQRKFDNLTK AERGGLSEL 
               
               
                   
                   
               
               
                   
                 
                   DKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEN 
                 
               
               
                   
                   
               
               
                   
                 
                   DKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY 
                 
               
               
                   
                   
               
               
                   
                 
                   HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY 
                 
               
               
                   
                   
               
               
                   
                 
                   DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT 
                 
               
               
                   
                   
               
               
                   
                 
                   LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKV 
                 
               
               
                   
                   
               
               
                   
                   LSMPQVNIVKKTEVQT GGFSKESILPKRNSDKLIA 
               
               
                   
                   
               
               
                   
                 RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK 
               
               
                   
                   
               
               
                   
                 LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVK 
               
               
                   
                   
               
               
                   
                 KDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL 
               
               
                   
                   
               
               
                   
                 ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ 
               
               
                   
                   
               
               
                   
                 HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNK 
               
               
                   
                   
               
               
                   
                 HRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTI 
               
               
                   
                   
               
               
                   
                 DRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG 
               
               
                   
                   
               
               
                   
                 GD 
               
               
                   
                 (single underline: HNH domain; 
               
               
                   
                 double underline:RuvC domain) 
               
            
           
         
       
     
     A nuclease-inactivated Cas9 protein may interchangeably be referred to as a “dCas9” protein (for nuclease-“dead” Cas9) or catalytically inactive Cas9. Methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known (See, e.g., Jinek et al.,  Science.  337:816-821(2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013)  Cell.  28; 152(5):1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of  S. pyogenes  Cas9 (Jinek et al.,  Science.  337:816-821(2012); Qi et al.,  Cell.  28; 152(5):1173-83 (2013)). In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase, referred to as an “nCas9” protein (for “nickase” Cas9). In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as “Cas9 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9. In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild-type Cas9. 
     In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length. 
     In some embodiments, wild-type Cas9 corresponds to Cas9 from  Streptococcus pyogenes  (NCBI Reference Sequence: NC 017053.1, nucleotide and amino acid sequences as follows). 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 43) 
               
               
                   
                 ATGGATAAGAAATACTCAATAGGCTTAGATATCGG 
               
               
                   
                   
               
               
                   
                 CACAAATAGCGTCGGATGGGCGGTGATCACTGATG 
               
               
                   
                   
               
               
                   
                 ATTATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTG 
               
               
                   
                   
               
               
                   
                 GGAAATACAGACCGCCACAGTATCAAAAAAAATCT 
               
               
                   
                   
               
               
                   
                 TATAGGGGCTCTTTTATTTGGCAGTGGAGAGACAG 
               
               
                   
                   
               
               
                   
                 CGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGA 
               
               
                   
                   
               
               
                   
                 AGGTATACACGTCGGAAGAATCGTATTTGTTATCT 
               
               
                   
                   
               
               
                   
                 ACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAG 
               
               
                   
                   
               
               
                   
                 ATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTT 
               
               
                   
                   
               
               
                   
                 TTGGTGGAAGAAGACAAGAAGCATGAACGTCATCC 
               
               
                   
                   
               
               
                   
                 TATTTTTGGAAATATAGTAGATGAAGTTGCTTATC 
               
               
                   
                   
               
               
                   
                 ATGAGAAATATCCAACTATCTATCATCTGCGAAAA 
               
               
                   
                   
               
               
                   
                 AAATTGGCAGATTCTACTGATAAAGCGGATTTGCG 
               
               
                   
                   
               
               
                   
                 CTTAATCTATTTGGCCTTAGCGCATATGATTAAGT 
               
               
                   
                   
               
               
                   
                 TTCGTGGTCATTTTTTGATTGAGGGAGATTTAAAT 
               
               
                   
                   
               
               
                   
                 CCTGATAATAGTGATGTGGACAAACTATTTATCCA 
               
               
                   
                   
               
               
                   
                 GTTGGTACAAATCTACAATCAATTATTTGAAGAAA 
               
               
                   
                   
               
               
                   
                 ACCCTATTAACGCAAGTAGAGTAGATGCTAAAGCG 
               
               
                   
                   
               
               
                   
                 ATTCTTTCTGCACGATTGAGTAAATCAAGACGATT 
               
               
                   
                   
               
               
                   
                 AGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGA 
               
               
                   
                   
               
               
                   
                 GAAATGGCTTGTTTGGGAATCTCATTGCTTTGTCA 
               
               
                   
                   
               
               
                   
                 TTGGGATTGACCCCTAATTTTAAATCAAATTTTGA 
               
               
                   
                   
               
               
                   
                 TTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAG 
               
               
                   
                   
               
               
                   
                 ATACTTACGATGATGATTTAGATAATTTATTGGCG 
               
               
                   
                   
               
               
                   
                 CAAATTGGAGATCAATATGCTGATTTGTTTTTGGC 
               
               
                   
                   
               
               
                   
                 AGCTAAGAATTTATCAGATGCTATTTTACTTTCAG 
               
               
                   
                   
               
               
                   
                 ATATCCTAAGAGTAAATAGTGAAATAACTAAGGCT 
               
               
                   
                   
               
               
                   
                 CCCCTATCAGCTTCAATGATTAAGCGCTACGATGA 
               
               
                   
                   
               
               
                   
                 ACATCATCAAGACTTGACTCTTTTAAAAGCTTTAG 
               
               
                   
                   
               
               
                   
                 TTCGACAACAACTTCCAGAAAAGTATAAAGAAATC 
               
               
                   
                   
               
               
                   
                 TTTTTTGATCAATCAAAAAACGGATATGCAGGTTA 
               
               
                   
                   
               
               
                   
                 TATTGATGGGGGAGCTAGCCAAGAAGAATTTTATA 
               
               
                   
                   
               
               
                   
                 AATTTATCAAACCAATTTTAGAAAAAATGGATGGT 
               
               
                   
                   
               
               
                   
                 ACTGAGGAATTATTGGTGAAACTAAATCGTGAAGA 
               
               
                   
                   
               
               
                   
                 TTTGCTGCGCAAGCAACGGACCTTTGACAACGGCT 
               
               
                   
                   
               
               
                   
                 CTATTCCCCATCAAATTCACTTGGGTGAGCTGCAT 
               
               
                   
                   
               
               
                   
                 GCTATTTTGAGAAGACAAGAAGACTTTTATCCATT 
               
               
                   
                   
               
               
                   
                 TTTAAAAGACAATCGTGAGAAGATTGAAAAAATCT 
               
               
                   
                   
               
               
                   
                 TGACTTTTCGAATTCCTTATTATGTTGGTCCATTG 
               
               
                   
                   
               
               
                   
                 GCGCGTGGCAATAGTCGTTTTGCATGGATGACTCG 
               
               
                   
                   
               
               
                   
                 GAAGTCTGAAGAAACAATTACCCCATGGAATTTTG 
               
               
                   
                   
               
               
                   
                 AAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCA 
               
               
                   
                   
               
               
                   
                 TTTATTGAACGCATGACAAACTTTGATAAAAATCT 
               
               
                   
                   
               
               
                   
                 TCCAAATGAAAAAGTACTACCAAAACATAGTTTGC 
               
               
                   
                   
               
               
                   
                 TTTATGAGTATTTTACGGTTTATAACGAATTGACA 
               
               
                   
                   
               
               
                   
                 AAGGTCAAATATGTTACTGAGGGAATGCGAAAACC 
               
               
                   
                   
               
               
                   
                 AGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTG 
               
               
                   
                   
               
               
                   
                 TTGATTTACTCTTCAAAACAAATCGAAAAGTAACC 
               
               
                   
                   
               
               
                   
                 GTTAAGCAATTAAAAGAAGATTATTTCAAAAAAAT 
               
               
                   
                   
               
               
                   
                 AGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTG 
               
               
                   
                   
               
               
                   
                 AAGATAGATTTAATGCTTCATTAGGCGCCTACCAT 
               
               
                   
                   
               
               
                   
                 GATTTGCTAAAAATTATTAAAGATAAAGATTTTTT 
               
               
                   
                   
               
               
                   
                 GGATAATGAAGAAAATGAAGATATCTTAGAGGATA 
               
               
                   
                   
               
               
                   
                 TTGTTTTAACATTGACCTTATTTGAAGATAGGGGG 
               
               
                   
                   
               
               
                   
                 ATGATTGAGGAAAGACTTAAAACATATGCTCACCT 
               
               
                   
                   
               
               
                   
                 CTTTGATGATAAGGTGATGAAACAGCTTAAACGTC 
               
               
                   
                   
               
               
                   
                 GCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAA 
               
               
                   
                   
               
               
                   
                 TTGATTAATGGTATTAGGGATAAGCAATCTGGCAA 
               
               
                   
                   
               
               
                   
                 AACAATATTAGATTTTTTGAAATCAGATGGTTTTG 
               
               
                   
                   
               
               
                   
                 CCAATCGCAATTTTATGCAGCTGATCCATGATGAT 
               
               
                   
                   
               
               
                   
                 AGTTTGACATTTAAAGAAGATATTCAAAAAGCACA 
               
               
                   
                   
               
               
                   
                 GGTGTCTGGACAAGGCCATAGTTTACATGAACAGA 
               
               
                   
                   
               
               
                   
                 TTGCTAACTTAGCTGGCAGTCCTGCTATTAAAAAA 
               
               
                   
                   
               
               
                   
                 GGTATTTTACAGACTGTAAAAATTGTTGATGAACT 
               
               
                   
                   
               
               
                   
                 GGTCAAAGTAATGGGGCATAAGCCAGAAAATATCG 
               
               
                   
                   
               
               
                   
                 TTATTGAAATGGCACGTGAAAATCAGACAACTCAA 
               
               
                   
                   
               
               
                   
                 AAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACG 
               
               
                   
                   
               
               
                   
                 AATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGA 
               
               
                   
                   
               
               
                   
                 TTCTTAAAGAGCATCCTGTTGAAAATACTCAATTG 
               
               
                   
                   
               
               
                   
                 CAAAATGAAAAGCTCTATCTCTATTATCTACAAAA 
               
               
                   
                   
               
               
                   
                 TGGAAGAGACATGTATGTGGACCAAGAATTAGATA 
               
               
                   
                   
               
               
                   
                 TTAATCGTTTAAGTGATTATGATGTCGATCACATT 
               
               
                   
                   
               
               
                   
                 GTTCCACAAAGTTTCATTAAAGACGATTCAATAGA 
               
               
                   
                   
               
               
                   
                 CAATAAGGTACTAACGCGTTCTGATAAAAATCGTG 
               
               
                   
                   
               
               
                   
                 GTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTC 
               
               
                   
                   
               
               
                   
                 AAAAAGATGAAAAACTATTGGAGACAACTTCTAAA 
               
               
                   
                   
               
               
                   
                 CGCCAAGTTAATCACTCAACGTAAGTTTGATAATT 
               
               
                   
                   
               
               
                   
                 TAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTT 
               
               
                   
                   
               
               
                   
                 GATAAAGCTGGTTTTATCAAACGCCAATTGGTTGA 
               
               
                   
                   
               
               
                   
                 AACTCGCCAAATCACTAAGCATGTGGCACAAATTT 
               
               
                   
                   
               
               
                   
                 TGGATAGTCGCATGAATACTAAATACGATGAAAAT 
               
               
                   
                   
               
               
                   
                 GATAAACTTATTCGAGAGGTTAAAGTGATTACCTT 
               
               
                   
                   
               
               
                   
                 AAAATCTAAATTAGTTTCTGACTTCCGAAAAGATT 
               
               
                   
                   
               
               
                   
                 TCCAATTCTATAAAGTACGTGAGATTAACAATTAC 
               
               
                   
                   
               
               
                   
                 CATCATGCCCATGATGCGTATCTAAATGCCGTCGT 
               
               
                   
                   
               
               
                   
                 TGGAACTGCTTTGATTAAGAAATATCCAAAACTTG 
               
               
                   
                   
               
               
                   
                 AATCGGAGTTTGTCTATGGTGATTATAAAGTTTAT 
               
               
                   
                   
               
               
                   
                 GATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGA 
               
               
                   
                   
               
               
                   
                 AATAGGCAAAGCAACCGCAAAATATTTCTTTTACT 
               
               
                   
                   
               
               
                   
                 CTAATATCATGAACTTCTTCAAAACAGAAATTACA 
               
               
                   
                   
               
               
                   
                 CTTGCAAATGGAGAGATTCGCAAACGCCCTCTAAT 
               
               
                   
                   
               
               
                   
                 CGAAACTAATGGGGAAACTGGAGAAATTGTCTGGG 
               
               
                   
                   
               
               
                   
                 ATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTA 
               
               
                   
                   
               
               
                   
                 TTGTCCATGCCCCAAGTCAATATTGTCAAGAAAAC 
               
               
                   
                   
               
               
                   
                 AGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAA 
               
               
                   
                   
               
               
                   
                 TTTTACCAAAAAGAAATTCGGACAAGCTTATTGCT 
               
               
                   
                   
               
               
                   
                 CGTAAAAAAGACTGGGATCCAAAAAAATATGGTGG 
               
               
                   
                   
               
               
                   
                 TTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAG 
               
               
                   
                   
               
               
                   
                 TGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAG 
               
               
                   
                   
               
               
                   
                 TTAAAATCCGTTAAAGAGTTACTAGGGATCACAAT 
               
               
                   
                   
               
               
                   
                 TATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTG 
               
               
                   
                   
               
               
                   
                 ACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAA 
               
               
                   
                   
               
               
                   
                 AAAGACTTAATCATTAAACTACCTAAATATAGTCT 
               
               
                   
                   
               
               
                   
                 TTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGG 
               
               
                   
                   
               
               
                   
                 CTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTG 
               
               
                   
                   
               
               
                   
                 GCTCTGCCAAGCAAATATGTGAATTTTTTATATTT 
               
               
                   
                   
               
               
                   
                 AGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAG 
               
               
                   
                   
               
               
                   
                 AAGATAACGAACAAAAACAATTGTTTGTGGAGCAG 
               
               
                   
                   
               
               
                   
                 CATAAGCATTATTTAGATGAGATTATTGAGCAAAT 
               
               
                   
                   
               
               
                   
                 CAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATG 
               
               
                   
                   
               
               
                   
                 CCAATTTAGATAAAGTTCTTAGTGCATATAACAAA 
               
               
                   
                   
               
               
                   
                 CATAGAGACAAACCAATACGTGAACAAGCAGAAAA 
               
               
                   
                   
               
               
                   
                 TATTATTCATTTATTTACGTTGACGAATCTTGGAG 
               
               
                   
                   
               
               
                   
                 CTCCCGCTGCTTTTAAATATTTTGATACAACAATT 
               
               
                   
                   
               
               
                   
                 GATCGTAAACGATATACGTCTACAAAAGAAGTTTT 
               
               
                   
                   
               
               
                   
                 AGATGCCACTCTTATCCATCAATCCATCACTGGTC 
               
               
                   
                   
               
               
                   
                 TTTATGAAACACGCATTGATTTGAGTCAGCTAGGA 
               
               
                   
                   
               
               
                   
                 GGTGACTGA 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 42) 
               
               
                   
                 MDKK YSIGLDIGTNSVGWAVITDDYKVPSKKFKVL   
               
               
                   
                   
               
               
                   
                   GNTDRHSIKKNLIGALLFGSGET AEATRLKRTARR 
               
               
                   
                   
               
               
                   
                 RYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF 
               
               
                   
                   
               
               
                   
                 LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRK 
               
               
                   
                   
               
               
                   
                 KLADSTDKADLRLIYLALAHMIKFRGHFLIEGDLN 
               
               
                   
                   
               
               
                   
                 PDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKA 
               
               
                   
                   
               
               
                   
                 ILSARLSKSRRLENLIAQLPGEKRNGLFGNLIALS 
               
               
                   
                   
               
               
                   
                 LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA 
               
               
                   
                   
               
               
                   
                 QIGDQYADLFLAAKNLSDAILLSDILRVNSEITKA 
               
               
                   
                   
               
               
                   
                 PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
                   
               
               
                   
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG 
               
               
                   
                   
               
               
                   
                 TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELH 
               
               
                   
                   
               
               
                   
                 AILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL 
               
               
                   
                   
               
               
                   
                 ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQS 
               
               
                   
                   
               
               
                   
                 FIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELT 
               
               
                   
                   
               
               
                   
                 KVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT 
               
               
                   
                   
               
               
                   
                 VKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYH 
               
               
                   
                   
               
               
                   
                 DLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRG 
               
               
                   
                   
               
               
                   
                 MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRK 
               
               
                   
                   
               
               
                   
                 LINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
                   
               
               
                   
                 SLTFKEDIQKAQVSGQG HSLHEQIANLAGSPAIKK   
               
               
                   
                   
               
               
                   
                   GILQTVKIVDELVKVMGHKPENIVIEMAR ENQTTQ 
               
               
                   
                   
               
               
                   
                 K GQKNSRERMKRIEEGIKELGSQILKEHPVENTQL   
               
               
                   
                   
               
               
                   
                 
                   QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHI 
                 
               
               
                   
                   
               
               
                   
                 
                   VPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVV 
                 
               
               
                   
                   
               
               
                   
                 
                   KKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEL 
                 
               
               
                   
                   
               
               
                   
                 
                   DKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEN 
                 
               
               
                   
                   
               
               
                   
                 
                   DKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY 
                 
               
               
                   
                   
               
               
                   
                 
                   HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY 
                 
               
               
                   
                   
               
               
                   
                 
                   DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT 
                 
               
               
                   
                   
               
               
                   
                 
                   LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKV 
                 
               
               
                   
                   
               
               
                   
                   LSMPQVNIVKKTEVQT GGFSKESILPKRNSDKLIA 
               
               
                   
                   
               
               
                   
                 RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK 
               
               
                   
                   
               
               
                   
                 LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVK 
               
               
                   
                   
               
               
                   
                 KDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL 
               
               
                   
                   
               
               
                   
                 ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ 
               
               
                   
                   
               
               
                   
                 HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNK 
               
               
                   
                   
               
               
                   
                 HRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTI 
               
               
                   
                   
               
               
                   
                 DRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG 
               
               
                   
                   
               
               
                   
                 GD 
               
               
                   
                 (single underline: HNH domain; 
               
               
                   
                 double underline: RuvC domain) 
               
            
           
         
       
     
     In some embodiments, wild-type Cas9 corresponds to, or comprises the following nucleotide and/or amino acid sequences: 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 44) 
                   
               
               
                 ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCAT 
                   
               
               
                   
               
               
                 AACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATT 
               
               
                   
               
               
                 CGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACT 
               
               
                   
               
               
                 CGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACA 
               
               
                   
               
               
                 AGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGT 
               
               
                   
               
               
                 CCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGAT 
               
               
                   
               
               
                 GAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTC 
               
               
                   
               
               
                 AACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTG 
               
               
                   
               
               
                 GGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATC 
               
               
                   
               
               
                 CAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGA 
               
               
                   
               
               
                 TGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCAC 
               
               
                   
               
               
                 AATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTG 
               
               
                   
               
               
                 ACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGA 
               
               
                   
               
               
                 CACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTAT 
               
               
                   
               
               
                 TTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACT 
               
               
                   
               
               
                 GAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGA 
               
               
                   
               
               
                 CTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCT 
               
               
                   
               
               
                 TTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTC 
               
               
                   
               
               
                 TACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACT 
               
               
                   
               
               
                 CAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAA 
               
               
                   
               
               
                 TCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAA 
               
               
                   
               
               
                 GACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCT 
               
               
                   
               
               
                 GGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCAT 
               
               
                   
               
               
                 GGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACC 
               
               
                   
               
               
                 AACTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTA 
               
               
                   
               
               
                 TTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCG 
               
               
                   
               
               
                 CCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAA 
               
               
                   
               
               
                 GTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGA 
               
               
                   
               
               
                 GATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGA 
               
               
                   
               
               
                 TAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTG 
               
               
                   
               
               
                 TTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCTCA 
               
               
                   
               
               
                 CCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGAT 
               
               
                   
               
               
                 TGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTT 
               
               
                   
               
               
                 CTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGACTCTTTAAC 
               
               
                   
               
               
                 CTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATA 
               
               
                   
               
               
                 TTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTG 
               
               
                   
               
               
                 GATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACG 
               
               
                   
               
               
                 CGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAG 
               
               
                   
               
               
                 AGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTG 
               
               
                   
               
               
                 CAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGA 
               
               
                   
               
               
                 ACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGA 
               
               
                   
               
               
                 AGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGAC 
               
               
                   
               
               
                 AATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGC 
               
               
                   
               
               
                 GAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTG 
               
               
                   
               
               
                 AACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCAT 
               
               
                   
               
               
                 GTTGCACAGATACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCG 
               
               
                   
               
               
                 GGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAAT 
               
               
                   
               
               
                 TCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTAATGCCGTC 
               
               
                   
               
               
                 GTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTA 
               
               
                   
               
               
                 CAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAG 
               
               
                   
               
               
                 CCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAAC 
               
               
                   
               
               
                 GGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGA 
               
               
                   
               
               
                 TAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAA 
               
               
                   
               
               
                 AGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGT 
               
               
                   
               
               
                 GATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCC 
               
               
                   
               
               
                 TACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGA 
               
               
                   
               
               
                 AGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCC 
               
               
                   
               
               
                 ATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACC 
               
               
                   
               
               
                 AAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGC 
               
               
                   
               
               
                 TTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCC 
               
               
                   
               
               
                 CATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCA 
               
               
                   
               
               
                 GCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCC 
               
               
                   
               
               
                 TAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATA 
               
               
                   
               
               
                 CGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGC 
               
               
                   
               
               
                 ATTCAAGTATTTTGACACAACGATAGATCGCAAACGATACACTTCTACCAAGGAGGTGCTAG 
               
               
                   
               
               
                 ACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAG 
               
               
                   
               
               
                 CTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGA 
               
               
                   
               
               
                 CGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCTGCAGGA 
               
               
                   
               
               
                 (SEQ ID NO: 1) 
                   
               
               
                 MDKK YSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET AEAT 
                   
               
               
                   
               
               
                 RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD 
               
               
                   
               
               
                 EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGL 
               
               
                   
               
               
                 TPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNT 
               
               
                   
               
               
                 EITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF 
               
               
                   
               
               
                 YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLK 
               
               
                   
               
               
                 DNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT 
               
               
                   
               
               
                 NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRK 
               
               
                   
               
               
                 VTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIV 
               
               
                   
               
               
                 LTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
               
               
                   
               
               
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGSPAIKKGILQTVKVV   
               
               
                   
               
               
                   DELVKVMGRHKPENIVIEMA RENQTTQK GQKNSRERMKRIEEGIKELGSQILKEHPVENTQL   
               
               
                   
               
               
                 
                   QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD 
                 
               
               
                   
               
               
                   NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK AERG GLSELDKAGFIKRQLVETRQITKH   
               
               
                   
               
               
                 
                   VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAV 
                 
               
               
                   
               
               
                 
                   VGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN 
                 
               
               
                   
               
               
                   GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQT GGFSKESILPKRNS 
               
               
                   
               
               
                 DKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP 
               
               
                   
               
               
                 IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLAS 
               
               
                   
               
               
                 HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI 
               
               
                   
               
               
                 REQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 (single underline: HNH domain; double underline: RuvC domain) 
               
            
           
         
       
     
     In some embodiments, wild-type Cas9 corresponds to Cas9 from  Streptococcus pyogenes  (NCBI Reference Sequence: NC_002737.2 (nucleotide sequence as follows); and Uniprot Reference Sequence: Q99ZW2 (amino acid sequence as follows). 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 45) 
                   
               
               
                 ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGAT 
                   
               
               
                   
               
               
                 CACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACA 
               
               
                   
               
               
                 GTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACT 
               
               
                   
               
               
                 CGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACA 
               
               
                   
               
               
                 GGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGT 
               
               
                   
               
               
                 CTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGAT 
               
               
                   
               
               
                 GAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTC 
               
               
                   
               
               
                 TACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTG 
               
               
                   
               
               
                 GTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATC 
               
               
                   
               
               
                 CAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGA 
               
               
                   
               
               
                 TGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTC 
               
               
                   
               
               
                 AGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTG 
               
               
                   
               
               
                 ACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGA 
               
               
                   
               
               
                 TACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGT 
               
               
                   
               
               
                 TTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACT 
               
               
                   
               
               
                 GAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGA 
               
               
                   
               
               
                 CTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTT 
               
               
                   
               
               
                 TTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTT 
               
               
                   
               
               
                 TATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACT 
               
               
                   
               
               
                 AAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAA 
               
               
                   
               
               
                 TTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAA 
               
               
                   
               
               
                 GACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATT 
               
               
                   
               
               
                 GGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCAT 
               
               
                   
               
               
                 GGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACA 
               
               
                   
               
               
                 AACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTA 
               
               
                   
               
               
                 TTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAG 
               
               
                   
               
               
                 CATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAA 
               
               
                   
               
               
                 GTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGA 
               
               
                   
               
               
                 AATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAA 
               
               
                   
               
               
                 TTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTT 
               
               
                   
               
               
                 TTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCA 
               
               
                   
               
               
                 CCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTT 
               
               
                   
               
               
                 TGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTT 
               
               
                   
               
               
                 TTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGAC 
               
               
                   
               
               
                 ATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATA 
               
               
                   
               
               
                 TTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTT 
               
               
                   
               
               
                 GATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACG 
               
               
                   
               
               
                 TGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAG 
               
               
                   
               
               
                 AAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTG 
               
               
                   
               
               
                 CAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGA 
               
               
                   
               
               
                 ATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTA 
               
               
                   
               
               
                 AAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGAT 
               
               
                   
               
               
                 AACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGC 
               
               
                   
               
               
                 CAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTG 
               
               
                   
               
               
                 AACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCAT 
               
               
                   
               
               
                 GTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCG 
               
               
                   
               
               
                 AGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAAT 
               
               
                   
               
               
                 TCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTC 
               
               
                   
               
               
                 GTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTA 
               
               
                   
               
               
                 TAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCG 
               
               
                   
               
               
                 CAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAAT 
               
               
                   
               
               
                 GGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGA 
               
               
                   
               
               
                 TAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCA 
               
               
                   
               
               
                 AGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCG 
               
               
                   
               
               
                 GACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCC 
               
               
                   
               
               
                 AACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAA 
               
               
                   
               
               
                 AATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCG 
               
               
                   
               
               
                 ATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACC 
               
               
                   
               
               
                 TAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAAT 
               
               
                   
               
               
                 TACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGT 
               
               
                   
               
               
                 CATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCA 
               
               
                   
               
               
                 GCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTT 
               
               
                   
               
               
                 TAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATA 
               
               
                   
               
               
                 CGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGC 
               
               
                   
               
               
                 TTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAG 
               
               
                   
               
               
                 ATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAG 
               
               
                   
               
               
                 CTAGGAGGTGACTGA 
               
               
                   
               
               
                 (SEQ ID NO: 1) 
                   
               
               
                 MDKK YSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET AEAT 
                   
               
               
                   
               
               
                 RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD 
               
               
                   
               
               
                 EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGL 
               
               
                   
               
               
                 TPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNT 
               
               
                   
               
               
                 EITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF 
               
               
                   
               
               
                 YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLK 
               
               
                   
               
               
                 DNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT 
               
               
                   
               
               
                 NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRK 
               
               
                   
               
               
                 VTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIV 
               
               
                   
               
               
                 LTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
               
               
                   
               
               
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGSPAIKKGILQTVKVV   
               
               
                   
               
               
                   DELVKVMGRHKPENIVIEMA RENQTTQK GQKNSRERMKRIEEGIKELGSQILKEHPVENTQL   
               
               
                   
               
               
                 
                   QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD 
                 
               
               
                   
               
               
                   NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK AERG GLSELDKAGFIKRQLVETRQITKH   
               
               
                   
               
               
                 
                   VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAV 
                 
               
               
                   
               
               
                 
                   VGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEOEIGKATAKYFFYSNIMNFFKTEITLAN 
                 
               
               
                   
               
               
                   GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQT GGFSKESILPKRNS 
               
               
                   
               
               
                 DKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP 
               
               
                   
               
               
                 IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLAS 
               
               
                   
               
               
                 HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI 
               
               
                   
               
               
                 REQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 (single underline: HNH domain; double underline: RuvC domain) 
               
            
           
         
       
     
     In some embodiments, Cas9 refers to Cas9 from:  Corynebacterium ulcerans  (NCBI Refs: NC_015683.1, NC_017317.1);  Corynebacterium diphtheria  (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1);  Prevotella intermedia  (NCBI Ref: NC_017861.1);  Spiroplasma taiwanense  (NCBI Ref: NC_021846.1);  Streptococcus iniae  (NCBI Ref: NC_021314.1);  Belliella baltica  (NCBI Ref: NC_018010.1);  Psychroflexus torquis  I (NCBI Ref: NC_018721.1);  Streptococcus thermophilus  (NCBI Ref: YP_820832.1),  Listeria innocua  (NCBI Ref: NP_472073.1),  Campylobacter jejuni  (NCBI Ref: YP_002344900.1) or  Neisseria meningitidis  (NCBI Ref: YP_002342100.1) or to a Cas9 from any other organism. 
     In some embodiments, dCas9 corresponds to, or comprises in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. For example, in some embodiments, a dCas9 domain comprises D10A and an H840A mutation as numbered in SEQ ID NO: 1 or corresponding mutations in another Cas9. In some embodiments, the dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A): 
     
       
         
           
               
            
               
                 (SEQ ID NO: 46) 
               
               
                 MDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA   
               
               
                   
               
               
                   LLFDSGET AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQG DSLHEHIANLAGSPAIKKGILQTVKVVDELVKV   
               
               
                   
               
               
                   MGRHKPENIVIEMA RENQTTQK GQKNSRERMKRIEEGIKELGSQILKEHP   
               
               
                   
               
               
                 
                   VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDD 
                 
               
               
                   
               
               
                 
                   SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
                 
               
               
                   
               
               
                   TK AERG GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI   
               
               
                   
               
               
                 
                   REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
                 
               
               
                   
               
               
                 
                   YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
                 
               
               
                   
               
               
                 
                   TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
                 
               
               
                   
               
               
                   QT GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE 
               
               
                   
               
               
                 DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK 
               
               
                   
               
               
                 PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ 
               
               
                   
               
               
                 SITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 (single underline: HNH domain; double underline: 
               
               
                   
               
               
                 RuvC domain). 
               
            
           
         
       
     
     In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 remains a histidine in the amino acid sequence provided above, or at corresponding positions in any of the amino acid sequences provided herein. 
     In other embodiments, dCas9 variants having mutations other than D10A and H840A are provided, which, e.g., result in nuclease inactivated Cas9 (dCas9). Such mutations, by way of example, include other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and/or the RuvC1 subdomain). In some embodiments, variants or homologues of dCas9 are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, variants of dCas9 are provided having amino acid sequences which are shorter, or longer, by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more. 
     In some embodiments, Cas9 fusion proteins as provided herein comprise the full-length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided herein. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length Cas9 sequence, but only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable sequences of Cas9 domains and fragments will be apparent to those of skill in the art. 
     It should be appreciated that additional Cas9 proteins (e.g., a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9), including variants and homologs thereof, are within the scope of this disclosure. Exemplary Cas9 proteins include, without limitation, those provided below. In some embodiments, the Cas9 protein is a nuclease dead Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a nuclease active Cas9. 
     Exemplary Catalytically Inactive Cas9 (dCas9): 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 47) 
                   
               
               
                 DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATR 
                   
               
               
                   
               
               
                 LKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDE 
               
               
                   
               
               
                 VAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQ 
               
               
                   
               
               
                 LVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLT 
               
               
                   
               
               
                 PNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTE 
               
               
                   
               
               
                 ITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFY 
               
               
                   
               
               
                 KFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD 
               
               
                   
               
               
                 NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTN 
               
               
                   
               
               
                 FDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKV 
               
               
                   
               
               
                 TVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVL 
               
               
                   
               
               
                 TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFL 
               
               
                   
               
               
                 KSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVD 
               
               
                   
               
               
                 ELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQ 
               
               
                   
               
               
                 NEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDN 
               
               
                   
               
               
                 VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV 
               
               
                   
               
               
                 AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQEYKVREINNYHHAHDAYLNAVV 
               
               
                   
               
               
                 GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANG 
               
               
                   
               
               
                 EIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSD 
               
               
                   
               
               
                 KLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI 
               
               
                   
               
               
                 DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH 
               
               
                   
               
               
                 YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIR 
               
               
                   
               
               
                 EQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQL 
               
               
                   
               
               
                 GGD 
               
               
                   
               
               
                 Exemplary catalytically Cas9 nickase (nCas9): 
               
               
                 (SEQ ID NO: 48) 
                   
               
               
                 DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATR 
                   
               
               
                   
               
               
                 LKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDE 
               
               
                   
               
               
                 VAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQ 
               
               
                   
               
               
                 LVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLT 
               
               
                   
               
               
                 PNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTE 
               
               
                   
               
               
                 ITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFY 
               
               
                   
               
               
                 KFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD 
               
               
                   
               
               
                 NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTN 
               
               
                   
               
               
                 FDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKV 
               
               
                   
               
               
                 TVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVL 
               
               
                   
               
               
                 TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFL 
               
               
                   
               
               
                 KSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVD 
               
               
                   
               
               
                 ELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQ 
               
               
                   
               
               
                 NEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDN 
               
               
                   
               
               
                 VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV 
               
               
                   
               
               
                 AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVV 
               
               
                   
               
               
                 GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANG 
               
               
                   
               
               
                 EIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSD 
               
               
                   
               
               
                 KLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI 
               
               
                   
               
               
                 DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH 
               
               
                   
               
               
                 YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIR 
               
               
                   
               
               
                 EQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQL 
               
               
                   
               
               
                 GGD 
               
               
                   
               
               
                 Exemplary catalytically active Cas9: 
               
               
                 (SEQ ID NO: 49) 
                   
               
               
                 DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATR 
                   
               
               
                   
               
               
                 LKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDE 
               
               
                   
               
               
                 VAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQ 
               
               
                   
               
               
                 LVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLT 
               
               
                   
               
               
                 PNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTE 
               
               
                   
               
               
                 ITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFY 
               
               
                   
               
               
                 KFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD 
               
               
                   
               
               
                 NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTN 
               
               
                   
               
               
                 FDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKV 
               
               
                   
               
               
                 TVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVL 
               
               
                   
               
               
                 TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFL 
               
               
                   
               
               
                 KSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVD 
               
               
                   
               
               
                 ELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQ 
               
               
                   
               
               
                 NEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDN 
               
               
                   
               
               
                 VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV 
               
               
                   
               
               
                 AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVV 
               
               
                   
               
               
                 GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANG 
               
               
                   
               
               
                 EIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSD 
               
               
                   
               
               
                 KLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI 
               
               
                   
               
               
                 DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH 
               
               
                   
               
               
                 YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIR 
               
               
                   
               
               
                 EQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQL 
               
               
                   
               
               
                 GGD. 
               
            
           
         
       
     
     In some embodiments, Cas9 refers to a Cas9 from archaea (e.g., nanoarchaea), which constitute a domain and kingdom of single-celled prokaryotic microbes. In some embodiments, Cas9 refers to CasX or CasY, which have been described in, for example, Burstein et al., “New CRISPR-Cas systems from uncultivated microbes.” Cell Res. 2017 Feb. 21. doi: 10.1038/cr.2017.21, the entire contents of which is hereby incorporated by reference. Using genome-resolved metagenomics, a number of CRISPR-Cas systems were identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems were discovered, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. In some embodiments, Cas9 refers to CasX, or a variant of CasX. In some embodiments, Cas9 refers to a CasY, or a variant of CasY. It should be appreciated that other RNA-guided DNA binding proteins may be used as a nucleic acid programmable DNA binding protein (napDNAbp), and are within the scope of this disclosure. 
     In particular embodiments, napDNAbps useful in the methods of the invention include circular permutants, which are known in the art and described, for example, by Oakes et al., Cell 176, 254-267, 2019. An exemplary circular permutant follows where the bold sequence indicates sequence derived from Cas9, the italics sequence denotes a linker sequence, and the underlined sequence denotes a bipartite nuclear localization sequence, CP5 (with MSP “NGC=Pam Variant with mutations Regular Cas9 likes NGG” PID=Protein Interacting Domain and “D10A” nickase): 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 38) 
                   
               
               
                 
                   EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM 
                 
                   
               
               
                   
               
               
                 
                   PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFMQPTVAYSVLVVAKVEK 
                 
               
               
                   
               
               
                 
                   GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRM 
                 
               
               
                   
               
               
                 
                   LASAKFLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE 
                 
               
               
                   
               
               
                 
                   FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIARKEYR 
                 
               
               
                   
               
               
                 
                   STKEVLDATLIHQSITGLYETRIDLSQLGGD 
                   GGSGGSGGSGGSGGSGGSGGM 
                   DKKYSIGLAI 
                 
               
               
                   
               
               
                 
                   GTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYT 
                 
               
               
                   
               
               
                 
                   RRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTI 
                 
               
               
                   
               
               
                 
                   YHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE 
                 
               
               
                   
               
               
                 
                   ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLA 
                 
               
               
                   
               
               
                 
                   EDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM 
                 
               
               
                   
               
               
                 
                   IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKM 
                 
               
               
                   
               
               
                 
                   DGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILT 
                 
               
               
                   
               
               
                 
                   FRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV 
                 
               
               
                   
               
               
                 
                   LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYF 
                 
               
               
                   
               
               
                 
                   KKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREM 
                 
               
               
                   
               
               
                 
                   IEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNF 
                 
               
               
                   
               
               
                 MQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHK 
               
               
                   
               
               
                 
                   PENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ 
                 
               
               
                   
               
               
                 
                   NGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKM 
                 
               
               
                   
               
               
                 
                   KNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNT 
                 
               
               
                   
               
               
                 
                   KYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPK 
                 
               
               
                   
               
               
                   LESEFVYGDYKVYDVRKMIAKSEQ   EGADKRTADGSEFESPKKKRKV * 
               
            
           
         
       
     
     Non-limiting examples of a polynucleotide programmable nucleotide binding domain which can be incorporated into a base editor include a CRISPR protein-derived domain, a restriction nuclease, a meganuclease, TAL nuclease (TALEN), and a zinc finger nuclease (ZFN). 
     In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein may be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to a naturally-occurring CasX or CasY protein. In some embodiments, the napDNAbp is a naturally-occurring CasX or CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to any CasX or CasY protein described herein. It should be appreciated that Cas12b/C2c1, CasX and CasY from other bacterial species may also be used in accordance with the present disclosure. 
     
       
         
           
               
               
            
               
                 Cas12b/C2c1 (uniprot.org/uniprot/T0D7A2#2) 
                   
               
               
                 sp|TOD7A2|C2C1_ALIAG CRISPR-associated endo-nuclease C2c1 OS = 
               
               
                   Alicyclobacillus acido-terrestris  (strain ATCC 49025/DSM 3922/ 
               
               
                 CIP 106132/NCIMB 13137/GD3B) GN = c2c1 PE = 1 SV = 1 
               
               
                 (SEQ ID NO: 50) 
                   
               
               
                 MAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECD 
                   
               
               
                   
               
               
                 KTAEECKAELLERLRARQVENGHRGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKF 
               
               
                   
               
               
                 LSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKEKAETRKSADRTADVLRALADFG 
               
               
                   
               
               
                 LKPLMRVYTDSEMSSVEWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGQEYAKLVEQ 
               
               
                   
               
               
                 KNRFEQKNFVGQEHLVHLVNQLQQDMKEASPGLESKEQTAHYVTGRALRGSDKVFEKWGKLA 
               
               
                   
               
               
                 PDAPFDLYDAEIKNVQRRNTRRFGSHDLFAKLAEPEYQALWREDASFLTRYAVYNSILRKLN 
               
               
                   
               
               
                 HAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGERRHAIRFHKLLKVENGVAREV 
               
               
                   
               
               
                 DDVTVPISMSEQLDNLLPRDPNEPIALYFRDYGAEQHFTGEFGGAKIQCRRDQLAHMHRRRG 
               
               
                   
               
               
                 ARDVYLNVSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSE 
               
               
                   
               
               
                 GLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSKGRVPFFFPIKGNDNLVAVHERSQLL 
               
               
                   
               
               
                 KLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPVDAAN 
               
               
                   
               
               
                 HMTPDWREAFENELQKLKSLHGICSDKEWMDAVYESVRRVWRHMGKQVRDWRKDVRSGERPK 
               
               
                   
               
               
                 IRGYAKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKE 
               
               
                   
               
               
                 DRLKKLADRIIMEALGYVYALDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLM 
               
               
                   
               
               
                 QWSHRGVFQELINQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCTQEHNPEPFPW 
               
               
                   
               
               
                 WLNKFVVEHTLDACPLRADDLIPTGEGEIFVSPFSAEEGDFHQIHADLNAAQNLQQRLWSDF 
               
               
                   
               
               
                 DISQIRLRCDWGEVDGELVLIPRLTGKRTADSYSNKVFYTNTGVTYYERERGKKRRKVFAQE 
               
               
                   
               
               
                 KLSEEEAELLVEADEAREKSVVLMRDPSGIINRGNWTRQKEFWSMV NQRIEGYLVKQIRSR 
               
               
                   
               
               
                 VPLQDSACENTGDI 
               
               
                   
               
               
                 CasX (uniprot.org/uniprot/F0NN87; uniprot.org/uniprot/F0NH53) 
               
               
                 &gt;tr|F0NN87|F0NN87_SULIH CRISPR-associated Casx protein OS = 
               
               
                   Sulfolobus   islandicus  (strain HVE10/4) GN = SiH_0402 PE = 4 
               
               
                 SV = 1 
               
               
                 (SEQ ID NO: 51) 
                   
               
               
                 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGK 
                   
               
               
                   
               
               
                 AKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAP 
               
               
                   
               
               
                 SFVKPEFYEFGRSPGMVERTRRVKLEVEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTP 
               
               
                   
               
               
                 TRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTIN 
               
               
                   
               
               
                 GGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLY 
               
               
                   
               
               
                 FANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG 
               
               
                   
               
               
                 &gt;tr|F0NH53|F0NH53_SULIR CRISPR associated protein, Casx OS = 
               
               
                   Sulfolobus   islandicus  (strain REY15A) GN = SiRe_0771 PE = 4 
               
               
                 SV = 1 
               
               
                 (SEQ ID NO: 52) 
                   
               
               
                 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGK 
                   
               
               
                   
               
               
                 AKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAP 
               
               
                   
               
               
                 SFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTP 
               
               
                   
               
               
                 TRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTIN 
               
               
                   
               
               
                 GGFSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTGSKRLEDLLYF 
               
               
                   
               
               
                 ANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG 
               
               
                   
               
               
                 Deltaproteobacteria CasX 
               
               
                 (SEQ ID NO: 53) 
                   
               
               
                 MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAA 
                   
               
               
                   
               
               
                 NNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTA 
               
               
                   
               
               
                 GFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLG 
               
               
                   
               
               
                 KFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIII 
               
               
                   
               
               
                 EHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVRMWVNLNLWQ 
               
               
                   
               
               
                 KLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEK 
               
               
                   
               
               
                 RNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERID 
               
               
                   
               
               
                 KKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQKWYG 
               
               
                   
               
               
                 DLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRF 
               
               
                   
               
               
                 TDGTDIKKSGKWQGLLYGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETG 
               
               
                   
               
               
                 LIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIA 
               
               
                   
               
               
                 LTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLA 
               
               
                   
               
               
                 DDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLT 
               
               
                   
               
               
                 SKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYN 
               
               
                   
               
               
                 RYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGH 
               
               
                   
               
               
                 EVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA 
               
               
                   
               
               
                 CasY (ncbi.nlm.nih.gov/protein/APG80656.1)  
               
               
                 &gt;APG80656.1 CRISPR-associated protein CasY [uncultured 
               
               
                 Parcubacteria group bacterium] 
               
               
                 (SEQ ID NO: 54) 
                   
               
               
                 MSKRHPRISGVKGYRLHAQRLEYTGKSGAMRTIKYPLYSSPSGGRTVPREIVSAINDDYVGL 
                   
               
               
                   
               
               
                 YGLSNFDDLYNAEKRNEEKVYSVLDFWYDCVQYGAVFSYTAPGLLKNVAEVRGGSYELTKTL 
               
               
                   
               
               
                 KGSHLYDELQIDKVIKFLNKKEISRANGSLDKLKKDIIDCFKAEYRERHKDQCNKLADDIKN 
               
               
                   
               
               
                 AKKDAGASLGERQKKLFRDFFGISEQSENDKPSFTNPLNLTCCLLPFDTVNNNRNRGEVLFN 
               
               
                   
               
               
                 KLKEYAQKLDKNEGSLEMWEYIGIGNSGTAFSNFLGEGFLGRLRENKITELKKAMMDITDAW 
               
               
                   
               
               
                 RGQEQEEELEKRLRILAALTIKLREPKFDNHWGGYRSDINGKLSSWLQNYINQTVKIKEDLK 
               
               
                   
               
               
                 GHKKDLKKAKEMINRFGESDTKEEAVVSSLLESIEKIVPDDSADDEKPDIPAIAIYRRFLSD 
               
               
                   
               
               
                 GRLTLNRFVQREDVQEALIKERLEAEKKKKPKKRKKKSDAEDEKETIDFKELFPHLAKPLKL 
               
               
                   
               
               
                 VPNFYGDSKRELYKKYKNAAIYTDALWKAVEKIYKSAFSSSLKNSFFDTDFDKDFFIKRLQK 
               
               
                   
               
               
                 IFSVYRRFNTDKWKPIVKNSFAPYCDIVSLAENEVLYKPKQSRSRKSAAIDKNRVRLPSTEN 
               
               
                   
               
               
                 IAKAGIALARELSVAGFDWKDLLKKEEHEEYIDLIELHKTALALLLAVTETQLDISALDFVE 
               
               
                   
               
               
                 NGTVKDFMKTRDGNLVLEGRFLEMFSQSIVFSELRGLAGLMSRKEFITRSAIQTMNGKQAEL 
               
               
                   
               
               
                 LYIPHEFQSAKITTPKEMSRAFLDLAPAEFATSLEPESLSEKSLLKLKQMRYYPHYFGYELT 
               
               
                   
               
               
                 RTGQGIDGGVAENALRLEKSPVKKREIKCKQYKTLGRGQNKIVLYVRSSYYQTQFLEWFLHR 
               
               
                   
               
               
                 PKNVQTDVAVSGSFLIDEKKVKTRWNYDALTVALEPVSGSERVFVSQPFTIFPEKSAEEEGQ 
               
               
                   
               
               
                 RYLGIDIGEYGIAYTALEITGDSAKILDQNFISDPQLKTLREEVKGLKLDQRRGTFAMPSTK 
               
               
                   
               
               
                 IARIRESLVHSLRNRIHHLALKHKAKIVYELEVSRFEEGKQKIKKVYATLKKADVYSEIDAD 
               
               
                   
               
               
                 KNLQTTVWGKLAVASEISASYTSQFCGACKKLWRAEMQVDETITTQELIGTVRVIKGGTLID 
               
               
                   
               
               
                 AIKDFMRPPIFDENDTPFPKYRDFCDKHHISKKMRGNSCLFICPFCRANADADIQASQTIAL 
               
               
                   
               
               
                 LRYVKEEKKVEDYFERFRKLKNIKVLGQMKKI 
               
            
           
         
       
     
     The term “Cas12” or “Cas12 domain” refers to an RNA guided nuclease comprising a Cas12 protein or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas12, and/or the gRNA binding domain of Cas12). Cas12 belongs to the class 2, Type V CRISPR/Cas system. A Cas12 nuclease is also referred to sometimes as a CRISPR (clustered regularly interspaced short palindromic repeat) associated nuclease. The sequence of an exemplary  Bacillus hisashii  Cas 12b (BhCas12b) Cas 12 domain is provided below: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 55) 
               
               
                 MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYY 
               
               
                   
               
               
                 MNILKLIRQEAIYEHHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTH 
               
               
                   
               
               
                 EVDKDEVFNILRELYEELVPSSVEKKGEANQLSNKFLYPLVDPNSQSGKG 
               
               
                   
               
               
                 TASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAEYGLI 
               
               
                   
               
               
                 PLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWN 
               
               
                   
               
               
                 LKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTN 
               
               
                   
               
               
                 EYRLSKRGLRGWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYS 
               
               
                   
               
               
                 VYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKKDAKQQATFTLADPIN 
               
               
                   
               
               
                 HPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTVQLDRLIYPTESGGW 
               
               
                   
               
               
                 EEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGTLGGA 
               
               
                   
               
               
                 RVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDF 
               
               
                   
               
               
                 PKVVNFKPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAAS 
               
               
                   
               
               
                 IFEVVDQKPDIEGKLFFPIKGTELYAVHRASFNIKLPGETLVKSREVLRK 
               
               
                   
               
               
                 AREDNLKLMNQKLNFLRNVLHFQQFEDITEREKRVTKWISRQENSDVPLV 
               
               
                   
               
               
                 YQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKSLSDGRK 
               
               
                   
               
               
                 GLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHL 
               
               
                   
               
               
                 NALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYN 
               
               
                   
               
               
                 PYEERSRFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAK 
               
               
                   
               
               
                 TGSPGIRCSVVTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGG 
               
               
                   
               
               
                 EKFISLSKDRKCVTTHADINAAQNLQKRFWTRTHGFYKVYCKAYQVDGQT 
               
               
                   
               
               
                 VYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIKKGSSKQSSSE 
               
               
                   
               
               
                 LVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLER 
               
               
                   
               
               
                 ILISKLTNQYSISTIEDDSSKQSMKRPAATKKAGQAKKKK. 
               
            
           
         
       
     
     Amino acid sequences having at least 85% or greater identity to the BhCas12b amino acid sequence are also useful in the methods of the invention. 
     By “cytidine deaminase” is meant a polypeptide or fragment thereof capable of catalyzing a deamination reaction that converts an amino group to a carbonyl group. In one embodiment, the cytidine deaminase converts cytosine to uracil or 5-methylcytosine to thymine. PmCDA1, which is derived from  Petromyzon marinus  ( Petromyzon marinus  cytosine deaminase 1, “PmCDA1”), AID (Activation-induced cytidine deaminase; AICDA), which is derived from a mammal (e.g., human, swine, bovine, horse, monkey etc.), and APOBEC are exemplary cytidine deaminases. 
     The term “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and Schirmer, R. H., Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids can be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and Schirmer, R. H., supra). Non-limiting examples of conservative mutations include amino acid substitutions of amino acids, for example, lysine for arginine and vice versa such that a positive charge can be maintained; glutamic acid for aspartic acid and vice versa such that a negative charge can be maintained; serine for threonine such that a free —OH can be maintained; and glutamine for asparagine such that a free —NH 2  can be maintained. 
     The term “coding sequence” or “protein coding sequence” as used interchangeably herein refers to a segment of a polynucleotide that codes for a protein. The region or sequence is bounded nearer the 5′ end by a start codon and nearer the 3′ end with a stop codon. Coding sequences can also be referred to as open reading frames. 
     The term “deaminase” or “deaminase domain,” as used herein, refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenine to hypoxanthine. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenosine or adenine (A) to inosine (I). In some embodiments, the deaminase or deaminase domain is an adenosine deaminase catalyzing the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases (e.g., engineered adenosine deaminases, evolved adenosine deaminases) provided herein can be from any organism, such as a bacterium. In some embodiments, the adenosine deaminase is from a bacterium, such as  Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae , or  Caulobacter crescentus.    
     In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is TadA variant. In some embodiments, the TadA variant is a TadA*8. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to a naturally occurring deaminase. For example, deaminase domains are described in International PCT Application Nos. PCT/2017/045381 (WO 2018/027078) and PCT/US2016/058344 (WO 2017/070632), each of which is incorporated herein by reference for its entirety. Also, see Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017)), and Rees, H. A., et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 December; 19(12):770-788. doi: 10.1038/s41576-018-0059-1, the entire contents of which are hereby incorporated by reference. 
     “Detect” refers to identifying the presence, absence or amount of the analyte to be detected. In one embodiment, a sequence alteration in a polynucleotide or polypeptide is detected. In another embodiment, the presence of indels is detected. 
     By “detectable label” is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an enzyme linked immunosorbent assay (ELISA)), biotin, digoxigenin, or haptens. 
     By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. 
     The term “effective amount,” as used herein, refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response. The effective amount of an active agent(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount. In one embodiment, an effective amount is the amount of a base editor of the invention (e.g., a fusion protein comprising a programable DNA binding protein, a nucleobase editor and gRNA) sufficient to introduce an alteration in a gene of interest in a cell (e.g., a cell in vitro or in vivo). In some embodiments, an effective amount of a fusion protein provided herein, e.g., of a nucleobase editor comprising a nCas9 domain and a deaminase domain (e.g., adenosine deaminase or cytidine deaminase) may refer to the amount of the fusion protein that is sufficient to induce editing of a target site specifically bound and edited by the nucleobase editor. In one embodiment, an effective amount is the amount of a base editor required to achieve a therapeutic effect (e.g., to reduce or control a disease or a symptom or condition thereof). Such therapeutic effect need not be sufficient to alter a gene of interest in all cells of a subject, tissue or organ, but only to alter a gene of interest in about 1%, 5%, 10%, 25%, 50%, 75% or more of the cells present in a subject, tissue or organ. 
     In some embodiments, an effective amount of a fusion protein provided herein, e.g., of a nucleobase editor comprising a nCas9 domain and a deaminase domain (e.g., adenosine deaminase or cytidine deaminase) refers to the amount of the fusion protein that is sufficient to induce editing of a target site specifically bound and edited by the nucleobase editors described herein. As will be appreciated by the skilled artisan, the effective amount of an agent, e.g., a fusion protein, a nuclease, a hybrid protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a polynucleotide, may vary depending on various factors as, for example, on the desired biological response, e.g., on the specific allele, genome, or target site to be edited, on the cell or tissue being targeted, and/or on the agent being used. 
     By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. 
     By “guide RNA” or “gRNA” is meant a polynucleotide which can be specific for a target sequence and can form a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cpf1). In an embodiment, the guide polynucleotide is a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule. gRNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs), though “gRNA” is used interchangeably to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules. Typically, gRNAs that exist as single RNA species comprise two domains: (1) a domain that shares homology to a target nucleic acid (e.g., and directs binding of a Cas9 complex to the target); and (2) a domain that binds a Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as a tracrRNA, and comprises a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to a tracrRNA as provided in Jinek et al., Science 337:816-821(2012), the entire contents of which is incorporated herein by reference. Other examples of gRNAs (e.g., those including domain 2) can be found in U.S. Provisional Patent Application, U.S. Ser. No. 61/874,682, filed Sep. 6, 2013, entitled “Switchable Cas9 Nucleases and Uses Thereof,” and U.S. Provisional Patent Application, U.S. Ser. No. 61/874,746, filed Sep. 6, 2013, entitled “Delivery System For Functional Nucleases,” the entire contents of each are hereby incorporated by reference in their entirety. In some embodiments, a gRNA comprises two or more of domains (1) and (2), and may be referred to as an “extended gRNA.” An extended gRNA will bind two or more Cas9 proteins and bind a target nucleic acid at two or more distinct regions, as described herein. The gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to said target site, providing the sequence specificity of the nuclease:RNA complex. 
     “Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. 
     The term “inhibitor of base repair” or “IBR” refers to a protein that is capable in inhibiting the activity of a nucleic acid repair enzyme, for example a base excision repair (BER) enzyme. In some embodiments, the IBR is an inhibitor of inosine base excision repair. Exemplary inhibitors of base repair include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGGl, hNEILl, T7 Endol, T4PDG, UDG, hSMUGl, and hAAG. In some embodiments, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is a catalytically inactive EndoV or a catalytically inactive hAAG. In some embodiments, the base repair inhibitor is an inhibitor of Endo V or hAAG. In some embodiments, the base repair inhibitor is a catalytically inactive EndoV or a catalytically inactive hAAG. 
     In some embodiments, the base repair inhibitor is uracil glycosylase inhibitor (UGI). UGI refers to a protein that is capable of inhibiting a uracil-DNA glycosylase base-excision repair enzyme. In some embodiments, a UGI domain comprises a wild-type UGI or a fragment of a wild-type UGI. In some embodiments, the UGI proteins provided herein include fragments of UGI and proteins homologous to a UGI or a UGI fragment. In some embodiments, the base repair inhibitor is an inhibitor of inosine base excision repair. In some embodiments, the base repair inhibitor is a “catalytically inactive inosine specific nuclease” or “dead inosine specific nuclease. Without wishing to be bound by any particular theory, catalytically inactive inosine glycosylases (e.g., alkyl adenine glycosylase (AAG)) can bind inosine, but cannot create an abasic site or remove the inosine, thereby sterically blocking the newly formed inosine moiety from DNA damage/repair mechanisms. In some embodiments, the catalytically inactive inosine specific nuclease can be capable of binding an inosine in a nucleic acid but does not cleave the nucleic acid. Non-limiting exemplary catalytically inactive inosine specific nucleases include catalytically inactive alkyl adenosine glycosylase (AAG nuclease), for example, from a human, and catalytically inactive endonuclease V (EndoV nuclease), for example, from  E. coli . In some embodiments, the catalytically inactive AAG nuclease comprises an E125Q mutation or a corresponding mutation in another AAG nuclease. 
     By “increases” is meant a positive alteration of at least 10%, 25%, 50%, 75%, or 100%. 
     An “intein” is a fragment of a protein that is able to excise itself and join the remaining fragments (the exteins) with a peptide bond in a process known as protein splicing. Inteins are also referred to as “protein introns.” The process of an intein excising itself and joining the remaining portions of the protein is herein termed “protein splicing” or “intein-mediated protein splicing.” In some embodiments, an intein of a precursor protein (an intein containing protein prior to intein-mediated protein splicing) comes from two genes. Such intein is referred to herein as a split intein (e.g., split intein-N and split intein-C). For example, in cyanobacteria, DnaE, the catalytic subunit a of DNA polymerase III, is encoded by two separate genes, dnaE-n and dnaE-c. The intein encoded by the dnaE-n gene may be herein referred as “intein-N.” The intein encoded by the dnaE-c gene may be herein referred as “intein-C.” 
     Other intein systems may also be used. For example, a synthetic intein based on the dnaE intein, the Cfa-N (e.g., split intein-N) and Cfa-C (e.g., split intein-C) intein pair, has been described (e.g., in Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5, incorporated herein by reference). Non-limiting examples of intein pairs that may be used in accordance with the present disclosure include: Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein and Cne Prp8 intein (e.g., as described in U.S. Pat. No. 8,394,604, incorporated herein by reference. 
     Exemplary nucleotide and amino acid sequences of inteins are provided. 
     
       
         
           
               
            
               
                 DnaE Intein-N DNA: 
               
               
                 (SEQ ID NO: 56) 
               
               
                 TGCCTGTCATACGAAACCGAGATACTGACAGTAGAATATGGCCTTCTGCC 
               
               
                   
               
               
                 AATCGGGAAGATTGTGGAGAAACGGATAGAATGCACAGTTTACTCTGTCG 
               
               
                   
               
               
                 ATAACAATGGTAACATTTATACTCAGCCAGTTGCCCAGTGGCACGACCGG 
               
               
                   
               
               
                 GGAGAGCAGGAAGTATTCGAATACTGTCTGGAGGATGGAAGTCTCATTAG 
               
               
                   
               
               
                 GGCCACTAAGGACCACAAATTTATGACAGTCGATGGCCAGATGCTGCCTA 
               
               
                   
               
               
                 TAGACGAAATCTTTGAGCGAGAGTTGGACCTCATGCGAGTTGACAACCTT 
               
               
                   
               
               
                 CCTAAT 
               
               
                   
               
               
                 DnaE Intein-N Protein: 
               
               
                 (SEQ ID NO: 57) 
               
               
                 CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDR 
               
               
                   
               
               
                 GEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNL 
               
               
                   
               
               
                 PN 
               
               
                   
               
               
                 DnaE Intein-C DNA: 
               
               
                 (SEQ ID NO: 58) 
               
               
                 ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAAACGTTTATGA 
               
               
                   
               
               
                 TATTGGAGTCGAAAGAGATCACAACTTTGCTCTGAAGAACGGATTCATAG 
               
               
                   
               
               
                 CTTCTAAT 
               
               
                   
               
               
                 Intein-C: 
               
               
                 (SEQ ID NO: 59) 
               
               
                 MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN 
               
               
                   
               
               
                 Cfa-N DNA: 
               
               
                 (SEQ ID NO: 60) 
               
               
                 TGCCTGTCTTATGATACCGAGATACTTACCGTTGAATATGGCTTCTTGCC 
               
               
                   
               
               
                 TATTGGAAAGATTGTCGAAGAGAGAATTGAATGCACAGTATATACTGTAG 
               
               
                   
               
               
                 ACAAGAATGGTTTCGTTTACACACAGCCCATTGCTCAATGGCACAATCGC 
               
               
                   
               
               
                 GGCGAACAAGAAGTATTTGAGTACTGTCTCGAGGATGGAAGCATCATACG 
               
               
                   
               
               
                 AGCAACTAAAGATCATAAATTCATGACCACTGACGGGCAGATGTTGCCAA 
               
               
                   
               
               
                 TAGATGAGATATTCGAGCGGGGCTTGGATCTCAAACAAGTGGATGGATTG 
               
               
                   
               
               
                 CCA 
               
               
                   
               
               
                 Cfa-N Protein: 
               
               
                 (SEQ ID NO: 61) 
               
               
                 CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNR 
               
               
                   
               
               
                 GEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGL 
               
               
                   
               
               
                 P 
               
               
                   
               
               
                 Cfa-C DNA: 
               
               
                 (SEQ ID NO: 62) 
               
               
                 ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCTCCCAAGAAGAAGAG 
               
               
                   
               
               
                 GAAAGTAAAGATAATATCTCGAAAAAGTCTTGGTACCCAAAATGTCTATG 
               
               
                   
               
               
                 ATATTGGAGTGGAGAAAGATCACAACTTCCTTCTCAAGAACGGTCTCGTA 
               
               
                   
               
               
                 GCCAGCAAC 
               
               
                   
               
               
                 Cfa-C Protein: 
               
               
                 (SEQ ID NO: 63) 
               
               
                 MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLV 
               
               
                   
               
               
                 ASN 
               
            
           
         
       
     
     Intein-N and intein-C may be fused to the N-terminal portion of the split Cas9 and the C-terminal portion of the split Cas9, respectively, for the joining of the N-terminal portion of the split Cas9 and the C-terminal portion of the split Cas9. For example, in some embodiments, an intein-N is fused to the C-terminus of the N-terminal portion of the split Cas9, i.e., to form a structure of N—[N-terminal portion of the split Cas9]-[intein-N]—C. In some embodiments, an intein-C is fused to the N-terminus of the C-terminal portion of the split Cas9, i.e., to form a structure of N-[intein-C]-[C-terminal portion of the split Cas9]-C. The mechanism of intein-mediated protein splicing for joining the proteins the inteins are fused to (e.g., split Cas9) is known in the art, e.g., as described in Shah et al., Chem Sci. 2014; 5(1):446-461, incorporated herein by reference. Methods for designing and using inteins are known in the art and described, for example by WO2014004336, WO2017132580, US20150344549, and US20180127780, each of which is incorporated herein by reference in their entirety. 
     The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified. 
     By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence. 
     By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis. 
     The term “linker”, as used herein, can refer to a covalent linker (e.g., covalent bond), a non-covalent linker, a chemical group, or a molecule linking two molecules or moieties, e.g., two components of a protein complex or a ribonucleocomplex, or two domains of a fusion protein, such as, for example, a polynucleotide programmable DNA binding domain (e.g., dCas9) and a deaminase domain (e.g., an adenosine deaminase, a cytidine deaminase, or an adenosine deaminase and a cytidine deaminase) or a napDNAbp domain (e.g., Cas12b) and a deaminase domain (e.g., an adenosine deaminase or a cytidine deaminase). In particular embodiments, linkers flank a deaminase domain that is inserted within a Cas protein or fragment thereof. A linker can join different components of, or different portions of components of, a base editor system. For example, in some embodiments, a linker can join a guide polynucleotide binding domain of a polynucleotide programmable nucleotide binding domain and a catalytic domain of a deaminase. In some embodiments, a linker can join a CRISPR polypeptide and a deaminase. In some embodiments, a linker can join a Cas9 and a deaminase. In some embodiments, a linker can join a dCas9 and a deaminase. In some embodiments, a linker can join a nCas9 and a deaminase. For example, in some embodiments, a linker can join a Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12 h, or Cas12i and a deaminase. In some embodiments, a linker can join a guide polynucleotide and a deaminase. In some embodiments, a linker can join a deaminating component and a polynucleotide programmable nucleotide binding component of a base editor system. In some embodiments, a linker can join an RNA-binding portion of a deaminating component and a napDNAbp component of a base editor system. In some embodiments, a linker can join an RNA-binding portion of a deaminating component and a polynucleotide programmable nucleotide binding component of a base editor system. In some embodiments, a linker can join an RNA-binding portion of a deaminating component and an RNA-binding portion of a polynucleotide programmable nucleotide binding component of a base editor system. A linker can be positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond or non-covalent interaction, thus connecting the two. In some embodiments, the linker can be an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be a DNA linker. In some embodiments, the linker can be an RNA linker. In some embodiments, a linker can comprise an aptamer capable of binding to a ligand. In some embodiments, the ligand may be carbohydrate, a peptide, a protein, or a nucleic acid. In some embodiments, the linker may comprise an aptamer may be derived from a riboswitch. The riboswitch from which the aptamer is derived may be selected from a theophylline riboswitch, a thiamine pyrophosphate (TPP) riboswitch, an adenosine cobalamin (AdoCbl) riboswitch, an S-adenosyl methionine (SAM) riboswitch, an SAH riboswitch, a flavin mononucleotide (FMN) riboswitch, a tetrahydrofolate riboswitch, a lysine riboswitch, a glycine riboswitch, a purine riboswitch, a GlmS riboswitch, or a pre-queosinel (PreQ1) riboswitch. In some embodiments, a linker may comprise an aptamer bound to a polypeptide or a protein domain, such as a polypeptide ligand. In some embodiments, the polypeptide ligand may be a K Homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif. In some embodiments, the polypeptide ligand may be a portion of a base editor system component. For example, a nucleobase editing component may comprise a deaminase domain and an RNA recognition motif. 
     In some embodiments, the linker can be an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker can be about 5-100 amino acids in length, for example, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 amino acids in length. In some embodiments, the linker can be about 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, or 450-500 amino acids in length. Longer or shorter linkers can be also contemplated. 
     In some embodiments, a linker joins a gRNA binding domain of an RNA-programmable nuclease, including a Cas9 nuclease domain, and the catalytic domain of a nucleic-acid editing protein (e.g., cytidine or adenosine deaminase). In some embodiments, a linker joins a dCas9 and a nucleic-acid editing protein. For example, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-200 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 50, 55, 60, 60, 65, 70, 70, 75, 80, 85, 90, 90, 95, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 175, 180, 190, or 200 amino acids in length. Longer or shorter linkers are also contemplated. 
     In some embodiments, the domains of the nucleobase editor are fused via a linker that comprises the amino acid sequence of SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 64), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 65), or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 66). In some embodiments, domains of the nucleobase editor are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 67), which may also be referred to as the XTEN linker. In some embodiments, a linker comprises the amino acid sequence SGGS (SEQ ID NO: 68). In some embodiments, a linker comprises (SGGS)n (SEQ ID NO: 69), (GGGS) n  (SEQ ID NO: 70), (GGGGS) n  (SEQ ID NO: 71), (G) n  (SEQ ID NO: 72), (EAAAK) n  (SEQ ID NO: 73), (GGS) n  (SEQ ID NO: 74), SGSETPGTSESATPES (SEQ ID NO: 67), or (XP) n  motif (SEQ ID NO: 75), or a combination of any of these, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. 
     In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 76). In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 77). In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS SGGS (SEQ ID NO: 78). In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence 
     
       
         
           
               
            
               
                 (SEQ ID NO: 79) 
               
               
                 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG 
               
               
                   
               
               
                 TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS. 
               
            
           
         
       
     
     By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder. 
     The term “mutation,” as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). In some embodiments, the presently disclosed base editors can efficiently generate an “intended mutation,” such as a point mutation, in a nucleic acid (e.g., a nucleic acid within a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations. In some embodiments, an intended mutation is a mutation that is generated by a specific base editor (e.g., cytidine base editor or adenosine base editor) bound to a guide polynucleotide (e.g., gRNA), specifically designed to generate the intended mutation. 
     In general, mutations made or identified in a sequence (e.g., an amino acid sequence as described herein) are numbered in relation to a reference (or wild-type) sequence, i.e., a sequence that does not contain the mutations. The skilled practitioner in the art would readily understand how to determine the position of mutations in amino acid and nucleic acid sequences relative to a reference sequence. 
     The term “non-conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with, or inhibit the biological activity of, the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the wild-type protein. 
     The term “nuclear localization sequence,” “nuclear localization signal,” or “NLS” refers to an amino acid sequence that promotes import of a protein into the cell nucleus. Nuclear localization sequences are known in the art and described, for example, in Plank et al., International PCT application, PCT/EP2000/011690, filed Nov. 23, 2000, published as WO/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS described, for example, by Koblan et al., Nature Biotech. 2018 doi:10.1038/nbt.4172. In some embodiments, an NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV (SEQ ID NO: 80), KRPAATKKAGQAKKKK (SEQ ID NO: 81), KKTELQTTNAENKTKKL (SEQ ID NO: 82), KRGINDRNFWRGENGRKTR (SEQ ID NO: 83), RKSGKIAAIVVKRPRK (SEQ ID NO: 84), PKKKRKV (SEQ ID NO: 85), or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 86). 
     The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5′ to 3′ direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (2′—e.g., fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages). 
     The term “nucleic acid programmable DNA binding protein” or “napDNAbp” may be used interchangeably with “polynucleotide programmable nucleotide binding domain” to refer to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or guide polynucleotide (e.g., gRNA), that guides the napDNAbp to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 protein. A Cas9 protein can associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, for example a nuclease active Cas9, a Cas9 nickase (nCas9), or a nuclease inactive Cas9 (dCas9). Non-limiting examples of nucleic acid programmable DNA binding proteins include, Cas9 (e.g., dCas9 and nCas9), Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12 h, and Cas12i. Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5 h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12 h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, homologues thereof, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, although they may not be specifically listed in this disclosure. See, e.g., Makarova et al. “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?”  CRISPR J.  2018 October; 1:325-336. doi: 10.1089/crispr.2018.0033; Yan et al., “Functionally diverse type V CRISPR-Cas systems”  Science.  2019 Jan. 4; 363(6422):88-91. doi: 10.1126/science.aav7271, the entire contents of each are hereby incorporated by reference. 
     The term “nucleobase,” “nitrogenous base,” or “base,” used interchangeably herein, refers to a nitrogen-containing biological compound that forms a nucleoside, which in turn is a component of a nucleotide. The ability of nucleobases to form base pairs and to stack one upon another leads directly to long-chain helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Five nucleobases—adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U)—are called primary or canonical. Adenine and guanine are derived from purine, and cytosine, uracil, and thymine are derived from pyrimidine. DNA and RNA can also contain other (non-primary) bases that are modified. Non-limiting exemplary modified nucleobases can include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydromethylcytosine. Hypoxanthine and xanthine can be created through mutagen presence, both of them through deamination (replacement of the amine group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can result from deamination of cytosine. A “nucleoside” consists of a nucleobase and a five carbon sugar (either ribose or deoxyribose). Examples of a nucleoside include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of a nucleoside with a modified nucleobase includes inosine (I), xanthosine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Ψ). A “nucleotide” consists of a nucleobase, a five carbon sugar (either ribose or deoxyribose), and at least one phosphate group. 
     The term “nucleic acid programmable DNA binding protein” or “napDNAbp” refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid, that guides the napDNAbp to a specific nucleic acid sequence. For example, a Cas12 protein can associate with a guide RNA that guides the Cas12 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas12 domain, for example a nuclease active Cas12 domain. Examples of napDNAbps include, Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12 h, and Cas12i. Other napDNAbps are also within the scope of this disclosure, although they may not be specifically listed in this disclosure. See, e.g., Makarova et al. “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 October; 1:325-336. doi: 10.1089/crispr.2018.0033; Yan et al., “Functionally diverse type V CRISPR-Cas systems” Science. 2019 Jan. 4; 363(6422):88-91. doi: 10.1126/science.aav7271, the entire contents of each are hereby incorporated by reference. 
     The terms “nucleobase editing domain” or “nucleobase editing protein,” as used herein, refers to a protein or enzyme that can catalyze a nucleobase modification in RNA or DNA, such as cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine), and adenine (or adenosine) to hypoxanthine (or inosine) deaminations, as well as non-templated nucleotide additions and insertions. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., an adenine deaminase or an adenosine deaminase; or a cytidine deaminase or a cytosine deaminase). In some embodiments, the nucleobase editing domain is more than one deaminase domain (e.g., an adenine deaminase or an adenosine deaminase and a cytidine or a cytosine deaminase). In some embodiments, the nucleobase editing domain can be a naturally occurring nucleobase editing domain. In some embodiments, the nucleobase editing domain can be an engineered or evolved nucleobase editing domain from the naturally occurring nucleobase editing domain. The nucleobase editing domain can be from any organism, such as a bacterium, human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. For example, nucleobase editing proteins are described in International PCT Application Nos. PCT/2017/045381 (WO 2018/027078) and PCT/US2016/058344 (WO 2017/070632), each of which is incorporated herein by reference for its entirety. Also see, Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), the entire contents of which are hereby incorporated by reference. 
     As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent. 
     A “patient” or “subject” as used herein refers to a mammalian subject or individual diagnosed with, at risk of having or developing, or suspected of having or developing a disease or a disorder. In some embodiments, the term “patient” refers to a mammalian subject with a higher than average likelihood of developing a disease or a disorder. Exemplary patients can be humans, non-human primates, cats, dogs, pigs, cattle, cats, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs) and other mammalians that can benefit from the therapies disclosed herein. Exemplary human patients can be male and/or female. 
     “Patient in need thereof” or “subject in need thereof” is referred to herein as a patient diagnosed with, at risk or having, predetermined to have, or suspected of having a disease or disorder. 
     The terms “pathogenic mutation,” “pathogenic variant,” “disease casing mutation,” “disease causing variant,” “deleterious mutation,” or “predisposing mutation” refers to a genetic alteration or mutation that increases an individual&#39;s susceptibility or predisposition to a certain disease or disorder. In some embodiments, the pathogenic mutation comprises at least one wild-type amino acid substituted by at least one pathogenic amino acid in a protein encoded by a gene. 
     The term “pharmaceutically-acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body). A pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.). The terms such as “excipient,” “carrier,” “pharmaceutically acceptable carrier,” “vehicle,” or the like are used interchangeably herein. 
     The term “pharmaceutical composition” can refer to a composition formulated for pharmaceutical use. 
     The terms “protein,” “peptide,” “polypeptide,” and their grammatical equivalents are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide can refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide can be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modifications, etc. A protein, peptide, or polypeptide can also be a single molecule or can be a multi-molecular complex. A protein, peptide, or polypeptide can be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide can be naturally occurring, recombinant, or synthetic, or any combination thereof. The term “fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein can be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an amino-terminal fusion protein or a carboxy-terminal fusion protein, respectively. A protein can comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain, or a catalytic domain of a nucleic acid editing protein. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, e.g., a compound that can act as a nucleic acid cleavage agent. In some embodiments, a protein is in a complex with, or is in association with, a nucleic acid, e.g., RNA or DNA. Any of the proteins provided herein can be produced by any method known in the art. For example, the proteins provided herein can be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference. 
     Polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) can comprise synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and include, for example, aminocyclohexane carboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine O-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine, N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptane carboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine. The polypeptides and proteins can be associated with post-translational modifications of one or more amino acids of the polypeptide constructs. Non-limiting examples of post-translational modifications include phosphorylation, acylation including acetylation and formylation, glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation including methylation and ethylation, ubiquitylation, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, isoprenylation, farnesylation, geranylation, glypiation, lipoylation and iodination. 
     The term “polynucleotide programmable nucleotide binding domain” or “nucleic acid programmable DNA binding protein (napDNAbp)” refers to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide polynucleotide (e.g., guide RNA), that guides the polynucleotide programmable nucleotide binding domain to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas12 protein. 
     The term “recombinant” as used herein in the context of proteins or nucleic acids refers to proteins or nucleic acids that do not occur in nature, but are the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations as compared to any naturally occurring sequence. 
     By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%. 
     By “reference” is meant a standard or control condition. In one embodiment, the reference is a wild-type or healthy cell. In other embodiments and without limitation, a reference is an untreated cell that is not subjected to a test condition, or is subjected to placebo or normal saline, medium, buffer, and/or a control vector that does not harbor a polynucleotide of interest. 
     A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween. In some embodiments, a reference sequence is a wild-type sequence of a protein of interest. In other embodiments, a reference sequence is a polynucleotide sequence encoding a wild-type protein. 
     The term “RNA-programmable nuclease,” and “RNA-guided nuclease” are used with (e.g., binds or associates with) one or more RNA(s) that is not a target for cleavage. In some embodiments, an RNA-programmable nuclease, when in a complex with an RNA, may be referred to as a nuclease:RNA complex. Typically, the bound RNA(s) is referred to as a guide RNA (gRNA). gRNAs can exist as a complex of two or more RNAs, or as a single RNA molecule. gRNAs that exist as a single RNA molecule may be referred to as single-guide RNAs (sgRNAs), though “gRNA” is used interchangeably to refer to guide RNAs that exist as either single molecules or as a complex of two or more molecules. Typically, gRNAs that exist as single RNA species comprise two domains: (1) a domain that shares homology to a target nucleic acid (e.g., and directs binding of a Cas9 complex to the target); and (2) a domain that binds a Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as a tracrRNA, and comprises a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to a tracrRNA as provided in Jinek et ah, Science 337:816-821(2012), the entire contents of which is incorporated herein by reference. Other examples of gRNAs (e.g., those including domain 2) can be found in U.S. Provisional Patent Application, U.S. Ser. No. 61/874,682, filed Sep. 6, 2013, entitled “Switchable Cas9 Nucleases and Uses Thereof,” and U.S. Provisional Patent Application, U.S. Ser. No. 61/874,746, filed Sep. 6, 2013, entitled “Delivery System For Functional Nucleases,” the entire contents of each are hereby incorporated by reference in their entirety. In some embodiments, a gRNA comprises two or more of domains (1) and (2), and may be referred to as an “extended gRNA.” For example, an extended gRNA will, e.g., bind two or more Cas9 proteins and bind a target nucleic acid at two or more distinct regions, as described herein. The gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to said target site, providing the sequence specificity of the nuclease:RNA complex. 
     In some embodiments, the RNA-programmable nuclease is the (CRISPR-associated system) Cas9 endonuclease, for example, Cas9 (Casnl) from  Streptococcus pyogenes  (see, e.g., “Complete genome sequence of an M1 strain of  Streptococcus pyogenes .” Ferretti J. J., et al., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., et al., Nature 471:602-607(2011). 
     Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNA hybridization to target DNA cleavage sites, these proteins are able to be targeted, in principle, to any sequence specified by the guide RNA. Methods of using RNA-programmable nucleases, such as Cas9, for site-specific cleavage (e.g., to modify a genome) are known in the art (see e.g., Cong, L. et al., Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823 (2013); Mali, P. et al., RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, W. Y. et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al., RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, J. E. et al., Genome engineering in  Saccharomyces cerevisiae  using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et al., RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013); the entire contents of each of which are incorporated herein by reference). 
     The term “single nucleotide polymorphism (SNP)” is a variation in a single nucleotide that occurs at a specific position in the genome, where each variation is present to some appreciable degree within a population (e.g., &gt;1%). For example, at a specific base position in the human genome, the C nucleotide can appear in most individuals, but in a minority of individuals, the position is occupied by an A. This means that there is a SNP at this specific position, and the two possible nucleotide variations, C or A, are said to be alleles for this position. SNPs underlie differences in susceptibility to disease. The severity of illness and the way our body responds to treatments are also manifestations of genetic variations. SNPs can fall within coding regions of genes, non-coding regions of genes, or in the intergenic regions (regions between genes). In some embodiments, SNPs within a coding sequence do not necessarily change the amino acid sequence of the protein that is produced, due to degeneracy of the genetic code. SNPs in the coding region are of two types: synonymous and nonsynonymous SNPs. Synonymous SNPs do not affect the protein sequence, while nonsynonymous SNPs change the amino acid sequence of protein. The nonsynonymous SNPs are of two types: missense and nonsense. SNPs that are not in protein-coding regions can still affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of noncoding RNA. Gene expression affected by this type of SNP is referred to as an eSNP (expression SNP) and can be upstream or downstream from the gene. A single nucleotide variant (SNV) is a variation in a single nucleotide without any limitations of frequency and can arise in somatic cells. A somatic single nucleotide variation can also be called a single-nucleotide alteration. 
     By “specifically binds” is meant a nucleic acid molecule, polypeptide, or complex thereof (e.g., a nucleic acid programmable DNA binding domain and guide nucleic acid), compound, or molecule that recognizes and binds a polypeptide and/or nucleic acid molecule of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample. 
     Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). 
     For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a one: embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In another embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art. 
     For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C., more preferably of at least about 42° C., and even more preferably of at least about 68° C. In an embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. 
     By “split” is meant divided into two or more fragments. 
     A “split Cas9 protein” or “split Cas9” refers to a Cas9 protein that is provided as an N-terminal fragment and a C-terminal fragment encoded by two separate nucleotide sequences. 
     The polypeptides corresponding to the N-terminal portion and the C-terminal portion of the Cas9 protein may be spliced to form a “reconstituted” Cas9 protein. In particular embodiments, the Cas9 protein is divided into two fragments within a disordered region of the protein, e.g., as described in Nishimasu et al., Cell, Volume 156, Issue 5, pp. 935-949, 2014, or as described in Jiang et al. (2016) Science 351: 867-871. PDB file: 5F9R, each of which is incorporated herein by reference. In some embodiments, the protein is divided into two fragments at any C, T, A, or S within a region of SpCas9 between about amino acids A292-G364, F445-K483, or E565-T637, or at corresponding positions in any other Cas9, Cas9 variant (e.g., nCas9, dCas9), or other napDNAbp. In some embodiments, protein is divided into two fragments at SpCas9 T310, T313, A456, 5469, or C574. In some embodiments, the process of dividing the protein into two fragments is referred to as “splitting” the protein. 
     In other embodiments, the N-terminal portion of the Cas9 protein comprises amino acids 1-573 or 1-637 S. pyogenes Cas9 wild-type (SpCas9) (NCBI Reference Sequence: NC_002737.2, Uniprot Reference Sequence: Q99ZW2) and the C-terminal portion of the Cas9 protein comprises a portion of amino acids 574-1368 or 638-1368 of SpCas9 wild-type. 
     The C-terminal portion of the split Cas9 can be joined with the N-terminal portion of the split Cas9 to form a complete Cas9 protein. In some embodiments, the C-terminal portion of the Cas9 protein starts from where the N-terminal portion of the Cas9 protein ends. As such, in some embodiments, the C-terminal portion of the split Cas9 comprises a portion of amino acids (551-651)-1368 of spCas9. “(551-651)-1368” means starting at an amino acid between amino acids 551-651 (inclusive) and ending at amino acid 1368. For example, the C-terminal portion of the split Cas9 may comprise a portion of any one of amino acid 551-1368, 552-1368, 553-1368, 554-1368, 555-1368, 556-1368, 557-1368, 558-1368, 559-1368, 560-1368, 561-1368, 562-1368, 563-1368, 564-1368, 565-1368, 566-1368, 567-1368, 568-1368, 569-1368, 570-1368, 571-1368, 572-1368, 573-1368, 574-1368, 575-1368, 576-1368, 577-1368, 578-1368, 579-1368, 580-1368, 581-1368, 582-1368, 583-1368, 584-1368, 585-1368, 586-1368, 587-1368, 588-1368, 589-1368, 590-1368, 591-1368, 592-1368, 593-1368, 594-1368, 595-1368, 596-1368, 597-1368, 598-1368, 599-1368, 600-1368, 601-1368, 602-1368, 603-1368, 604-1368, 605-1368, 606-1368, 607-1368, 608-1368, 609-1368, 610-1368, 611-1368, 612-1368, 613-1368, 614-1368, 615-1368, 616-1368, 617-1368, 618-1368, 619-1368, 620-1368, 621-1368, 622-1368, 623-1368, 624-1368, 625-1368, 626-1368, 627-1368, 628-1368, 629-1368, 630-1368, 631-1368, 632-1368, 633-1368, 634-1368, 635-1368, 636-1368, 637-1368, 638-1368, 639-1368, 640-1368, 641-1368, 642-1368, 643-1368, 644-1368, 645-1368, 646-1368, 647-1368, 648-1368, 649-1368, 650-1368, or 651-1368 of spCas9. In some embodiments, the C-terminal portion of the split Cas9 protein comprises a portion of amino acids 574-1368 or 638-1368 of SpCas9. 
     By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline. Subjects include livestock, domesticated animals raised to produce labor and to provide commodities, such as food, including without limitation, cattle, goats, chickens, horses, pigs, rabbits, and sheep. 
     By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). In one embodiment, such a sequence is at least 60%, 80% or 85%, 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison. 
     Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e −3  and e −100  indicating a closely related sequence. 
     COBALT is used, for example, with the following parameters:
         a) alignment parameters: Gap penalties-11, -1 and End-Gap penalties-5, -1,   b) CDD Parameters: Use RPS BLAST on; Blast E-value 0.003; Find Conserved columns and Recompute on, and   c) Query Clustering Parameters: Use query clusters on; Word Size 4; Max cluster distance 0.8; Alphabet Regular.
 
EMBOSS Needle is used, for example, with the following parameters:
   a) Matrix: BLOSUM62;   b) GAP OPEN: 10;   c) GAP EXTEND: 0.5;   d) OUTPUT FORMAT: pair;   e) END GAP PENALTY: false;   f) END GAP OPEN: 10; and   g) END GAP EXTEND: 0.5.       

     The term “target site” refers to a sequence within a nucleic acid molecule that is modified by a nucleobase editor. In one embodiment, the target site is deaminated by a deaminase or a fusion protein comprising a deaminase (e.g., cytidine or adenine deaminase). 
     As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith or obtaining a desired pharmacologic and/or physiologic effect. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. In some embodiments, the effect is therapeutic, i.e., without limitation, the effect partially or completely reduces, diminishes, abrogates, abates, alleviates, decreases the intensity of, or cures a disease and/or adverse symptom attributable to the disease. In some embodiments, the effect is preventative, i.e., the effect protects or prevents an occurrence or reoccurrence of a disease or condition. To this end, the presently disclosed methods comprise administering a therapeutically effective amount of a compositions as described herein. 
     By “uracil glycosylase inhibitor” or “UGI” is meant an agent that inhibits the uracil-excision repair system. In one embodiment, the agent is a protein or fragment thereof that binds a host uracil-DNA glycosylase and prevents removal of uracil residues from DNA. In an embodiment, a UGI is a protein, a fragment thereof, or a domain that is capable of inhibiting a uracil-DNA glycosylase base-excision repair enzyme. In some embodiments, a UGI domain comprises a wild-type UGI or a modified version thereof. In some embodiments, a UGI domain comprises a fragment of the exemplary amino acid sequence set forth below. In some embodiments, a UGI fragment comprises an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the exemplary UGI sequence provided below. In some embodiments, a UGI comprises an amino acid sequence that is homologous to the exemplary UGI amino acid sequence or fragment thereof, as set forth below. In some embodiments, the UGI, or a portion thereof, is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a wild-type UGI or a UGI sequence, or portion thereof, as set forth below. An exemplary UGI comprises an amino acid sequence as follows: 
     
       
         
           
               
            
               
                 &gt;splP14739IUNGI_BPPB2 Uracil-DNA glycosylase 
               
               
                 inhibitor 
               
               
                 (SEQ ID NO: 87) 
               
               
                 MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDES 
               
               
                   
               
               
                 TDENVMLLTSDAPEYKPWALVIQDSNGENKIKML. 
               
            
           
         
       
     
     The term “vector” refers to a means of introducing a nucleic acid sequence into a cell, resulting in a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, and episome. “Expression vectors” are nucleic acid sequences comprising the nucleotide sequence to be expressed in the recipient cell. Expression vectors may include additional nucleic acid sequences to promote and/or facilitate the expression of the of the introduced sequence such as start, stop, enhancer, promoter, and secretion sequences. 
     Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. 
     DNA editing has emerged as a viable means to modify disease states by correcting pathogenic mutations at the genetic level. Until recently, all DNA editing platforms have functioned by inducing a DNA double strand break (DSB) at a specified genomic site and relying on endogenous DNA repair pathways to determine the product outcome in a semi-stochastic manner, resulting in complex populations of genetic products. Though precise, user-defined repair outcomes can be achieved through the homology directed repair (HDR) pathway, a number of challenges have prevented high efficiency repair using HDR in therapeutically-relevant cell types. In practice, this pathway is inefficient relative to the competing, error-prone non-homologous end joining pathway. Further, HDR is tightly restricted to the G1 and S phases of the cell cycle, preventing precise repair of DSBs in post-mitotic cells. As a result, it has proven difficult or impossible to alter genomic sequences in a user-defined, programmable manner with high efficiencies in these populations. 
     Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. 
     DNA editing has emerged as a viable means to modify disease states by correcting pathogenic mutations at the genetic level. Until recently, all DNA editing platforms have functioned by inducing a DNA double strand break (DSB) at a specified genomic site and relying on endogenous DNA repair pathways to determine the product outcome in a semi-stochastic manner, resulting in complex populations of genetic products. Though precise, user-defined repair outcomes can be achieved through the homology directed repair (HDR) pathway, a number of challenges have prevented high efficiency repair using HDR in therapeutically-relevant cell types. In practice, this pathway is inefficient relative to the competing, error-prone non-homologous end joining pathway. Further, HDR is tightly restricted to the G1 and S phases of the cell cycle, preventing precise repair of DSBs in post-mitotic cells. As a result, it has proven difficult or impossible to alter genomic sequences in a user-defined, programmable manner with high efficiencies in these populations. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS.  1 A- 1 C  depict plasmids.  FIG.  1 A  is an expression vector encoding a TadA7.10-dCas9 base editor.  FIG.  1 B  is a plasmid comprising nucleic acid molecules encoding proteins that confer chloramphenicol resistance (CamR) and spectinomycin resistance (SpectR). The plasmid also comprises a kanamycin resistance gene disabled by two point mutations.  FIG.  1 C  is a plasmid comprising nucleic acid molecules encoding proteins that confer chloramphenicol resistance (CamR) and spectinomycin resistance (SpectR). The plasmid also comprises a kanamycin resistance gene disabled by three point mutations. 
         FIG.  2    is an image of bacterial colonies transduced with the expression vectors depicted in  FIG.  1   , which included a defective kanamycin resistance gene. The vectors contained ABE7.10 variants that were generated using error prone PCR. Bacterial cells expressing these “evolved” ABE7.10 variants were selected for kanamycin resistance using increasing concentrations of kanamycin. Bacteria expressing ABE7.10 variants having adenosine deaminase activity were capable of correcting the mutations introduced into the kanamycin resistance gene, thereby restoring kanamycin resistance. The kanamycin resistant cells were selected for further analysis. 
         FIGS.  3 A and  3 B  illustrate editing of a regulatory region of the hemoglobin subunit gamma (HGB1) locus, which is a therapeutically relevant site for upregulation of fetal hemoglobin.  FIG.  3 A  is a drawing of a portion of the regulatory region for the HGB1 gene.  FIG.  3 A  discloses SEQ ID NO: 440.  FIG.  3 B  quantifies the efficiency and specificity of adenosine deaminase variants. Editing is assayed at the hemoglobin subunit gamma 1 (HGB1) locus in HEK293T cells, which is therapeutically relevant site for upregulation of fetal hemoglobin. The top panel depicts nucleotide residues in the target region of the regulatory sequence of the HGB1 gene. A5, A8, A9, and A11 denote the edited adenosine residues in HGB1.  FIG.  3 B  discloses SEQ ID NO: 441. 
         FIG.  4    illustrates the relative effectiveness of adenosine base editors comprising a dCas9 that recognizes a noncanonical PAM sequence. The top panel depicts the coding sequence of the hemoglobin subunit. The bottom panel is a graph demonstrating the efficiency of adenosine deaminase variant base editors with guide RNAs of varying lengths.  FIG.  4    discloses SEQ ID NOS 442-443, respectively, in order of appearance. 
         FIG.  5    is a graph illustrating the efficiency and specificity of ABE8 base editors. The percent editing at intended target nucleotides and unintended target nucleotides (bystanders) is quantified.  FIG.  5    discloses SEQ ID NOS 444-445, respectively, in order of appearance. 
         FIG.  6    is a graph illustrating the efficiency and specificity of ABE8 base editors. The percent editing at intended target nucleotides and unintended target nucleotides (bystanders) is quantified. 
         FIGS.  7 A- 7 D  depict eighth generation adenine base editors mediate superior A•T to G•C conversion in human cells.  FIG.  7 A  illustrates an overview of adenine base editing: i) ABE8 creates an R-loop at a sgRNA-targeted site in the genome; ii) TadA* deaminase chemically converts adenine to inosine via hydrolytic deamination on the ss-DNA portion of the R-loop; iii) D10A nickase of Cas9 nicks the strand opposite of the inosine containing strand; iv) the inosine containing strand can be used as a template during DNA replication; v) inosine preferentially base pairs with cytosine in the context of DNA polymerases; and vi) following replication, inosine may be replaced by guanosine.  FIG.  7 B  illustrates the architecture of ABE8.x-m and ABE8.x-d.  FIG.  7 C  illustrates three perspectives of the  E. coli  TadA deaminase (PDB 1Z3A) aligned with the  S. aureus  TadA (not shown) complexed with tRNAArg2 (PDB 2B3J). Mutations identified in eighth round of evolution are highlighted.  FIG.  7 D  are graphs depicting A•T to G•C base editing efficiencies of core ABE8 constructs relative to ABE7.10 constructs in Hek293T cells across eight genomic sites. Values and error bars reflect the mean and s.d. of three independent biological replicates performed on different days. 
         FIGS.  8 A- 8 C  depict Cas9 PAM-variant ABE8s and catalytically dead Cas9 ABE8 variants mediate higher A•T to G•C conversion than corresponding ABE7.10 variants in human cells. Values and error bars reflect the mean and s.d. of three independent biological replicates performed on different days.  FIG.  8 A  is a graph depicting A•T to G•C conversion in Hek293T cells with NG-Cas9 ABE8s (-NG PAM).  FIG.  8 B  is a graph depiecting A•T to G•C conversion in Hek293T cells with Sa-Cas9 ABE8s (-NNGRRT PAM).  FIG.  8 C  is a graph depiecting A•T to G•C conversion in Hek293T cells with catalytically inactivated, dCas9-ABE8s (D10A, H840A in  S. pyogenes  Cas9). 
         FIGS.  9 A- 9 E  depict the comparison between the on- and off-target editing frequencies between ABE7.10, ABEmax and ABEmax with one BPNLS in Hek293T cells. Individual data points are shown and error bars represent s.d. for n=3 independent biological replicates, performed on different days.  FIGS.  9 A and  9 B  are graphs that depict on-target DNA editing frequencies.  FIGS.  9 B and  9 C  are graphs that depict sgRNA-guided DNA-off-target editing frequencies.  FIG.  9 E  is a graph depicting RNA off-target editing frequencies. 
         FIGS.  10 A- 10 B  depict the median A•T to G•C conversion and corresponding INDEL formation of TadA, C-terminal alpha-helix truncation ABE constructs in HEK293T cells.  FIG.  10 A  is a heat map depicting A•T to G•C median editing conversion across 8 genomic sites.  FIG.  10 B  is a heat map depicting INDEL formation. Delta residue value corresponds to deletion position in TadA. Median value generated from n=3 biological replicate. 
         FIG.  11    are heat maps depicting the median A•T to G•C conversion of 40 ABE8 constructs in HEK293T cells across 8 genomic sites. Median values were determined from two or greater biological replicates. 
         FIG.  12    is a heat map depicting median INDEL % of 40 ABE8 constructs in HEK293T cells across 8 genomic sites. Median values were determined from two or greater biological replicates. 
         FIG.  13    is a graph depicting fold change in editing, ABE8:ABE7. Representation of average ABE8:ABE7 A•T to G•C editing in Hek293T cells across all A positions within the target of eight different genomic sites. Positions 2-12 denote location of a target adenine within the 20-nt protospacer with position 20 directly 5′ of the -NGG PAM. 
         FIG.  14    depicts a dendrogram of ABE8s. Core ABE8 constructs selected for further studies highlighted in in black. 
         FIG.  15    are heat maps depicting median A•T to G•C conversion of core eight ABE8 constructs in HEK293T cells across 8 genomic sites. Median values were determined from three or greater biological replicates. 
         FIG.  16    is a heat map depicting median INDEL frequency of core 8 ABE8s tested at 8 genomic sites in HEK293T cells. 
         FIG.  17    are heat maps depicting median A•T to G•C conversion of core NG-ABE8 constructs 9 (-NG PAM) at six genomic sites in HEK293T cells. Median value generated from n=3 biological replicate. 
         FIG.  18    is a heat map depicting median INDEL frequency of core NG-ABE8s tested at six genomic sites in HEK293T cells. Median value generated from n=3 biological replicate. 
         FIG.  19    are heat maps depicting median A•T to G•C conversion of core Sa-ABE8 constructs (-NNGRRT PAM) at six genomic sites in HEK293T cells. Site positions are numbered −2 to 20 (5′ to 3′) within the 22-nt protospacer. Position 20 is 5′ to the NNGRRT PAM. Median value generated from n=3 biological replicate. 
         FIG.  20    is a heat map depicting median INDEL frequency of core Sa-ABE8s tested at 8 genomic sites in HEK293T cells. Median value generated from n=3 biological replicate. 
         FIG.  21    are heat maps depicting median A•T to G•C conversion of core dC9-ABE8-m constructs at eight genomic sites in HEK293T cells. Dead Cas9 (dC9) is defined as D10A and H840A mutations within  S. pyogenes  Cas9. Median value generated from n&gt;3 biological replicate. 
         FIG.  22    are heat maps depicting median A•T to G•C conversion of core dC9-ABE8-d constructs at eight genomic sites in HEK293T cells. Dead Cas9 (dC9) is defined as D10A and H840A mutations within  S. pyogenes  Cas9. Median value generated from n&gt;3 biological replicate. 
         FIGS.  23 A and  23 B  depict Median INDEL frequency of core dC9-ABE8s tested at 8 genomic sites in HEK293T cells. Median value generated from n&gt;3 biological replicate.  FIG.  23 A  is a heat map depicting indel frequency shown for dC9-ABE8-m variants relative to ABE7.10.  FIG.  23 B  is a heat map depicting indel frequency shown for dC9-ABE8-d variants relative to ABE7.10. 
         FIG.  24    is a graph depicting C•G to T•A editing with Hek293T cells treated with ABE8s and ABE7.10. Editing frequencies for each site averaged across all C positions within the target. Cytosines within the protospacer are indicted with shading.  FIG.  24    discloses SEQ ID NOS 446-451, respectively, in order of appearance. 
         FIGS.  25 A- 25 H  depict DNA on-target and sgRNA-dependent DNA off-target editing by ABE8 constructs and ABE8 constructs with TadA mutations to improve specificity for DNA. Individual data points are shown and error bars represent s.d. for n=3 independent biological replicates, performed on different days.  FIGS.  25 A and  25 B  are graph depicting on-target DNA editing frequencies for core ABE8 constructs as compared to ABE7.  FIGS.  25 C and  25 D  are graphs depicting on-target DNA editing frequencies for ABE8 with mutations that improve RNA off-target editing.  FIGS.  25 E and  25 F  are graphs depicting sgRNA-guided DNA-off-target editing frequencies for core ABE 8 constructs as compared to ABE7.  FIGS.  25 G and  25 H  are graphs depicting sgRNA-guided DNA-off-target editing frequencies for ABE 8 constructs with mutations that improve RNA off-target editing. 
         FIG.  26    is a graph depicting indel frequencies at 12 previously identified sgRNA-dependent Cas9 off-target loci in human cells Individual data points are shown and error bars represent s.d. for n=3 independent biological replicates, performed on different days. 
         FIGS.  27 A and  27 B  depict A•T to G•C conversion and phenotypic outcomes in primary cells.  FIG.  27 A  is a graph depicting A•T to G•C conversion at −198 HBG1/2 site in CD34+ cells treated with ABE from two separate donors. NGS analysis conducted at 48 and 144 h post treatment. −198 HBG1/2 target sequence shown with A7 highlighted. Percent A•T to G•C plotted for A7.  FIG.  27 A  discloses SEQ ID NO: 452.  FIG.  27 B  is a graph depicting percentage of γ-globin formed as a fraction of alpha-globin. Values shown from two different donors, post ABE treatment and erythroid differentiation. 
         FIGS.  28 A and  28 B  depict A•T to G•C conversion of CD34+ cells treated with ABE8 at the −198 promoter site upstream of HBG1/2.  FIG.  28 A  is a heat map depicting A to G editing frequency of ABE8s in CD34+ cells from two donors, where Donor 2 is heterozygous for sickle cell disease, at 48 and 144 h post editor treatment.  FIG.  28 B  is a graphical representation of distribution of total sequencing reads which contain either A7 only edits or combined (A7+A8) edits. 
         FIG.  29    is a heat map depicting INDEL frequency of CD34+ cells treated with ABE8 at the −198 site of the gamma-globin promoter. Frequencies shown from two donors at 48 h and 144 h time points. 
         FIG.  30    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of untreated differentiated CD34+ cells (donor 1). 
         FIG.  31    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells treated with ABE7.10-m (donorl) 
         FIG.  32    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells treated with ABE7.10-d (donorl). 
         FIG.  33    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells treated with ABE8.8-m (donorl)  FIG.  34    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells treated with ABE8.8-d (donorl). 
         FIG.  35    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells treated with ABE8.13-m (donorl). 
         FIG.  36    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells treated with ABE8.13-d (donorl). 
         FIG.  37    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells treated with ABE8.17-m (donorl). 
         FIG.  38    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells treated with ABE8.17-d (donorl). 
         FIG.  39    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells treated with ABE8.20-m (donorl). 
         FIG.  40    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells treated with ABE8.20-d (donor 1). 
         FIG.  41    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells untreated (donor 2). Note: donor 2 is heterozygous for sickle cell disease. 
         FIG.  42    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells treated with ABE7.10-m (donor 2). Note: donor 2 is heterozygous for sickle cell disease. 
         FIG.  43    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells treated with ABE7.10-d (donor 2). Note: donor 2 is heterozygous for sickle cell disease. 
         FIG.  44    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells treated with ABE8.8-m (donor 2). Note: donor 2 is heterozygous for sickle cell disease. 
         FIG.  45    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells treated with ABE8.8-d (donor 2). Note: donor 2 is heterozygous for sickle cell disease. 
         FIG.  46    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells treated with ABE8.13-m (donor 2). Note: donor 2 is heterozygous for sickle cell disease. 
         FIG.  47    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells treated with ABE8.13-d (donor 2). Note: donor 2 is heterozygous for sickle cell disease. 
         FIG.  48    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells treated with ABE8.17-m (donor 2). Note: donor 2 is heterozygous for sickle cell disease. 
         FIG.  49    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells treated with ABE8.17-d (donor 2). Note: donor 2 is heterozygous for sickle cell disease. 
         FIG.  50    depicts an UHPLC UV-Vis trace (220 nm) and integration of globin chain levels of differentiated CD34+ cells treated with ABE8.20-m (donor 2). Note: donor 2 is heterozygous for sickle cell disease. 
         FIG.  51 A- 51 E  depict editing with ABE8.8 at two independent sites reached over 90% editing on day 11 post erythroid differentiation before enucleation and about 60% of gamma globin over alpha globin or total beta family globin on day 18 post erythroid differentiation.  FIG.  51 A  is a graph depicting an average of ABE8.8 editing in 2 healthy donors in 2 independent experiments. Editing efficiency was measured with primers that distinguish HBG1 and HBG2.  FIG.  51 B  is a graph depicting an average of 1 healthy donor in 2 independent experiments. Editing efficiency was measured with primers that recognize both HBG1 and HBG2.  FIG.  51 C  is a graph depicting editing of ABE8.8 in a donor with heterozygous E6V mutation.  FIGS.  51 D and  51 E  are graphs depicting gamma globin increase in the ABE8.8 edited cells. 
         FIGS.  52 A and  52 B  depict percent editing using ABE variants to correct sickle cell mutations.  FIG.  52 A  is a graph depicting a screen of different editor variants with about 70% editing in SCD patient fibroblasts.  FIG.  52 B  is a graph depicting CD34 cells from healthy donors edited with a lead ABE variant, targeting a synonymous mutation A13 in an adjacent proline that resides within the editing window and serves as a proxy for editing the SCD mutation. ABE8 variants showed an average editing frequency around 40% at the proxy A13. 
         FIGS.  53 A and  53 B  depict RNA amplicon sequencing to detect cellular A-to-I editing in RNA associated with ABE treatment. Individual data points are shown and error bars represent s.d. for n=3 independent biological replicates, performed on different days.  FIG.  53 A  is a graph depicting A-to-I editing frequencies in targeted RNA amplicons for core ABE 8 constructs as compared to ABET and Cas9(D10A) nickase control.  FIG.  53 B  is a graph depicting A-to-I editing frequencies in targeted RNA amplicons for ABE8 with mutations that have been reported to improve RNA off-target editing. 
         FIG.  54    is a schematic diagram illustrating the loss of dopamine that results from the loss of dopaminergic neurons in Parkinson Disease. 
         FIG.  55    is a schematic diagram showing a guide RNA and target sequences for the correction of R1441C and R1441H mutations in LRRK2 associated with Parkinson&#39;s Disease.  FIG.  55    discloses SEQ ID NOS 453-454, respectively, in order of appearance. 
         FIG.  56    is a schematic diagram showing target sequences for correction of the Y1699C, G2019S, and 12020 mutations in LRRK2 associated with Parkinson&#39;s Disease.  FIG.  56    discloses SEQ ID NOS 290 and 455-457, respectively, in order of appearance. 
         FIG.  57 A- 57 C  provides a graph, a schematic diagram, and a table.  FIG.  57 A  quantifies the percent conversion of A to G at nucleic acid position 7 of the LRRK2 target sequence. The editors used are designated PV1-PV14, a description of this which is provided below. pCMV designates the CMV promoter; bpNLS designates a bipartite Nuclear Localization Signal; monoABE8.1 designates a monomeric form of the ABE8.1 base editor. 
         FIG.  57 B  depicts target sequences and guide RNA for correction of the R1441C mutation in LRRK2 associated with Parkinson&#39;s Disease.  FIG.  57 B  discloses SEQ ID NOS 458-459, respectively, in order of appearance.  FIG.  57 C  shows the percent conversion of A to G at nucleic acid position 7 of the LRRK2 target sequence. Editors PV1-14 were used to edit LRRK2 R1441C. Editors (15-28) were used to edit G2109. The editors (PV1-28) used for correction of the LRRK2 mutations follows: 
       
         
           
             
                 
              
                 
                   PV1 (also termed PV15).  
                 
                 
                   pCMV_monoABE8.1_bpNLS + Y147T 
                 
                 
                   (SEQ ID NO: 21) 
                 
                 
                     MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
                 
                 
                     
                 
                 
                   LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG 
                 
                 
                     
                 
                 
                   RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFR 
                 
                 
                     
                 
                 
                   MPRQVFNAQKKAQSSTD 
                 
                 
                     
                 
                 
                   PV2 (also termed PV16).  
                 
                 
                   pCMV_monoABE8.1_bpNLS + Y147R 
                 
                 
                   (SEQ ID NO: 88) 
                 
                 
                     MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
                 
                 
                     
                 
                 
                   LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG 
                 
                 
                     
                 
                 
                   RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCRFFR 
                 
                 
                     
                 
                 
                   MPRQVFNAQKKAQSSTD 
                 
                 
                     
                 
                 
                   PV3 (also termed PV17).  
                 
                 
                   pCMV_monoABE8.1_bpNLS + Q154S 
                 
                 
                   (SEQ ID NO: 89) 
                 
                 
                     MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
                 
                 
                     
                 
                 
                   LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG 
                 
                 
                     
                 
                 
                   RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFR 
                 
                 
                     
                 
                 
                   MPRSVFNAQKKAQSSTD 
                 
                 
                     
                 
                 
                   PV4 (also termed PV18).  
                 
                 
                   pCMV_monoABE8.1_bpNLS + Y123H 
                 
                 
                   (SEQ ID NO: 90) 
                 
                 
                     MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
                 
                 
                     
                 
                 
                   LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG 
                 
                 
                     
                 
                 
                   RVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCYFFR 
                 
                 
                     
                 
                 
                   MPRQVFNAQKKAQSSTD 
                 
                 
                     
                 
                 
                   PV5 (also termed PV19).  
                 
                 
                   pCMV_monoABE8.1_bpNLS + V82S 
                 
                 
                   (SEQ ID NO: 91) 
                 
                 
                     MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
                 
                 
                     
                 
                 
                   LHDPTAHAEIMALRQGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIG 
                 
                 
                     
                 
                 
                   RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFR 
                 
                 
                     
                 
                 
                   MPRQVFNAQKKAQSSTD 
                 
                 
                     
                 
                 
                   PV6 (also termed PV20).  
                 
                 
                   pCMV_monoABE8.1_bpNLS + T166R 
                 
                 
                   (SEQ ID NO: 92) 
                 
                 
                     MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
                 
                 
                     
                 
                 
                   LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG 
                 
                 
                     
                 
                 
                   RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFR 
                 
                 
                     
                 
                 
                   MPRQVFNAQKKAQSSRD 
                 
                 
                     
                 
                 
                   PV7 (also termed PV21).  
                 
                 
                   pCMV_monoABE8.1_bpNLS + Q154R 
                 
                 
                   (SEQ ID NO: 93) 
                 
                 
                     MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
                 
                 
                     
                 
                 
                   LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG 
                 
                 
                     
                 
                 
                   RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFR 
                 
                 
                     
                 
                 
                   MPRRVFNAQKKAQSSTD 
                 
                 
                     
                 
                 
                   PV8 (also termed PV22).  
                 
                 
                   pCMV_monoABE8.1_bpNLS + Y147R_Q154R_Y123H 
                 
                 
                   (SEQ ID NO: 94) 
                 
                 
                     MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
                 
                 
                     
                 
                 
                   LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG 
                 
                 
                     
                 
                 
                   RVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFR 
                 
                 
                     
                 
                 
                   MPRRVFNAQKKAQSSTD 
                 
                 
                     
                 
                 
                   PV9 (also termed PV23).  
                 
                 
                   pCMV_monoABE8.1_bpNLS + Y147R_Q154R_I76Y 
                 
                 
                   (SEQ ID NO: 95) 
                 
                 
                     MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
                 
                 
                     
                 
                 
                   LHDPTAHAEIMALRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIG 
                 
                 
                     
                 
                 
                   RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCRFFR 
                 
                 
                     
                 
                 
                   MPRRVFNAQKKAQSSTD 
                 
                 
                     
                 
                 
                   PV10 (also termed PV24).  
                 
                 
                   pCMV_monoABE8.1_bpNLS + Y147R_Q154R_T166R 
                 
                 
                   (SEQ ID NO: 96) 
                 
                 
                     MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
                 
                 
                     
                 
                 
                   LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG 
                 
                 
                     
                 
                 
                   RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCRFFR 
                 
                 
                     
                 
                 
                   MPRRVFNAQKKAQSSRD 
                 
                 
                     
                 
                 
                   PV11 (also termed PV25).  
                 
                 
                   pCMV_monoABE8.1_bpNLS + Y147T_Q154R 
                 
                 
                   (SEQ ID NO: 97) 
                 
                 
                     MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
                 
                 
                     
                 
                 
                   LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG 
                 
                 
                     
                 
                 
                   RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFR 
                 
                 
                     
                 
                 
                   MPRRVFNAQKKAQSSTD 
                 
                 
                     
                 
                 
                   PV12 (also termed PV26).  
                 
                 
                   pCMV_monoABE8.1_bpNLS + Y147T_Q154S 
                 
                 
                   (SEQ ID NO: 98) 
                 
                 
                     MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
                 
                 
                     
                 
                 
                   LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG 
                 
                 
                     
                 
                 
                   RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFR 
                 
                 
                     
                 
                 
                   MPRSVFNAQKKAQSSTD 
                 
                 
                     
                 
                 
                   PV13 (also termed PV27).  
                 
                 
                   pCMV_monoABE8.1_bpNLS + H123Y123H_Y147R_Q154R_I76Y 
                 
                 
                   (SEQ ID NO: 99) 
                 
                 
                     MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
                 
                 
                     
                 
                 
                   LHDPTAHAEIMALRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIG 
                 
                 
                     
                 
                 
                   RVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFR 
                 
                 
                     
                 
                 
                   MPRRVFNAQKKAQSSTD 
                 
                 
                     
                 
                 
                   PV14 (also termed PV28).  
                 
                 
                   pCMV_monoABE8.1_bpNLS + V82S + Q154R 
                 
                 
                   (SEQ ID NO: 100) 
                 
                 
                     MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
                 
                 
                     
                 
                 
                   LHDPTAHAEIMALRQGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIG 
                 
                 
                     
                 
                 
                   RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFR 
                 
                 
                     
                 
                 
                   MPRRVFNAQKKAQSSTD 
                 
              
             
           
         
       
         FIG.  58 A- 58 C  provides a graph, a schematic diagram, and a table.  FIG.  58 A  quantifies the percent conversion of A to G at nucleic acid position 6 of the LRRK2 target sequence. The editors used are designated PV15-PV28, a description of this which is provided above. pCMV designates the CMV promoter; bpNLS designates a bipartite Nuclear Localization Signal; monoABE8.1 designates a monomeric form of the ABE8.1 base editor.  FIG.  58 B  depicts target sequences and guide RNAs for correction of the G2019S mutation in LRRK2 associated with Parkinson&#39;s Disease.  FIG.  58 B  discloses SEQ ID NOS 460-461, respectively, in order of appearance.  FIG.  58 C  shows the percent conversion of A to G at nucleic acid positions 4 and 6 of the LRRK2 target sequence. The A to G transition at position 4 is a bystander effect. 
         FIGS.  59 A- 59 L  depicts the sequence reads for the A to G transition at position 7 of the LRRK2 target sequence, which encodes R1441C (See  FIG.  57 A- 57 C ). The editor is indicated (PV1-14). A description of PV1-28 is provided at  FIG.  56   . 
         FIGS.  60 A- 60 W  depicts the sequence reads for the A to G transition at positions 4 and 6 of the LRRK2 target sequence, which encodes G2019S (See  FIG.  58 A- 58 C ). 
         FIG.  61 A  provides a schematic diagram depicting the target sequence for correction of a pathogenic mutation A419V in LRRK2, which is encoded by an antisense strand G&gt;A mutation. The mutation is corrected using an ABE targeting the A at position 12 using an SpCas9 variant that has specificity for a TGG PAM.  FIG.  61 A  discloses SEQ ID NOS 462-463, respectively, in order of appearance. 
         FIG.  61 B  provides a schematic diagram depicting the target sequence for correction of a pathogenic mutation L1114L in LRRK2, which is associated with Parkinson Disease. The mutation is an antisense strand T&gt;C, which is corrected using a base editor having cytidine deaminase activity (CBE).  FIG.  61 B  discloses SEQ ID NOS 464-465, respectively, in order of appearance. 
         FIG.  61 C  provides a schematic diagram depicting the target sequence for correction of a pathogenic mutation I1122V in LRRK2, which is associated with Parkinson Disease. The mutation is an antisense strand T&gt;C, which is corrected using a base editor having cytidine deaminase activity (CBE).  FIG.  61 C  discloses SEQ ID NOS 466-467, respectively, in order of appearance. 
         FIG.  61 D  provides a schematic diagram depicting the target sequence for correction of a pathogenic mutation M1869V in LRRK2, which is associated with Parkinson Disease. The mutation is an antisense strand T&gt;C, which is corrected using a base editor having cytidine deaminase activity (CBE).  FIG.  61 D  discloses SEQ ID NOS 468-469, respectively, in order of appearance. 
         FIGS.  62 A and  62 B  depict the precise base editing correction of the  Mus musculus  IDUA W401X mutation in HEK293T cells.  FIG.  62 A  is a graph depicting the percentage of base editing of the  Mus musculus  IDUA W401X mutation using ABE8 base editor variants using a 21-nucleotide guide RNA.  FIG.  62 B  is a graph depicting the percent indels for the ABE8 base editor variants using a 21-nucleotide guide RNA. 
         FIG.  63    is a graph depicting the percentage of base editing of the  Mus musculus  IDUA W401X mutation using ABE8 base editor variants using either a 20-nucleotide guide RNA or a 21-nucleotide guide RNA. 
         FIG.  64    depicts a diagramic illustration of the  Homo sapiens  IDUA genomic nucleic acid and amino acid sequence as a target for A-to-G nucleotide base editing to correct the W402X mutation. Also shown in the figure is the nucleic acid sequence of a corresponding guide RNA (gRNA). Noted in the figure is the target adenosine (A) nucleobase (boxed) in the IDUA nucleic acid sequence.  FIG.  64    discloses SEQ ID NOS 470-472, respectively, in order of appearance. 
         FIGS.  65 A and  65 B  depict the precise base editing correction of the  Homo sapiens  IDUA W402X mutation in HEK293T cells.  FIG.  65 A  is a graph depicting the percentage of base editing of the  Homo sapiens  IDUA W402X mutation using ABE8 base editor variants using a 20-nucleotide guide RNA.  FIG.  65 B  is a graph depicting the percent indels for the ABE8 base editor variants using a 20-nucleotide guide RNA. 
         FIGS.  66 A through  66 O  are tables depicting the efficiency of percentage of A-to-G nucleotide change in the IDUA nucleic acid sequence using ABE8 base editor variants, as detected by deep sequencing (MySeq) following PCR of the genomic DNA in cells in which base editing had occurred.  FIGS.  66 A through  66 M  depict the percent of A to G base editing at position 6 in the IDUA nucleic acid target site using three samples of each ABE8 base editor variants ABE8.1 through ABE8.13, respectively.  FIG.  66 N  depicts the percent of A to G base editing at position 6 in the IDUA nucleic acid target site using three samples of positive control base editor ABE7.10.  FIG.  66 O  depicts the percent of A to G base editing at position 6 in the IDUA nucleic acid target site using two samples of negative control. 
         FIG.  67    illustrates Rett/MECP2: Mutation correction. MECP2 loss of function—can result from many different de novo mutations. X-linked: XX patients are mosaic for MECP2 loss; XY usually results in infant mortality.  FIG.  67    discloses SEQ ID NOS 473 and 306-308, respectively, in order of appearance. 
         FIG.  68    illustrates Rett Syndrome R106W mutation correction for top 3 guide sequences. 
         FIG.  69    illustrates Rett Syndrome R255X mutation correction with editors having NGTT PAM optimization. 
         FIGS.  70 A-C : Hurler/IDUA mutation correction.  FIG.  70 A  illustrates experiment design of IDUA W402X mutation correction.  FIG.  70 A  discloses SEQ ID NO: 474.  FIG.  70 B  illustrates the percent editing for each editor construct.  FIG.  70 C  illustrates specific activity (nmol/mg/h) for edited and unedited constructs. 
         FIG.  71    depicts In vivo base editing with ABE 8.8. From left to right for each of each sample: Guide 11 (AAV9), Guide 12 (AAV9), Guide 11 (PHP.eB), Guide 12 (PHP.eB), and Control. 
         FIGS.  72 A- 72 B . A•T to G•C conversion by ABE7.10 and ABE8 variants at the ABCA4 G1961E allele in a model cell line.  FIG.  72 A : A•T to G•C conversion in HEK293T cells at an integrated disease allele and wobble base of the ABCA4 G1961E codon after plasmid lipofection of the 21-nt spacer sgRNA and base editor variant. Cells incubated for 5 days after lipofection and were then assessed for editing.  FIG.  72 B : The DNA sequence at the site of interest including the ABCA4 G1961E disease allele, the wobble base of the codon, and the -NGG PAM used by the 21-nt spacer sgRNA. Error bars represent the s.d. of three replicates. In each data set, the disease allele is on the left and the wobble base is on the right.  FIG.  72 B  discloses SEQ ID NOS 291 and 475, respectively, in order of appearance. 
         FIG.  73   . A•T to G•C conversion by sgRNA spacer-length variants at the ABCA4 G1961E allele in a model cell line. A•T to G•C conversion in HEK293T cells at an integrated disease allele and wobble base of the ABCA4 G1961E codon after plasmid lipofection of the sgRNA of varied spacer lengths and ABE7.10. Cells incubated for 5 days after lipofection and were then assessed for editing. hRz=inclusion of a self-cleaving hammer head ribozyme at the 5′-end of the sgRNA. Error bars represent the s.d. of three replicates. In each data set, the disease allele is on the left and the wobble base is on the right. 
         FIG.  74   . Schematic of the dual AAV delivery of a split base editor using split intein reconstitution. Two AAV particles are packaged separately with the components required for base editing. One virus encodes the C-terminal region of the base editor with an N-terminal split intein fusion, and a complementary virus encodes the N-terminal region of the base editor with a C-terminal split intein fusion as well as the sgRNA. Upon co-transduction of the complementary viruses, the sgRNA is transcribed and each half of the base editor is expressed and recombined through protein trans-splicing via the split intein. 
         FIGS.  75 A- 75 B . A•T to G•C conversion by dual AAV delivery of split ABE variants at the ABCA4 G1961 in wild type cells.  FIG.  75 A : A•T to G•C and C•G to T•A conversion in wild type ARPE-19 cells at the wild type ABCA4 G1961 target site, in which editing at position 8A serves as a surrogate target for editing in these cells. Cells infected at a MOI of 5E+4 viral genomes per virus per cell. Cells were incubated for 2 weeks post infection and were then assessed for editing. Error bars represent the s.d. of six replicates. For each data point, samples treated with Pos. 8 (A&gt;G)-surrogate site are shown on the left and Pos. 5 (C&gt;T) are shown on the right. 
         FIG.  75 B : The DNA sequence at the wild type target site including the ABCA4 G1961 allele and the -NGG PAM used by the 21-nt spacer sgRNA targeting the wild type sequence.  FIG.  75 B  discloses SEQ ID NOS 292 and 476, respectively, in order of appearance. 
         FIGS.  76 A- 76 B . Off target base editing in wild type ARPE-19 cells dual infected with AAV2 expressing split ABE7.10 and sgRNA targeting the disease allele of ABCA4 G1961E.  FIG.  76 A : Maximum A•T to G•C conversion across the target or off-target protospacers 2 weeks after co-infection with the dual AAV (teal) compared to untreated controls (gray).  FIG.  76 B : Maximum non-A•T to G•C conversion across the target or off-target protospacers 2 weeks after co-infection with the dual AAV (teal) compared to untreated controls (gray). For each data point, samples treated with wild type (wt) ARPE-19 cells are shown on the left and untreated wt ARPE-19 cells are shown on the right. 
         FIG.  77   . Indel formation due to base editing in wild type ARPE-19 cells dual infected with AAV2 expressing split ABE7.10 and sgRNA targeting the disease allele of ABCA4 G1961E. Percentage of indels formed within or proximal to the target or off-target protospacers 2 weeks after co-infection with the dual AAV (teal) compared to untreated controls (gray). For each data point, samples treated with wild type (wt) ARPE-19 cells are shown on the left and untreated wt ARPE-19 cells are shown on the right. 
         FIG.  78   : Primate Retina Integrity and GFP expression at Day 22 post-culture. Sections were immunolabeled with anti-Rhodopsin, anti-GFP, and biotinylated peanut agglutinin antibodies overnight at 4° C. Anc80L65.hGRK.eGFP showed GFP to be observed exclusively in the photoreceptor-containing outer nuclear layer (ONL) confirming photoreceptor-specific activity of the GRK promoter. Top row is Day 0, untransduced. The second row is Day 22, untransduced. Third row is Day 22, GRK. Fourth row is Day 22, CMB. Columns are unstained (1 st  column), DAPI (2 nd  column), GFP (3rd column), PNA (4th column), and rhodopsin (5th column). 
         FIG.  79   : Cas9 Expression in NHP. Cas9 expression is detected in primate retina as early as day 6 post-culture. Results are shown for ABE7.10 (columns 1 and 2), ABE8.5 (columns 2 and 3), and ABE8.9 (columns 3 and 4). Top row: day 6 post-culture. Bottom row: day 17 post-culture. The results demonstrate that the AAV system delivers split-inteins that express Cas9. Scale Bar: 100 μm 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The invention provides compositions comprising novel adenine base editors (e.g., ABE8) that have increased efficiency and methods of using them to generate modifications in target nucleobase sequences. 
     Nucleobase Editor 
     Disclosed herein is a base editor or a nucleobase editor for editing, modifying or altering a target nucleotide sequence of a polynucleotide. Described herein is a nucleobase editor or a base editor comprising a polynucleotide programmable nucleotide binding domain (e.g., Cas9) and a nucleobase editing domain (e.g., adenosine deaminase). A polynucleotide programmable nucleotide binding domain (e.g., Cas9), when in conjunction with a bound guide polynucleotide (e.g., gRNA), can specifically bind to a target polynucleotide sequence (i.e., via complementary base pairing between bases of the bound guide nucleic acid and bases of the target polynucleotide sequence) and thereby localize the base editor to the target nucleic acid sequence desired to be edited. In some embodiments, the target polynucleotide sequence comprises single-stranded DNA or double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises RNA. In some embodiments, the target polynucleotide sequence comprises a DNA-RNA hybrid. 
     Polynucleotide Programmable Nucleotide Binding Domain 
     It should be appreciated that polynucleotide programmable nucleotide binding domains can also include nucleic acid programmable proteins that bind RNA. For example, the polynucleotide programmable nucleotide binding domain can be associated with a nucleic acid that guides the polynucleotide programmable nucleotide binding domain to an RNA. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, though they are not specifically listed in this disclosure. 
     A polynucleotide programmable nucleotide binding domain of a base editor can itself comprise one or more domains. For example, a polynucleotide programmable nucleotide binding domain can comprise one or more nuclease domains. In some embodiments, the nuclease domain of a polynucleotide programmable nucleotide binding domain can comprise an endonuclease or an exonuclease. Herein the term “exonuclease” refers to a protein or polypeptide capable of digesting a nucleic acid (e.g., RNA or DNA) from free ends, and the term “endonuclease” refers to a protein or polypeptide capable of catalyzing (e.g., cleaving) internal regions in a nucleic acid (e.g., DNA or RNA). In some embodiments, an endonuclease can cleave a single strand of a double-stranded nucleic acid. In some embodiments, an endonuclease can cleave both strands of a double-stranded nucleic acid molecule. In some embodiments a polynucleotide programmable nucleotide binding domain can be a deoxyribonuclease. In some embodiments a polynucleotide programmable nucleotide binding domain can be a ribonuclease. 
     In some embodiments, a nuclease domain of a polynucleotide programmable nucleotide binding domain can cut zero, one, or two strands of a target polynucleotide. In some embodiments, the polynucleotide programmable nucleotide binding domain can comprise a nickase domain. Herein the term “nickase” refers to a polynucleotide programmable nucleotide binding domain comprising a nuclease domain that is capable of cleaving only one strand of the two strands in a duplexed nucleic acid molecule (e.g., DNA). In some embodiments, a nickase can be derived from a fully catalytically active (e.g., natural) form of a polynucleotide programmable nucleotide binding domain by introducing one or more mutations into the active polynucleotide programmable nucleotide binding domain. For example, where a polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain can include a D10A mutation and a histidine at position 840. In such embodiments, the residue H840 retains catalytic activity and can thereby cleave a single strand of the nucleic acid duplex. In another example, a Cas9-derived nickase domain can comprise an H840A mutation, while the amino acid residue at position 10 remains a D. In some embodiments, a nickase can be derived from a fully catalytically active (e.g., natural) form of a polynucleotide programmable nucleotide binding domain by removing all or a portion of a nuclease domain that is not required for the nickase activity. For example, where a polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain can comprise a deletion of all or a portion of the RuvC domain or the HNH domain. 
     The amino acid sequence of an exemplary catalytically active Cas9 is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 1) 
               
               
                 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE 
               
               
                   
               
               
                 DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK 
               
               
                   
               
               
                 PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ 
               
               
                   
               
               
                 SITGLYETRIDLSQLGGD. 
               
            
           
         
       
     
     A base editor comprising a polynucleotide programmable nucleotide binding domain comprising a nickase domain is thus able to generate a single-strand DNA break (nick) at a specific polynucleotide target sequence (e.g., determined by the complementary sequence of a bound guide nucleic acid). In some embodiments, the strand of a nucleic acid duplex target polynucleotide sequence that is cleaved by a base editor comprising a nickase domain (e.g., Cas9-derived nickase domain) is the strand that is not edited by the base editor (i.e., the strand that is cleaved by the base editor is opposite to a strand comprising a base to be edited). In other embodiments, a base editor comprising a nickase domain (e.g., Cas9-derived nickase domain) can cleave the strand of a DNA molecule which is being targeted for editing. In such embodiments, the non-targeted strand is not cleaved. 
     Also provided herein are base editors comprising a polynucleotide programmable nucleotide binding domain which is catalytically dead (i.e., incapable of cleaving a target polynucleotide sequence). Herein the terms “catalytically dead” and “nuclease dead” are used interchangeably to refer to a polynucleotide programmable nucleotide binding domain which has one or more mutations and/or deletions resulting in its inability to cleave a strand of a nucleic acid. In some embodiments, a catalytically dead polynucleotide programmable nucleotide binding domain base editor can lack nuclease activity as a result of specific point mutations in one or more nuclease domains. For example, in the case of a base editor comprising a Cas9 domain, the Cas9 can comprise both a D10A mutation and an H840A mutation. Such mutations inactivate both nuclease domains, thereby resulting in the loss of nuclease activity. In other embodiments, a catalytically dead polynucleotide programmable nucleotide binding domain can comprise one or more deletions of all or a portion of a catalytic domain (e.g., RuvC1 and/or HNH domains). In further embodiments, a catalytically dead polynucleotide programmable nucleotide binding domain comprises a point mutation (e.g., D10A or H840A) as well as a deletion of all or a portion of a nuclease domain. 
     Also contemplated herein are mutations capable of generating a catalytically dead polynucleotide programmable nucleotide binding domain from a previously functional version of the polynucleotide programmable nucleotide binding domain. For example, in the case of catalytically dead Cas9 (“dCas9”), variants having mutations other than D10A and H840A are provided, which result in nuclease inactivated Cas9. Such mutations, by way of example, include other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and/or the RuvC1 subdomain). Additional suitable nuclease-inactive dCas9 domains can be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A/H840A, D10A/D839A/H840A, and D10A/D839A/H840A/N863A mutant domains (See, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference). 
     Non-limiting examples of a polynucleotide programmable nucleotide binding domain which can be incorporated into a base editor include a CRISPR protein-derived domain, a restriction nuclease, a meganuclease, TAL nuclease (TALEN), and a zinc finger nuclease (ZFN). In some embodiments, a base editor comprises a polynucleotide programmable nucleotide binding domain comprising a natural or modified protein or portion thereof which via a bound guide nucleic acid is capable of binding to a nucleic acid sequence during CRISPR (i.e., Clustered Regularly Interspaced Short Palindromic Repeats)-mediated modification of a nucleic acid. Such a protein is referred to herein as a “CRISPR protein.” Accordingly, disclosed herein is a base editor comprising a polynucleotide programmable nucleotide binding domain comprising all or a portion of a CRISPR protein (i.e. a base editor comprising as a domain all or a portion of a CRISPR protein, also referred to as a “CRISPR protein-derived domain” of the base editor). A CRISPR protein-derived domain incorporated into a base editor can be modified compared to a wild-type or natural version of the CRISPR protein. For example, as described below a CRISPR protein-derived domain can comprise one or more mutations, insertions, deletions, rearrangements and/or recombinations relative to a wild-type or natural version of the CRISPR protein. 
     CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (mc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, and then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA,” or simply “gNRA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., et al., Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. 
     In some embodiments, the methods described herein can utilize an engineered Cas protein. A guide RNA (gRNA) is a short synthetic RNA composed of a scaffold sequence necessary for Cas-binding and a user-defined ˜20 nucleotide spacer that defines the genomic target to be modified. Thus, a skilled artisan can change the genomic target of the Cas protein specificity is partially determined by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome. 
     In some embodiments, the gRNA scaffold sequence is as follows: GUUUUAGAGC UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU (SEQ ID NO: 101). 
     In some embodiments, a CRISPR protein-derived domain incorporated into a base editor is an endonuclease (e.g., deoxyribonuclease or ribonuclease) capable of binding a target polynucleotide when in conjunction with a bound guide nucleic acid. In some embodiments, a CRISPR protein-derived domain incorporated into a base editor is a nickase capable of binding a target polynucleotide when in conjunction with a bound guide nucleic acid. In some embodiments, a CRISPR protein-derived domain incorporated into a base editor is a catalytically dead domain capable of binding a target polynucleotide when in conjunction with a bound guide nucleic acid. In some embodiments, a target polynucleotide bound by a CRISPR protein derived domain of a base editor is DNA. In some embodiments, a target polynucleotide bound by a CRISPR protein-derived domain of a base editor is RNA. 
     Cas proteins that can be used herein include class 1 and class 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5 h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12 h, and Cas12i, CARF, DinG, homologues thereof, or modified versions thereof. An unmodified CRISPR enzyme can have DNA cleavage activity, such as Cas9, which has two functional endonuclease domains: RuvC and HNH. A CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within a target sequence and/or within a complement of a target sequence. For example, a CRISPR enzyme can direct cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. 
     A vector that encodes a CRISPR enzyme that is mutated to with respect, to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence can be used. Cas9 can refer to a polypeptide with at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and/or sequence homology to a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from  S. pyogenes ). Cas9 can refer to a polypeptide with at most or at most about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and/or sequence homology to a wild-type exemplary Cas9 polypeptide (e.g., from  S. pyogenes ). Cas9 can refer to the wild-type or a modified form of the Cas9 protein that can comprise an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof. 
     In some embodiments, a CRISPR protein-derived domain of a base editor can include all or a portion of Cas9 from  Corynebacterium ulcerans  (NCBI Refs: NC_015683.1, NC_017317.1);  Corynebacterium diphtheria  (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1);  Prevotella intermedia  (NCBI Ref: NC_017861.1);  Spiroplasma taiwanense  (NCBI Ref: NC_021846.1);  Streptococcus  iniae (NCBI Ref: NC_021314.1);  Belliella baltica  (NCBI Ref: NC_018010.1); Psychroflexus torquis (NCBI Ref: NC_018721.1);  Streptococcus thermophilus  (NCBI Ref: YP_820832.1);  Listeria innocua  (NCBI Ref: NP_472073.1);  Campylobacter jejuni  (NCBI Ref: YP_002344900.1);  Neisseria meningitidis  (NCBI Ref: YP_002342100.1),  Streptococcus pyogenes , or  Staphylococcus aureus.    
     Cas9 Domains of Nucleobase Editors 
     Cas9 nuclease sequences and structures are well known to those of skill in the art (See, e.g., “Complete genome sequence of an M1 strain of  Streptococcus pyogenes .” Ferretti et al., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., et al., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., et al., Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to,  S. pyogenes  and  S. thermophilus . Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. 
     In some embodiments, a nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. Non-limiting, exemplary Cas9 domains are provided herein. The Cas9 domain may be a nuclease active Cas9 domain, a nuclease inactive Cas9 domain (dCas9), or a Cas9 nickase (nCas9). In some embodiments, the Cas9 domain is a nuclease active domain. For example, the Cas9 domain may be a Cas9 domain that cuts both strands of a duplexed nucleic acid (e.g., both strands of a duplexed DNA molecule). In some embodiments, the Cas9 domain comprises any one of the amino acid sequences as set forth herein. In some embodiments the Cas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth herein. In some embodiments, the Cas9 domain comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more or more mutations compared to any one of the amino acid sequences set forth herein. In some embodiments, the Cas9 domain comprises an amino acid sequence that has at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth herein. 
     In some embodiments, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as “Cas9 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9. In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild-type Cas9. In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length. 
     In some embodiments, Cas9 fusion proteins as provided herein comprise the full-length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided herein. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length Cas9 sequence, but only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and additional suitable sequences of Cas9 domains and fragments will be apparent to those of skill in the art. 
     A Cas9 protein can associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence that has complementary to the guide RNA. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 domain, for example a nuclease active Cas9, a Cas9 nickase (nCas9), or a nuclease inactive Cas9 (dCas9). Examples of nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, Cas12b/C2C1, and Cas12c/C2C3. In some embodiments, wild-type Cas9 corresponds to Cas9 from  Streptococcus pyogenes  (NCBI Reference Sequence: NC_017053.1, nucleotide and amino acid sequences as follows). 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 43) 
                   
               
               
                 ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGAT 
                   
               
               
                   
               
               
                 CACTGATGATTATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACA 
               
               
                   
               
               
                 GTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGGCAGTGGAGAGACAGCGGAAGCGACT 
               
               
                   
               
               
                 CGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACA 
               
               
                   
               
               
                 GGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGT 
               
               
                   
               
               
                 CTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGAT 
               
               
                   
               
               
                 GAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGCAGATTC 
               
               
                   
               
               
                 TACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTG 
               
               
                   
               
               
                 GTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATC 
               
               
                   
               
               
                 CAGTTGGTACAAATCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTAGAGTAGA 
               
               
                   
               
               
                 TGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTC 
               
               
                   
               
               
                 AGCTCCCCGGTGAGAAGAGAAATGGCTTGTTTGGGAATCTCATTGCTTTGTCATTGGGATTG 
               
               
                   
               
               
                 ACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGA 
               
               
                   
               
               
                 TACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGT 
               
               
                   
               
               
                 TTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATAGT 
               
               
                   
               
               
                 GAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAGCGCTACGATGAACATCATCAAGA 
               
               
                   
               
               
                 CTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTT 
               
               
                   
               
               
                 TTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTT 
               
               
                   
               
               
                 TATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACT 
               
               
                   
               
               
                 AAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAA 
               
               
                   
               
               
                 TTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAA 
               
               
                   
               
               
                 GACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATT 
               
               
                   
               
               
                 GGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCAT 
               
               
                   
               
               
                 GGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACA 
               
               
                   
               
               
                 AACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTA 
               
               
                   
               
               
                 TTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAGGGAATGCGAAAACCAG 
               
               
                   
               
               
                 CATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAA 
               
               
                   
               
               
                 GTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGA 
               
               
                   
               
               
                 AATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGCGCCTACCATGATTTGCTAAAAA 
               
               
                   
               
               
                 TTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTT 
               
               
                   
               
               
                 TTAACATTGACCTTATTTGAAGATAGGGGGATGATTGAGGAAAGACTTAAAACATATGCTCA 
               
               
                   
               
               
                 CCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTT 
               
               
                   
               
               
                 TGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTT 
               
               
                   
               
               
                 TTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGAC 
               
               
                   
               
               
                 ATTTAAAGAAGATATTCAAAAAGCACAGGTGTCTGGACAAGGCCATAGTTTACATGAACAGA 
               
               
                   
               
               
                 TTGCTAACTTAGCTGGCAGTCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAATTGTT 
               
               
                   
               
               
                 GATGAACTGGTCAAAGTAATGGGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGA 
               
               
                   
               
               
                 AAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAG 
               
               
                   
               
               
                 GTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAA 
               
               
                   
               
               
                 AATGAAAAGCTCTATCTCTATTATCTACAAAATGGAAGAGACATGTATGTGGACCAAGAATT 
               
               
                   
               
               
                 AGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCATTAAAG 
               
               
                   
               
               
                 ACGATTCAATAGACAATAAGGTACTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAAC 
               
               
                   
               
               
                 GTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAA 
               
               
                   
               
               
                 GTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAAC 
               
               
                   
               
               
                 TTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTG 
               
               
                   
               
               
                 GCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGA 
               
               
                   
               
               
                 GGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCT 
               
               
                   
               
               
                 ATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTT 
               
               
                   
               
               
                 GGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAA 
               
               
                   
               
               
                 AGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAA 
               
               
                   
               
               
                 AATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGA 
               
               
                   
               
               
                 GAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAA 
               
               
                   
               
               
                 AGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGA 
               
               
                   
               
               
                 AAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGAC 
               
               
                   
               
               
                 AAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAAC 
               
               
                   
               
               
                 GGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAAT 
               
               
                   
               
               
                 CCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATT 
               
               
                   
               
               
                 GACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAA 
               
               
                   
               
               
                 ATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTAC 
               
               
                   
               
               
                 AAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCAT 
               
               
                   
               
               
                 TATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCA 
               
               
                   
               
               
                 TAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAG 
               
               
                   
               
               
                 CAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGT 
               
               
                   
               
               
                 GAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTT 
               
               
                   
               
               
                 TAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATG 
               
               
                   
               
               
                 CCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTA 
               
               
                   
               
               
                 GGAGGTGACTGA  
               
               
                   
               
               
                 (SEQ ID NO: 42) 
                   
               
               
                 MDKK YSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGALLFGSGET AEAT 
                   
               
               
                   
               
               
                 RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD 
               
               
                   
               
               
                 EVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQIYNQLFEENPINASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFGNLIALSLGL 
               
               
                   
               
               
                 TPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNS 
               
               
                   
               
               
                 EITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF 
               
               
                   
               
               
                 YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLK 
               
               
                   
               
               
                 DNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT 
               
               
                   
               
               
                 NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRK 
               
               
                   
               
               
                 VTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIV 
               
               
                   
               
               
                 LTLTLFEDRGMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
               
               
                   
               
               
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG HSLHEQIANLAGSPAIKKGILQTVKIV   
               
               
                   
               
               
                   DELVKVMGHKPENIVIEMAR ENQTTQK GQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQ   
               
               
                   
               
               
                 
                   NEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDN 
                 
               
               
                   
               
               
                   VPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK AERG GLSELDKAGFIKRQLVETRQITKHV   
               
               
                   
               
               
                 
                   AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVV 
                 
               
               
                   
               
               
                 
                   GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANG 
                 
               
               
                   
               
               
                   EIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQT GGFSKESILPKRNSD 
               
               
                   
               
               
                 KLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI 
               
               
                   
               
               
                 DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH 
               
               
                   
               
               
                 YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIR 
               
               
                   
               
               
                 EQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQL 
               
               
                   
               
               
                 GGD  
               
               
                 (single underline: HNH domain; double underline: RuvC domain) 
               
            
           
         
       
     
     In some embodiments, wild-type Cas9 corresponds to, or comprises the following nucleotide and/or amino acid sequences: 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 44) 
                   
               
               
                 ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCTGTCAT 
                   
               
               
                   
               
               
                 AACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATT 
               
               
                   
               
               
                 CGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACT 
               
               
                   
               
               
                 CGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACA 
               
               
                   
               
               
                 AGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGT 
               
               
                   
               
               
                 CCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGAT 
               
               
                   
               
               
                 GAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTC 
               
               
                   
               
               
                 AACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTG 
               
               
                   
               
               
                 GGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATC 
               
               
                   
               
               
                 CAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGA 
               
               
                   
               
               
                 TGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCAC 
               
               
                   
               
               
                 AATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTG 
               
               
                   
               
               
                 ACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGA 
               
               
                   
               
               
                 CACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTAT 
               
               
                   
               
               
                 TTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACT 
               
               
                   
               
               
                 GAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGA 
               
               
                   
               
               
                 CTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCT 
               
               
                   
               
               
                 TTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTC 
               
               
                   
               
               
                 TACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACT 
               
               
                   
               
               
                 CAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAA 
               
               
                   
               
               
                 TCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAA 
               
               
                   
               
               
                 GACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCT 
               
               
                   
               
               
                 GGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCAT 
               
               
                   
               
               
                 GGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACC 
               
               
                   
               
               
                 AACTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTA 
               
               
                   
               
               
                 TTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCG 
               
               
                   
               
               
                 CCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAA 
               
               
                   
               
               
                 GTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGA 
               
               
                   
               
               
                 GATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGA 
               
               
                   
               
               
                 TAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTG 
               
               
                   
               
               
                 TTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCTCA 
               
               
                   
               
               
                 CCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGAT 
               
               
                   
               
               
                 TGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTT 
               
               
                   
               
               
                 CTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGACTCTTTAAC 
               
               
                   
               
               
                 CTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATA 
               
               
                   
               
               
                 TTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTG 
               
               
                   
               
               
                 GATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACG 
               
               
                   
               
               
                 CGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAG 
               
               
                   
               
               
                 AGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTG 
               
               
                   
               
               
                 CAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGA 
               
               
                   
               
               
                 ACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGA 
               
               
                   
               
               
                 AGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGAC 
               
               
                   
               
               
                 AATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGC 
               
               
                   
               
               
                 GAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTG 
               
               
                   
               
               
                 AACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCAT 
               
               
                   
               
               
                 GTTGCACAGATACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCG 
               
               
                   
               
               
                 GGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAAT 
               
               
                   
               
               
                 TCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTAATGCCGTC 
               
               
                   
               
               
                 GTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTA 
               
               
                   
               
               
                 CAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAG 
               
               
                   
               
               
                 CCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAAC 
               
               
                   
               
               
                 GGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGA 
               
               
                   
               
               
                 TAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAA 
               
               
                   
               
               
                 AGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGT 
               
               
                   
               
               
                 GATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCC 
               
               
                   
               
               
                 TACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGA 
               
               
                   
               
               
                 AGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCC 
               
               
                   
               
               
                 ATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACC 
               
               
                   
               
               
                 AAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGC 
               
               
                   
               
               
                 TTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCC 
               
               
                   
               
               
                 CATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCA 
               
               
                   
               
               
                 GCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCC 
               
               
                   
               
               
                 TAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATA 
               
               
                   
               
               
                 CGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGC 
               
               
                   
               
               
                 ATTCAAGTATTTTGACACAACGATAGATCGCAAACGATACACTTCTACCAAGGAGGTGCTAG 
               
               
                   
               
               
                 ACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAG 
               
               
                   
               
               
                 CTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGA 
               
               
                   
               
               
                 CGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCTGCAGGA 
               
               
                   
               
               
                 (SEQ ID NO: 102) 
                   
               
               
                 MDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET AEAT 
                   
               
               
                   
               
               
                 RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD 
               
               
                   
               
               
                 EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGL 
               
               
                   
               
               
                 TPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNT 
               
               
                   
               
               
                 EITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF 
               
               
                   
               
               
                 YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLK 
               
               
                   
               
               
                 DNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT 
               
               
                   
               
               
                 NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRK 
               
               
                   
               
               
                 VTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIV 
               
               
                   
               
               
                 LTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
               
               
                   
               
               
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGSPAIKKGILQTVKVV   
               
               
                   
               
               
                   DELVKVMGRHKPENIVIEMA RENQTTQK GQKNSRERMKRIEEGIKELGSQILKEHPVENTQL   
               
               
                   
               
               
                 
                   QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD 
                 
               
               
                   
               
               
                   NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK AERG GLSELDKAGFIKRQLVETRQITKH   
               
               
                   
               
               
                 
                   VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAV 
                 
               
               
                   
               
               
                 
                   VGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN 
                 
               
               
                   
               
               
                   GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQT GGFSKESILPKRNS 
               
               
                   
               
               
                 DKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP 
               
               
                   
               
               
                 IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLAS 
               
               
                   
               
               
                 HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI 
               
               
                   
               
               
                 REQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD  
               
               
                 (single underline: HNH domain; double underline: RuvC domain). 
               
            
           
         
       
     
     In some embodiments, wild-type Cas9 corresponds to Cas9 from  Streptococcus pyogenes  (NCBI Reference Sequence: NC_002737.2 (nucleotide sequence as follows); and Uniprot Reference Sequence: Q99ZW2 (amino acid sequence as follows): 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 45) 
                   
               
               
                 ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGAT 
                   
               
               
                   
               
               
                 CACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACA 
               
               
                   
               
               
                 GTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACT 
               
               
                   
               
               
                 CGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACA 
               
               
                   
               
               
                 GGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGT 
               
               
                   
               
               
                 CTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGAT 
               
               
                   
               
               
                 GAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTC 
               
               
                   
               
               
                 TACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTG 
               
               
                   
               
               
                 GTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATC 
               
               
                   
               
               
                 CAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGA 
               
               
                   
               
               
                 TGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTC 
               
               
                   
               
               
                 AGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTG 
               
               
                   
               
               
                 ACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGA 
               
               
                   
               
               
                 TACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGT 
               
               
                   
               
               
                 TTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACT 
               
               
                   
               
               
                 GAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGA 
               
               
                   
               
               
                 CTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTT 
               
               
                   
               
               
                 TTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTT 
               
               
                   
               
               
                 TATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACT 
               
               
                   
               
               
                 AAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAA 
               
               
                   
               
               
                 TTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAA 
               
               
                   
               
               
                 GACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATT 
               
               
                   
               
               
                 GGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCAT 
               
               
                   
               
               
                 GGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACA 
               
               
                   
               
               
                 AACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTA 
               
               
                   
               
               
                 TTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAG 
               
               
                   
               
               
                 CATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAA 
               
               
                   
               
               
                 GTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGA 
               
               
                   
               
               
                 AATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAA 
               
               
                   
               
               
                 TTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTT 
               
               
                   
               
               
                 TTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCA 
               
               
                   
               
               
                 CCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTT 
               
               
                   
               
               
                 TGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTT 
               
               
                   
               
               
                 TTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGAC 
               
               
                   
               
               
                 ATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATA 
               
               
                   
               
               
                 TTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTT 
               
               
                   
               
               
                 GATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACG 
               
               
                   
               
               
                 TGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAG 
               
               
                   
               
               
                 AAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTG 
               
               
                   
               
               
                 CAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGA 
               
               
                   
               
               
                 ATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTA 
               
               
                   
               
               
                 AAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGAT 
               
               
                   
               
               
                 AACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGC 
               
               
                   
               
               
                 CAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTG 
               
               
                   
               
               
                 AACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCAT 
               
               
                   
               
               
                 GTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCG 
               
               
                   
               
               
                 AGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAAT 
               
               
                   
               
               
                 TCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTC 
               
               
                   
               
               
                 GTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTA 
               
               
                   
               
               
                 TAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCG 
               
               
                   
               
               
                 CAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAAT 
               
               
                   
               
               
                 GGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGA 
               
               
                   
               
               
                 TAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCA 
               
               
                   
               
               
                 AGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCG 
               
               
                   
               
               
                 GACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCC 
               
               
                   
               
               
                 AACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAA 
               
               
                   
               
               
                 AATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCG 
               
               
                   
               
               
                 ATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACC 
               
               
                   
               
               
                 TAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAAT 
               
               
                   
               
               
                 TACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGT 
               
               
                   
               
               
                 CATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCA 
               
               
                   
               
               
                 GCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTT 
               
               
                   
               
               
                 TAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATA 
               
               
                   
               
               
                 CGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGC 
               
               
                   
               
               
                 TTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAG 
               
               
                   
               
               
                 ATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAG 
               
               
                   
               
               
                 CTAGGAGGTGACTGA  
               
               
                   
               
               
                 (SEQ ID NO: 1) 
                   
               
               
                 MDKK YSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET AEAT 
                   
               
               
                   
               
               
                 RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD 
               
               
                   
               
               
                 EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGL 
               
               
                   
               
               
                 TPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNT 
               
               
                   
               
               
                 EITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF 
               
               
                   
               
               
                 YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLK 
               
               
                   
               
               
                 DNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT 
               
               
                   
               
               
                 NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRK 
               
               
                   
               
               
                 VTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIV 
               
               
                   
               
               
                 LTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
               
               
                   
               
               
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG DSLHEHIANLAGSPAIKKGILQTVKVV   
               
               
                   
               
               
                   DELVKVMGRHKPENIVIEMA RENQTTQK GQKNSRERMKRIEEGIKELGSQILKEHPVENTQL   
               
               
                   
               
               
                 
                   QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD 
                 
               
               
                   
               
               
                   NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK AERG GLSELDKAGFIKRQLVETRQITKH   
               
               
                   
               
               
                 
                   VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAV 
                 
               
               
                   
               
               
                 
                   VGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN 
                 
               
               
                   
               
               
                   GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQT GGFSKESILPKRNS 
               
               
                   
               
               
                 DKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP 
               
               
                   
               
               
                 IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLAS 
               
               
                   
               
               
                 HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPI 
               
               
                   
               
               
                 REQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                 (single underline: HNH domain; double underline: RuvC domain) 
               
            
           
         
       
     
     In some embodiments, Cas9 refers to Cas9 from:  Corynebacterium ulcerans  (NCBI Refs: NC_015683.1, NC_017317.1);  Corynebacterium diphtheria  (NCBI Refs: NC_016782.1, NC_016786.1);  Spiroplasma syrphidicola  (NCBI Ref: NC_021284.1);  Prevotella intermedia  (NCBI Ref: NC_017861.1);  Spiroplasma taiwanense  (NCBI Ref: NC_021846.1);  Streptococcus iniae  (NCBI Ref: NC_021314.1);  Belliella baltica  (NCBI Ref: NC_018010.1);  Psychroflexus torquis  I (NCBI Ref: NC_018721.1);  Streptococcus thermophilus  (NCBI Ref: YP_820832.1),  Listeria innocua  (NCBI Ref: NP_472073.1),  Campylobacter jejuni  (NCBI Ref: YP_002344900.1) or  Neisseria meningitidis  (NCBI Ref: YP_002342100.1) or to a Cas9 from any other organism. 
     It should be appreciated that additional Cas9 proteins (e.g., a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9), including variants and homologs thereof, are within the scope of this disclosure. Exemplary Cas9 proteins include, without limitation, those provided below. In some embodiments, the Cas9 protein is a nuclease dead Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a nuclease active Cas9. 
     In some embodiments, the Cas9 domain is a nuclease-inactive Cas9 domain (dCas9). For example, the dCas9 domain may bind to a duplexed nucleic acid molecule (e.g., via a gRNA molecule) without cleaving either strand of the duplexed nucleic acid molecule. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10X mutation and a H840X mutation of the amino acid sequence set forth herein, or a corresponding mutation in any of the amino acid sequences provided herein, wherein X is any amino acid change. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10A mutation and a H840A mutation of the amino acid sequence set forth herein, or a corresponding mutation in any of the amino acid sequences provided herein. As one example, a nuclease-inactive Cas9 domain comprises the amino acid sequence set forth in Cloning vector pPlatTET-gRNA2 (Accession No. BAV54124). 
     The amino acid sequence of an exemplary catalytically inactive Cas9 (dCas9) is as follows: 
                    (SEQ ID NO: 46)       MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL               LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE               ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL               IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS               GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN               FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL               RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN               GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG               SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN               SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK               HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV               KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN               EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS               RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS               GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR               ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL               QNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKS               DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK               RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD               FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK               MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGE               IVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR               KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS               FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN               ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE               FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY               FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD            
(see, e.g., Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.”  Cell.  2013; 152(5): 1173-83, the entire contents of which are incorporated herein by reference).
 
     Additional suitable nuclease-inactive dCas9 domains will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A/H840A, D10A/D839A/H840A, and D10A/D839A/H840A/N863A mutant domains (See, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.  Nature Biotechnology.  2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference). 
     In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase, referred to as an “nCas9” protein (for “nickase” Cas9). A nuclease-inactivated Cas9 protein may interchangeably be referred to as a “dCas9” protein (for nuclease-“dead” Cas9) or catalytically inactive Cas9. Methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known (See, e.g., Jinek et al.,  Science.  337:816-821(2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013)  Cell.  28; 152(5):1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H840A completely inactivate the nuclease activity of  S. pyogenes  Cas9 (Jinek et al.,  Science.  337:816-821(2012); Qi et al.,  Cell.  28; 152(5):1173-83 (2013)). 
     In some embodiments, the dCas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the dCas9 domains provided herein. In some embodiments, the Cas9 domain comprises an amino acid sequences that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more or more mutations compared to any one of the amino acid sequences set forth herein. In some embodiments, the Cas9 domain comprises an amino acid sequence that has at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth herein. 
     In some embodiments, dCas9 corresponds to, or comprises in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate the Cas9 nuclease activity. For example, in some embodiments, a dCas9 domain comprises D10A and an H840A mutation or corresponding mutations in another Cas9. 
     In some embodiments, the dCas9 comprises the amino acid sequence of dCas9 (D10A and H840A): 
     
       
         
           
               
            
               
                 (SEQ ID NO: 46) 
               
               
                 MDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL   
               
               
                   
               
               
                   LFDSGET AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE 
               
               
                   
               
               
                 ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL 
               
               
                   
               
               
                 IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS 
               
               
                   
               
               
                 GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN 
               
               
                   
               
               
                 FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL 
               
               
                   
               
               
                 RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN 
               
               
                   
               
               
                 GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG 
               
               
                   
               
               
                 SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN 
               
               
                   
               
               
                 SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK 
               
               
                   
               
               
                 HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV 
               
               
                   
               
               
                 KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN 
               
               
                   
               
               
                 EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS 
               
               
                   
               
               
                 RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS 
               
               
                   
               
               
                 GQG DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA R 
               
               
                   
               
               
                 ENQTTQK GQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL   
               
               
                   
               
               
                 
                   QNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKS 
                 
               
               
                   
               
               
                   DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK AERG GLSELDKAGFIK   
               
               
                   
               
               
                 
                   RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD 
                 
               
               
                   
               
               
                 
                   FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK 
                 
               
               
                   
               
               
                 
                   MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGE 
                 
               
               
                   
               
               
                   IVWDKGRDFATVRKVLSMPQVNIVKKTEVQT GGFSKESILPKRNSDKLIAR 
               
               
                   
               
               
                 KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS 
               
               
                   
               
               
                 FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN 
               
               
                   
               
               
                 ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE 
               
               
                   
               
               
                 FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY 
               
               
                   
               
               
                 FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                 (single underline: HNH domain; double underline:  
               
               
                 RuvC domain). 
               
            
           
         
       
     
     In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 remains a histidine in the amino acid sequence provided above, or at corresponding positions in any of the amino acid sequences provided herein. 
     In other embodiments, dCas9 variants having mutations other than D10A and H840A are provided, which, e.g., result in nuclease inactivated Cas9 (dCas9). Such mutations, by way of example, include other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and/or the RuvC1 subdomain). In some embodiments, variants or homologues of dCas9 are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, variants of dCas9 are provided having amino acid sequences which are shorter, or longer, by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more. 
     In some embodiments, the Cas9 domain is a Cas9 nickase. The Cas9 nickase may be a Cas9 protein that is capable of cleaving only one strand of a duplexed nucleic acid molecule (e.g., a duplexed DNA molecule). In some embodiments the Cas9 nickase cleaves the target strand of a duplexed nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is base paired to (complementary to) a gRNA (e.g., an sgRNA) that is bound to the Cas9. In some embodiments, a Cas9 nickase comprises a D10A mutation and has a histidine at position 840. In some embodiments the Cas9 nickase cleaves the non-target, non-base-edited strand of a duplexed nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is not base paired to a gRNA (e.g., an sgRNA) that is bound to the Cas9. In some embodiments, a Cas9 nickase comprises an H840A mutation and has an aspartic acid residue at position 10, or a corresponding mutation. In some embodiments the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 nickases provided herein. Additional suitable Cas9 nickases will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure. The amino acid sequence of an exemplary catalytically Cas9 nickase (nCas9) is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 103) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL 
               
               
                   
               
               
                 LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE 
               
               
                   
               
               
                 ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL 
               
               
                   
               
               
                 IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS 
               
               
                   
               
               
                 GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN 
               
               
                   
               
               
                 FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL 
               
               
                   
               
               
                 RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN 
               
               
                   
               
               
                 GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG 
               
               
                   
               
               
                 SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN 
               
               
                   
               
               
                 SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK 
               
               
                   
               
               
                 HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV 
               
               
                   
               
               
                 KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN 
               
               
                   
               
               
                 EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS 
               
               
                   
               
               
                 RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS 
               
               
                   
               
               
                 GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR 
               
               
                   
               
               
                 ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL 
               
               
                   
               
               
                 QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKS 
               
               
                   
               
               
                 DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK 
               
               
                   
               
               
                 RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD 
               
               
                   
               
               
                 FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK 
               
               
                   
               
               
                 MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGE 
               
               
                   
               
               
                 IVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
               
               
                   
               
               
                 KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS 
               
               
                   
               
               
                 FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN 
               
               
                   
               
               
                 ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE 
               
               
                   
               
               
                 FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY 
               
               
                   
               
               
                 FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
            
           
         
       
     
     In some embodiments, Cas9 refers to a Cas9 from archaea (e.g., nanoarchaea), which constitute a domain and kingdom of single-celled prokaryotic microbes. In some embodiments, the programmable nucleotide binding protein may be a CasX or CasY protein, which have been described in, for example, Burstein et al., “New CRISPR-Cas systems from uncultivated microbes.” Cell Res. 2017 Feb. 21. doi: 10.1038/cr.2017.21, the entire contents of which is hereby incorporated by reference. Using genome-resolved metagenomics, a number of CRISPR-Cas systems were identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems were discovered, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. In some embodiments, in a base editor system described herein Cas9 is replaced by CasX, or a variant of CasX. In some embodiments, in a base editor system described herein Cas9 is replaced by CasY, or a variant of CasY. It should be appreciated that other RNA-guided DNA binding proteins may be used as a nucleic acid programmable DNA binding protein (napDNAbp), and are within the scope of this disclosure. 
     In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein may be a CasX or CasY protein. In some embodiments, the napDNAbp is a CasX protein. In some embodiments, the napDNAbp is a CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to a naturally-occurring CasX or CasY protein. In some embodiments, the programmable nucleotide binding protein is a naturally-occurring CasX or CasY protein. In some embodiments, the programmable nucleotide binding protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to any CasX or CasY protein described herein. It should be appreciated that CasX and CasY from other bacterial species may also be used in accordance with the present disclosure. 
     In some embodiments, the Cas9 is a  Neisseria  menigitidis Cas9 (NmeCas9) or a variant thereof. NmeCas9 features and PAM sequences as described in Edraki et al. Mol. Cell. (2019) 73(4): 714-726 is incorporated herein by reference in its entirety. 
     An exemplary amino acid sequence of a Nme1Cas9 is provided below: 
     
       
         
           
               
               
            
               
                 type II CRISPR RNA-guided endonuclease Cas9 [ Neisseria meningitidis ] 
                   
               
               
                 WP_002235162.1 
               
               
                 (SEQ ID NO: 104) 
                   
               
            
           
           
               
               
               
            
               
                    1 
                 maafkpnpin yilgldigia svgwamveid edenpiclid lgvrvferae vpktgdslam 
                   
               
               
                   
               
               
                   61 
                 arrlarsvrr ltrrrahrll rarrllkreg vlqaadfden glikslpntp wqlraaaldr 
               
               
                   
               
               
                  121 
                 kltplewsav llhlikhrgy lsqrkneget adkelgallk gvadnahalq tgdfrtpael 
               
               
                   
               
               
                  181 
                 alnkfekesg hirnqrgdys htfsrkdlqa elillfekqk efgnphvsgg lkegietllm 
               
               
                   
               
               
                  241 
                 tqrpalsgda vqkmlghctf epaepkaakn tytaerfiwl tklnnlrile qgserpltdt 
               
               
                   
               
               
                  301 
                 eratlmdepy rkskltyaqa rkllgledta ffkglrygkd naeastlmem kayhaisral 
               
               
                   
               
               
                  361 
                 ekeglkdkks plnlspelqd eigtafslfk tdeditgrlk driqpeilea llkhisfdkf 
               
               
                   
               
               
                  421 
                 vqislkalrr ivplmeqgkr ydeacaeiyg dhygkkntee kiylppipad eirnpvvlra 
               
               
                   
               
               
                  481 
                 lsqarkving vvrrygspar ihietarevg ksfkdrkeie krqeenrkdr ekaaakfrey 
               
               
                   
               
               
                  541 
                 fpnfvgepks kdilklrlye qqhgkclysg keinlgrlne kgyveidhal pfsrtwddsf 
               
               
                   
               
               
                  601 
                 nnkvlvlgse nqnkgnqtpy eyfngkdnsr ewqefkarve tsrfprskkq rillqkfded 
               
               
                   
               
               
                  661 
                 gfkernlndt ryvnrflcqf vadrmrltgk gkkrvfasng qitnllrgfw glrkvraend 
               
               
                   
               
               
                  721 
                 rhhaldavvv acstvamqqk itrfvrykem nafdgktidk etgevlhqkt hfpqpweffa 
               
               
                   
               
               
                  781 
                 qevmirvfgk pdgkpefeea dtpeklrtll aeklssrpea vheyvtplfv srapnrkmsg 
               
               
                   
               
               
                  841 
                 qghmetvksa krldegvsvl rvpltqlklk dlekmvnrer epklyealka rleahkddpa 
               
               
                   
               
               
                  901 
                 kafaepfyky dkagnrtqqv kavrveqvqk tgvwvrnhng iadnatmvrv dvfekgdkyy 
               
               
                   
               
               
                  961 
                 lvpiyswqva kgilpdravv qgkdeedwql iddsfnfkfs lhpndlvevi tkkarmfgyf 
               
               
                   
               
               
                 1021 
                 aschrgtgni nirihdldhk igkngilegi gvktalsfqk yqidelgkei rpcrlkkrpp 
               
               
                   
               
               
                 1081 
                 vr  
               
            
           
         
       
     
     An exemplary amino acid sequence of a Nme2Cas9 is provided below: 
     
       
         
           
               
               
            
               
                 type II CRISPR RNA-guided endonuclease Cas9 [ Neisseria meningitidis ] 
                   
               
               
                 WP_002230835.1 
               
               
                 (SEQ ID NO: 105) 
                   
               
            
           
           
               
               
               
            
               
                    1 
                 maafkpnpin yilgldigia svgwamveid eeenpirlid lgvrvferae vpktgdslam 
                   
               
               
                   
               
               
                   61 
                 arrlarsvrr ltrrrahrll rarrllkreg vlqaadfden glikslpntp wqlraaaldr 
               
               
                   
               
               
                  121 
                 kltplewsav llhlikhrgy lsqrkneget adkelgallk gvannahalq tgdfrtpael 
               
               
                   
               
               
                  181 
                 alnkfekesg hirnqrgdys htfsrkdlqa elillfekqk efgnphvsgg lkegietllm 
               
               
                   
               
               
                  241 
                 tqrpalsgda vqkmlghctf epaepkaakn tytaerfiwl tklnnlrile qgserpltdt 
               
               
                   
               
               
                  301 
                 eratlmdepy rkskltyaqa rkllgledta ffkglrygkd naeastlmem kayhaisral 
               
               
                   
               
               
                  361 
                 ekeglkdkks plnlsselqd eigtafslfk tdeditgrlk drvqpeilea llkhisfdkf 
               
               
                   
               
               
                  421 
                 vqislkalrr ivplmeqgkr ydeacaeiyg dhygkkntee kiylppipad eirnpvvlra 
               
               
                   
               
               
                  481 
                 lsqarkving vvrrygspar ihietarevg ksfkdrkeie krqeenrkdr ekaaakfrey 
               
               
                   
               
               
                  541 
                 fpnfvgepks kdilklrlye qqhgkclysg keinlvrlne kgyveidhal pfsrtwddsf 
               
               
                   
               
               
                  601 
                 nnkvlvlgse nqnkgnqtpy eyfngkdnsr ewqefkarve tsrfprskkq rillqkfded 
               
               
                   
               
               
                  661 
                 gfkecnlndt ryvnrflcqf vadhilltgk gkrrvfasng qitnllrgfw glrkvraend 
               
               
                   
               
               
                  721 
                 rhhaldavvv acstvamqqk itrfvrykem nafdgktidk etgkvlhqkt hfpqpweffa 
               
               
                   
               
               
                  781 
                 qevmirvfgk pdgkpefeea dtpeklrtll aeklssrpea vheyvtplfv srapnrkmsg 
               
               
                   
               
               
                  841 
                 ahkdtlrsak rfvkhnekis vkrvwlteik ladlenmvny kngreielye alkarleayg 
               
               
                   
               
               
                  901 
                 gnakqafdpk dnpfykkggq lvkavrvekt qesgvllnkk naytiadngd mvrvdvfckv 
               
               
                   
               
               
                  961 
                 dkkgknqyfi vpiyawqvae nilpdidckg yriddsytfc fslhkydlia fqkdekskve 
               
               
                   
               
               
                 1021 
                 fayyincdss ngrfylawhd kgskeqqfri stqnlvliqk yqvnelgkei rpcrlkkrpp 
               
               
                   
               
               
                 1081 
                 vr  
               
            
           
         
       
     
     In some embodiments, the Cas protein is a CasX or CasY. An exemplary CasX ((uniprot.org/uniprot/FONN87; uniprot.org/uniprot/F0NH53) tr|F0NN87|F0NN87_SULIHCRISPR-associatedCasx protein OS= Sulfolobus islandicus  (strain HVE10/4) GN=SiH 0402 PE=4 SV=1) amino acid sequence is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 51) 
               
               
                 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAK 
               
               
                   
               
               
                 NNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFP 
               
               
                   
               
               
                 TTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLE 
               
               
                   
               
               
                 VEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNG 
               
               
                   
               
               
                 IVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGG 
               
               
                   
               
               
                 FSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG 
               
               
                   
               
               
                 SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG. 
               
            
           
         
       
     
     An exemplary CasX (&gt;tr|F0NH 53 |F0NH 53 _SULIR CRISPR associated protein, Casx OS= Sulfolobus islandicus  (strain REY15A) GN=SiRe 0771 PE=4 SV=1) amino acid sequence is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 52) 
               
               
                 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAK 
               
               
                   
               
               
                 NNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFP 
               
               
                   
               
               
                 TTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLE 
               
               
                   
               
               
                 VEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNG 
               
               
                   
               
               
                 IVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGG 
               
               
                   
               
               
                 FSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG 
               
               
                   
               
               
                 SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG. 
               
            
           
         
       
     
     Deltaproteobacteria CasX 
     
       
         
           
               
            
               
                 (SEQ ID NO: 106) 
               
               
                 MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKP 
               
               
                   
               
               
                 EVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQ 
               
               
                   
               
               
                 PASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAY 
               
               
                   
               
               
                 TNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHV 
               
               
                   
               
               
                 TKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEH 
               
               
                   
               
               
                 QKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDfAYNEVIAR 
               
               
                   
               
               
                 VRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINE 
               
               
                   
               
               
                 VKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENP 
               
               
                   
               
               
                 KKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEA 
               
               
                   
               
               
                 RNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQKWYGDLR 
               
               
                   
               
               
                 GNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMN 
               
               
                   
               
               
                 YGKKGRIRFTDGTDIKKSGKWQGLLYGGGKAKVIDLTFDPDDEQLIILPL 
               
               
                   
               
               
                 AFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVA 
               
               
                   
               
               
                 LTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSG 
               
               
                   
               
               
                 GPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVR 
               
               
                   
               
               
                 NSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAK 
               
               
                   
               
               
                 LAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGW 
               
               
                   
               
               
                 ATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWT 
               
               
                   
               
               
                 KGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFL 
               
               
                   
               
               
                 NSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA 
               
            
           
         
       
     
     An exemplary CasY ((ncbi.nlm.nih.gov/protein/APG80656.1)&gt;APG80656.1 CRISPR-associated protein CasY [uncultured Parcubacteria group bacterium]) amino acid sequence is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 54) 
               
               
                 MSKRHPRISGVKGYRLHAQRLEYTGKSGAMRTIKYPLYSSPSGGRTVPRE 
               
               
                   
               
               
                 IVSAINDDYVGLYGLSNFDDLYNAEKRNEEKVYSVLDFWYDCVQYGAVFS 
               
               
                   
               
               
                 YTAPGLLKNVAEVRGGSYELTKTLKGSHLYDELQIDKVIKFLNKKEISRA 
               
               
                   
               
               
                 NGSLDKLKKDIIDCFKAEYRERHKDQCNKLADDIKNAKKDAGASLGERQK 
               
               
                   
               
               
                 KLFRDFFGISEQSENDKPSFTNPLNLTCCLLPFDTVNNNRNRGEVLFNKL 
               
               
                   
               
               
                 KEYAQKLDKNEGSLEMWEYIGIGNSGTAFSNFLGEGFLGRLRENKITELK 
               
               
                   
               
               
                 KAMMDITDAWRGQEQEEELEKRLRILAALTIKLREPKFDNHWGGYRSDIN 
               
               
                   
               
               
                 GKLSSWLQNYINQTVKIKEDLKGHKKDLKKAKEMINRFGESDTKEEAVVS 
               
               
                   
               
               
                 SLLESIEKIVPDDSADDEKPDIPAIAIYRRFLSDGRLTLNRFVQREDVQE 
               
               
                   
               
               
                 ALIKERLEAEKKKKPKKRKKKSDAEDEKETIDFKELFPHLAKPLKLVPNF 
               
               
                   
               
               
                 YGDSKRELYKKYKNAAIYTDALWKAVEKIYKSAFSSSLKNSFFDTDFDKD 
               
               
                   
               
               
                 FFIKRLQKIFSVYRRFNTDKWKPIVKNSFAPYCDIVSLAENEVLYKPKQS 
               
               
                   
               
               
                 RSRKSAAIDKNRVRLPSTENIAKAGIALARELSVAGFDWKDLLKKEEHEE 
               
               
                   
               
               
                 YIDLIELHKTALALLLAVTETQLDISALDFVENGTVKDFMKTRDGNLVLE 
               
               
                   
               
               
                 GRFLEMFSQSIVFSELRGLAGLMSRKEFITRSAIQTMNGKQAELLYIPHE 
               
               
                   
               
               
                 FQSAKITTPKEMSRAFLDLAPAEFATSLEPESLSEKSLLKLKQMRYYPHY 
               
               
                   
               
               
                 FGYELTRTGQGIDGGVAENALRLEKSPVKKREIKCKQYKTLGRGQNKIVL 
               
               
                   
               
               
                 YVRSSYYQTQFLEWFLHRPKNVQTDVAVSGSFLIDEKKVKTRWNYDALTV 
               
               
                   
               
               
                 ALEPVSGSERVFVSQPFTIFPEKSAEEEGQRYLGIDIGEYGIAYTALEIT 
               
               
                   
               
               
                 GDSAKILDQNFISDPQLKTLREEVKGLKLDQRRGTFAMPSTKIARIRESL 
               
               
                   
               
               
                 VHSLRNRIHHLALKHKAKIVYELEVSRFEEGKQKIKKVYATLKKADVYSE 
               
               
                   
               
               
                 IDADKNLQTTVWGKLAVASEISASYTSQFCGACKKLWRAEMQVDETITTQ 
               
               
                   
               
               
                 ELIGTVRVIKGGTLIDAIKDFMRPPIFDENDTPFPKYRDFCDKHHISKKM 
               
               
                   
               
               
                 RGNSCLFICPFCRANADADIQASQTIALLRYVKEEKKVEDYFERFRKLKN 
               
               
                   
               
               
                 IKVLGQMKKI. 
               
            
           
         
       
     
     The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Cas9 undergoes a conformational change upon target binding that positions the nuclease domains to cleave opposite strands of the target DNA. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (˜3-4 nucleotides upstream of the PAM sequence). The resulting DSB is then repaired by one of two general repair pathways: (1) the efficient but error-prone non-homologous end joining (NHEJ) pathway; or (2) the less efficient but high-fidelity homology directed repair (HDR) pathway. 
     The “efficiency” of non-homologous end joining (NHEJ) and/or homology directed repair (HDR) can be calculated by any convenient method. For example, in some embodiments, efficiency can be expressed in terms of percentage of successful HDR. For example, a surveyor nuclease assay can be used to generate cleavage products and the ratio of products to substrate can be used to calculate the percentage. For example, a surveyor nuclease enzyme can be used that directly cleaves DNA containing a newly integrated restriction sequence as the result of successful HDR. More cleaved substrate indicates a greater percent HDR (a greater efficiency of HDR). As an illustrative example, a fraction (percentage) of HDR can be calculated using the following equation [(cleavage products)/(substrate plus cleavage products)] (e.g., (b+c)/(a+b+c), where “a” is the band intensity of DNA substrate and “b” and “c” are the cleavage products). 
     In some embodiments, efficiency can be expressed in terms of percentage of successful NHEJ. For example, a T7 endonuclease I assay can be used to generate cleavage products and the ratio of products to substrate can be used to calculate the percentage NHEJ. T7 endonuclease I cleaves mismatched heteroduplex DNA which arises from hybridization of wild-type and mutant DNA strands (NHEJ generates small random insertions or deletions (indels) at the site of the original break). More cleavage indicates a greater percent NHEJ (a greater efficiency of NHEJ). As an illustrative example, a fraction (percentage) of NHEJ can be calculated using the following equation: (1−(1−(b+c)/(a+b+c)) 1/2 )×100, where “a” is the band intensity of DNA substrate and “b” and “c” are the cleavage products (Ran et. al., Cell. 2013 Sep. 12; 154(6):1380-9; and Ran et al., Nat Protoc. 2013 November; 8(11): 2281-2308). 
     The NHEJ repair pathway is the most active repair mechanism, and it frequently causes small nucleotide insertions or deletions (indels) at the DSB site. The randomness of NHEJ-mediated DSB repair has important practical implications, because a population of cells expressing Cas9 and a gRNA or a guide polynucleotide can result in a diverse array of mutations. In most embodiments, NHEJ gives rise to small indels in the target DNA that result in amino acid deletions, insertions, or frameshift mutations leading to premature stop codons within the open reading frame (ORF) of the targeted gene. The ideal end result is a loss-of-function mutation within the targeted gene. 
     While NHEJ-mediated DSB repair often disrupts the open reading frame of the gene, homology directed repair (HDR) can be used to generate specific nucleotide changes ranging from a single nucleotide change to large insertions like the addition of a fluorophore or tag. 
     In order to utilize HDR for gene editing, a DNA repair template containing the desired sequence can be delivered into the cell type of interest with the gRNA(s) and Cas9 or Cas9 nickase. The repair template can contain the desired edit as well as additional homologous sequence immediately upstream and downstream of the target (termed left &amp; right homology arms). The length of each homology arm can be dependent on the size of the change being introduced, with larger insertions requiring longer homology arms. The repair template can be a single-stranded oligonucleotide, double-stranded oligonucleotide, or a double-stranded DNA plasmid. The efficiency of HDR is generally low (&lt;10% of modified alleles) even in cells that express Cas9, gRNA and an exogenous repair template. The efficiency of HDR can be enhanced by synchronizing the cells, since HDR takes place during the S and G2 phases of the cell cycle. Chemically or genetically inhibiting genes involved in NHEJ can also increase HDR frequency. 
     In some embodiments, Cas9 is a modified Cas9. A given gRNA targeting sequence can have additional sites throughout the genome where partial homology exists. These sites are called off-targets and need to be considered when designing a gRNA. In addition to optimizing gRNA design, CRISPR specificity can also be increased through modifications to Cas9. Cas9 generates double-strand breaks (DSBs) through the combined activity of two nuclease domains, RuvC and HNH. Cas9 nickase, a D10A mutant of SpCas9, retains one nuclease domain and generates a DNA nick rather than a DSB. The nickase system can also be combined with HDR-mediated gene editing for specific gene edits. 
     In some embodiments, Cas9 is a variant Cas9 protein. A variant Cas9 polypeptide has an amino acid sequence that is different by one amino acid (e.g., has a deletion, insertion, substitution, fusion) when compared to the amino acid sequence of a wild-type Cas9 protein. In some instances, the variant Cas9 polypeptide has an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nuclease activity of the Cas9 polypeptide. For example, in some instances, the variant Cas9 polypeptide has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 protein. In some embodiments, the variant Cas9 protein has no substantial nuclease activity. When a subject Cas9 protein is a variant Cas9 protein that has no substantial nuclease activity, it can be referred to as “dCas9.” 
     In some embodiments, a variant Cas9 protein has reduced nuclease activity. For example, a variant Cas9 protein exhibits less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1%, of the endonuclease activity of a wild-type Cas9 protein, e.g., a wild-type Cas9 protein. 
     In some embodiments, a variant Cas9 protein can cleave the complementary strand of a guide target sequence but has reduced ability to cleave the non-complementary strand of a double stranded guide target sequence. For example, the variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the RuvC domain. As a non-limiting example, in some embodiments, a variant Cas9 protein has a D10A (aspartate to alanine at amino acid position 10) and can therefore cleave the complementary strand of a double stranded guide target sequence but has reduced ability to cleave the non-complementary strand of a double stranded guide target sequence (thus resulting in a single strand break (SSB) instead of a double strand break (DSB) when the variant Cas9 protein cleaves a double stranded target nucleic acid) (see, for example, Jinek et al., Science. 2012 Aug. 17; 337(6096):816-21). 
     In some embodiments, a variant Cas9 protein can cleave the non-complementary strand of a double stranded guide target sequence but has reduced ability to cleave the complementary strand of the guide target sequence. For example, the variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC/HNH/RuvC domain motifs). As a non-limiting example, in some embodiments, the variant Cas9 protein has an H840A (histidine to alanine at amino acid position 840) mutation and can therefore cleave the non-complementary strand of the guide target sequence but has reduced ability to cleave the complementary strand of the guide target sequence (thus resulting in a SSB instead of a DSB when the variant Cas9 protein cleaves a double stranded guide target sequence). Such a Cas9 protein has a reduced ability to cleave a guide target sequence (e.g., a single stranded guide target sequence) but retains the ability to bind a guide target sequence (e.g., a single stranded guide target sequence). 
     In some embodiments, a variant Cas9 protein has a reduced ability to cleave both the complementary and the non-complementary strands of a double stranded target DNA. As a non-limiting example, in some embodiments, the variant Cas9 protein harbors both the D10A and the H840A mutations such that the polypeptide has a reduced ability to cleave both the complementary and the non-complementary strands of a double stranded target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g., a single stranded target DNA) but retains the ability to bind a target DNA (e.g., a single stranded target DNA). 
     As another non-limiting example, in some embodiments, the variant Cas9 protein harbors W476A and W1126A mutations such that the polypeptide has a reduced ability to cleave a target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g., a single stranded target DNA) but retains the ability to bind a target DNA (e.g., a single stranded target DNA). 
     As another non-limiting example, in some embodiments, the variant Cas9 protein harbors P475A, W476A, N477A, D1125A, W1126A, and D1127A mutations such that the polypeptide has a reduced ability to cleave a target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g., a single stranded target DNA) but retains the ability to bind a target DNA (e.g., a single stranded target DNA). 
     As another non-limiting example, in some embodiments, the variant Cas9 protein harbors H840A, W476A, and W1126A, mutations such that the polypeptide has a reduced ability to cleave a target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g., a single stranded target DNA) but retains the ability to bind a target DNA (e.g., a single stranded target DNA). As another non-limiting example, in some embodiments, the variant Cas9 protein harbors H840A, D10A, W476A, and W1126A, mutations such that the polypeptide has a reduced ability to cleave a target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g., a single stranded target DNA) but retains the ability to bind a target DNA (e.g., a single stranded target DNA). In some embodiments, the variant Cas9 has restored catalytic His residue at position 840 in the Cas9 HNH domain (A840H). 
     As another non-limiting example, in some embodiments, the variant Cas9 protein harbors, H840A, P475A, W476A, N477A, D1125A, W1126A, and D1127A mutations such that the polypeptide has a reduced ability to cleave a target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g., a single stranded target DNA) but retains the ability to bind a target DNA (e.g., a single stranded target DNA). As another non-limiting example, in some embodiments, the variant Cas9 protein harbors D10A, H840A, P475A, W476A, N477A, D1125A, W1126A, and D1127A mutations such that the polypeptide has a reduced ability to cleave a target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g., a single stranded target DNA) but retains the ability to bind a target DNA (e.g., a single stranded target DNA). In some embodiments, when a variant Cas9 protein harbors W476A and W1126A mutations or when the variant Cas9 protein harbors P475A, W476A, N477A, D1125A, W1126A, and D1127A mutations, the variant Cas9 protein does not bind efficiently to a PAM sequence. Thus, in some such embodiments, when such a variant Cas9 protein is used in a method of binding, the method does not require a PAM sequence. In other words, in some embodiments, when such a variant Cas9 protein is used in a method of binding, the method can include a guide RNA, but the method can be performed in the absence of a PAM sequence (and the specificity of binding is therefore provided by the targeting segment of the guide RNA). Other residues can be mutated to achieve the above effects (i.e., inactivate one or the other nuclease portions). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or A987 can be altered (i.e., substituted). Also, mutations other than alanine substitutions are suitable. 
     In some embodiments, a variant Cas9 protein that has reduced catalytic activity (e.g., when a Cas9 protein has a D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or a A987 mutation, e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and/or D986A), the variant Cas9 protein can still bind to target DNA in a site-specific manner (because it is still guided to a target DNA sequence by a guide RNA) as long as it retains the ability to interact with the guide RNA. 
     In some embodiments, the variant Cas protein can be spCas9, spCas9-VRQR, spCas9-VRER, xCas9 (sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9-LRVSQL. 
     In some embodiments, a modified SpCas9 including amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER) and having specificity for the altered PAM 5′-NGC-3′ was used. 
     Alternatives to  S. pyogenes  Cas9 can include RNA-guided endonucleases from the Cpf1 family that display cleavage activity in mammalian cells. CRISPR from  Prevotella  and  Francisella  1 (CRISPR/Cpf1) is a DNA-editing technology analogous to the CRISPR/Cas9 system. Cpf1 is an RNA-guided endonuclease of a class II CRISPR/Cas system. This acquired immune mechanism is found in  Prevotella  and  Francisella  bacteria. Cpf1 genes are associated with the CRISPR locus, coding for an endonuclease that use a guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR/Cas9 system limitations. Unlike Cas9 nucleases, the result of Cpf1-mediated DNA cleavage is a double-strand break with a short 3′ overhang. Cpf1&#39;s staggered cleavage pattern can open up the possibility of directional gene transfer, analogous to traditional restriction enzyme cloning, which can increase the efficiency of gene editing. Like the Cas9 variants and orthologues described above, Cpf1 can also expand the number of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack the NGG PAM sites favored by SpCas9. The Cpf1 locus contains a mixed alpha/beta domain, a RuvC-I followed by a helical region, a RuvC-II and a zinc finger-like domain. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9. Furthermore, Cpf1 does not have a HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alpha-helical recognition lobe of Cas9. Cpf1 CRISPR-Cas domain architecture shows that Cpf1 is functionally unique, being classified as Class 2, type V CRISPR system. The Cpf1 loci encode Cast, Cas2 and Cas4 proteins more similar to types I and III than from type II systems. Functional Cpf1 doesn&#39;t need the trans-activating CRISPR RNA (tracrRNA), therefore, only CRISPR (crRNA) is required. This benefits genome editing because Cpf1 is not only smaller than Cas9, but also it has a smaller sgRNA molecule (proximately half as many nucleotides as Cas9). The Cpf1-crRNA complex cleaves target DNA or RNA by identification of a protospacer adjacent motif 5′-YTN-3′ in contrast to the G-rich PAM targeted by Cas9. After identification of PAM, Cpf1 introduces a sticky-end-like DNA double-stranded break of 4 or 5 nucleotides overhang. 
     Cas12 Domains of Nucleobase Editors 
     Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multisubunit effector complexes, while Class 2 systems have a single protein effector. For example, Cas9 and Cpf1 are Class 2 effectors, albeit different types (Type II and Type V, respectively). In addition to Cpf1, Class 2, Type V CRISPR-Cas systems also comprise Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12 h, and Cas12i). See, e.g., Shmakov et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems,” Mol. Cell, 2015 Nov. 5; 60(3): 385-397; Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR Journal, 2018, 1(5): 325-336; and Yan et al., “Functionally Diverse Type V CRISPR-Cas Systems,” Science, 2019 Jan. 4; 363: 88-91; the entire contents of each is hereby incorporated by reference. Type V Cas proteins contain a RuvC (or RuvC-like) endonuclease domain. While production of mature CRISPR RNA (crRNA) is generally tracrRNA-independent, Cas12b/C2c1, for example, requires tracrRNA for production of crRNA. Cas12b/C2c1 depends on both crRNA and tracrRNA for DNA cleavage. 
     Nucleic acid programmable DNA binding proteins contemplated in the present invention include Cas proteins that are classified as Class 2, Type V (Cas12 proteins). Non-limiting examples of Cas Class 2, Type V proteins include Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12 h, and Cas12i, homologues thereof, or modified versions thereof. As used herein, a Cas12 protein can also be referred to as a Cas12 nuclease, a Cas12 domain, or a Cas12 protein domain. In some embodiments, the Cas12 proteins of the present invention comprise an amino acid sequence interrupted by an internally fused protein domain such as a deaminase domain. 
     In some embodiments, the Cas12 domain is a nuclease inactive Cas12 domain or a Cas12 nickase. In some embodiments, the Cas12 domain is a nuclease active domain. For example, the Cas12 domain may be a Cas12 domain that nicks one strand of a duplexed nucleic acid (e.g., duplexed DNA molecule). In some embodiments, the Cas12 domain comprises any one of the amino acid sequences as set forth herein. In some embodiments the Cas12 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth herein. In some embodiments, the Cas12 domain comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more mutations compared to any one of the amino acid sequences set forth herein. In some embodiments, the Cas12 domain comprises an amino acid sequence that has at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth herein. 
     In some embodiments, proteins comprising fragments of Cas12 are provided. For example, in some embodiments, a protein comprises one of two Cas12 domains: (1) the gRNA binding domain of Cas12; or (2) the DNA cleavage domain of Cas12. In some embodiments, proteins comprising Cas12 or fragments thereof are referred to as “Cas12 variants.” A Cas12 variant shares homology to Cas12, or a fragment thereof. For example, a Cas12 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild type Cas12. In some embodiments, the Cas12 variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild type Cas12. In some embodiments, the Cas12 variant comprises a fragment of Cas12 (e.g., a gRNA binding domain or a DNA cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas12. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas12. In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length. 
     In some embodiments, Cas12 corresponds to, or comprises in part or in whole, a Cas12 amino acid sequence having one or more mutations that alter the Cas12 nuclease activity. Such mutations, by way of example, include amino acid substitutions within the RuvC nuclease domain of Cas12. In some embodiments, variants or homologues of Cas12 are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to a wild type Cas12. In some embodiments, variants of Cas12 are provided having amino acid sequences which are shorter, or longer, by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more. 
     In some embodiments, Cas12 fusion proteins as provided herein comprise the full-length amino acid sequence of a Cas12 protein, e.g., one of the Cas12 sequences provided herein. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length Cas12 sequence, but only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas12 domains are provided herein, and additional suitable sequences of Cas12 domains and fragments will be apparent to those of skill in the art. 
     Generally, the class 2, Type V Cas proteins have a single functional RuvC endonuclease domain (See, e.g., Chen et al., “CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity,” Science 360:436-439 (2018)). In some cases, the Cas12 protein is a variant Cas12b protein. (See Strecker et al., Nature Communications, 2019, 10(1): Art. No.: 212). In one embodiment, a variant Cas12 polypeptide has an amino acid sequence that is different by 1, 2, 3, 4, 5 or more amino acids (e.g., has a deletion, insertion, substitution, fusion) when compared to the amino acid sequence of a wild type Cas12 protein. In some instances, the variant Cas12 polypeptide has an amino acid change (e.g., deletion, insertion, or substitution) that reduces the activity of the Cas12 polypeptide. For example, in some instances, the variant Cas12 is a Cas12b polypeptide that has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nickase activity of the corresponding wild-type Cas12b protein. In some cases, the variant Cas12b protein has no substantial nickase activity. 
     In some cases, a variant Cas12b protein has reduced nickase activity. For example, a variant Cas12b protein exhibits less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1%, of the nickase activity of a wild-type Cas12b protein. 
     In some embodiments, the Cas12 protein includes RNA-guided endonucleases from the Cas12a/Cpf1 family that displays activity in mammalian cells. CRISPR from  Prevotella  and  Francisella  1 (CRISPR/Cpf1) is a DNA editing technology analogous to the CRISPR/Cas9 system. Cpf1 is an RNA-guided endonuclease of a class II CRISPR/Cas system. This acquired immune mechanism is found in  Prevotella  and  Francisella  bacteria. Cpf1 genes are associated with the CRISPR locus, coding for an endonuclease that use a guide RNA to find and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the CRISPR/Cas9 system limitations. Unlike Cas9 nucleases, the result of Cpf1-mediated DNA cleavage is a double-strand break with a short 3′ overhang. Cpf1&#39;s staggered cleavage pattern can open up the possibility of directional gene transfer, analogous to traditional restriction enzyme cloning, which can increase the efficiency of gene editing. Like the Cas9 variants and orthologues described above, Cpf1 can also expand the number of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack the NGG PAM sites favored by SpCas9. The Cpf1 locus contains a mixed alpha/beta domain, a RuvC-I followed by a helical region, a RuvC-II and a zinc finger-like domain. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9. Furthermore, Cpf1, unlike Cas9, does not have a HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alpha-helical recognition lobe of Cas9. Cpf1 CRISPR-Cas domain architecture shows that Cpf1 is functionally unique, being classified as Class 2, type V CRISPR system. The Cpf1 loci encode Cast, Cas2, and Cas4 proteins are more similar to types I and III than type II systems. Functional Cpf1 does not require the trans-activating CRISPR RNA (tracrRNA), therefore, only CRISPR (crRNA) is required. This benefits genome editing because Cpf1 is not only smaller than Cas9, but also it has a smaller sgRNA molecule (approximately half as many nucleotides as Cas9). The Cpf1-crRNA complex cleaves target DNA or RNA by identification of a protospacer adjacent motif 5′-YTN-3′ or 5′-TTTN-3′ in contrast to the G-rich PAM targeted by Cas9. After identification of PAM, Cpf1 introduces a sticky-end-like DNA double-stranded break having an overhang of 4 or 5 nucleotides. 
     In some aspects of the present invention, a vector encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence can be used. Cas12 can refer to a polypeptide with at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and/or sequence homology to a wild type exemplary Cas12 polypeptide (e.g., Cas12 from  Bacillus hisashii ). Cas12 can refer to a polypeptide with at most or at most about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and/or sequence homology to a wild type exemplary Cas12 polypeptide (e.g., from  Bacillus hisashii  (BhCas12b),  Bacillus  sp. V3-13 (BvCas12b), and  Alicyclobacillus acidiphilus  (AaCas12b)). Cas12 can refer to the wild type or a modified form of the Cas12 protein that can comprise an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof. 
     Nucleic Acid Programmable DNA Binding Proteins 
     Some aspects of the disclosure provide fusion proteins comprising domains that act as nucleic acid programmable DNA binding proteins, which may be used to guide a protein, such as a base editor, to a specific nucleic acid (e.g., DNA or RNA) sequence. In particular embodiments, a fusion protein comprises a nucleic acid programmable DNA binding protein domain and a deaminase domain. Non-limiting examples of nucleic acid programmable DNA binding proteins include, Cas9 (e.g., dCas9 and nCas9), Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12 h, and Cas12i. Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5 h, CasSa, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12 h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, homologues thereof, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of this disclosure, although they may not be specifically listed in this disclosure. See, e.g., Makarova et al. “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 October; 1:325-336. doi: 10.1089/crispr.2018.0033; Yan et al., “Functionally diverse type V CRISPR-Cas systems” Science. 2019 Jan. 4; 363(6422):88-91. doi: 10.1126/science.aav7271, the entire contents of each are hereby incorporated by reference. 
     One example of a nucleic acid programmable DNA-binding protein that has different PAM specificity than Cas9 is Clustered Regularly Interspaced Short Palindromic Repeats from  Prevotella  and  Francisella  1 (Cpf1). Similar to Cas9, Cpf1 is also a class 2 CRISPR effector. It has been shown that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif (TTN, TTTN, or YTN). Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells. Cpf1 proteins are known in the art and have been described previously, for example Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA.” Cell (165) 2016, p. 949-962; the entire contents of which is hereby incorporated by reference. 
     Useful in the present compositions and methods are nuclease-inactive Cpf1 (dCpf1) variants that may be used as a guide nucleotide sequence-programmable DNA-binding protein domain. The Cpf1 protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9 but does not have a HNH endonuclease domain, and the N-terminal of Cpf1 does not have the alfa-helical recognition lobe of Cas9. It was shown in Zetsche et al., Cell, 163, 759-771, 2015 (which is incorporated herein by reference) that, the RuvC-like domain of Cpf1 is responsible for cleaving both DNA strands and inactivation of the RuvC-like domain inactivates Cpf1 nuclease activity. For example, mutations corresponding to D917A, E1006A, or D1255A in  Francisella novicida  Cpf1 inactivate Cpf1 nuclease activity. In some embodiments, the dCpf1 of the present disclosure comprises mutations corresponding to D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, or D917A/E1006A/D1255A. It is to be understood that any mutations, e.g., substitution mutations, deletions, or insertions that inactivate the RuvC domain of Cpf1, may be used in accordance with the present disclosure. 
     In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein may be a Cpf1 protein. In some embodiments, the Cpf1 protein is a Cpf1 nickase (nCpf1). In some embodiments, the Cpf1 protein is a nuclease inactive Cpf1 (dCpf1). In some embodiments, the Cpf1, the nCpf1, or the dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a Cpf1 sequence disclosed herein. In some embodiments, the dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to a Cpf1 sequence disclosed herein, and comprises mutations corresponding to D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, or D917A/E1006A/D1255A. It should be appreciated that Cpf1 from other bacterial species may also be used in accordance with the present disclosure. 
     Wild-Type  Francisella novicida  Cpf1 (D917, E1006, and D1255 are Bolded and Underlined) 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 107) 
                   
               
               
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFI 
                   
               
               
                   
               
               
                 EEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFN 
               
               
                   
               
               
                 QNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENR 
               
               
                   
               
               
                 KNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDY 
               
               
                   
               
               
                 KTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQ 
               
               
                   
               
               
                 INDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKE 
               
               
                   
               
               
                 TLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPS 
               
               
                   
               
               
                 KKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNK 
               
               
                   
               
               
                 DNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKD 
               
               
                   
               
               
                 EHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI 
               
               
                   
               
               
                 LFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFY 
               
               
                   
               
               
                 NPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDT 
               
               
                   
               
               
                 QRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTL 
               
               
                   
               
               
                 YWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLI 
               
               
                   
               
               
                 KDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI   D   RGERHLAYYTLVD 
               
               
                   
               
               
                 GKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEI 
               
               
                   
               
               
                 AKLVIEYNAIVVF   E   DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRA 
               
               
                   
               
               
                 YQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKIC 
               
               
                   
               
               
                 YNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLL 
               
               
                   
               
               
                 KDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNF 
               
               
                   
               
               
                 FDSRQAPKNMPQDA   D   ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN 
               
               
                   
               
               
                   Francisella novicida  Cpf1 D917A (A917, E1006, and D1255 are  
               
               
                 bolded and underlined) 
               
               
                 (SEQ ID NO: 108) 
                   
               
               
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFI 
                   
               
               
                   
               
               
                 EEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFN 
               
               
                   
               
               
                 QNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENR 
               
               
                   
               
               
                 KNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDY 
               
               
                   
               
               
                 KTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQ 
               
               
                   
               
               
                 INDKTLKKYKMSVLFKQILSDTESKSEVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKE 
               
               
                   
               
               
                 TLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPS 
               
               
                   
               
               
                 KKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNK 
               
               
                   
               
               
                 DNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKD 
               
               
                   
               
               
                 EHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI 
               
               
                   
               
               
                 LFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFY 
               
               
                   
               
               
                 NPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDT 
               
               
                   
               
               
                 QRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTL 
               
               
                   
               
               
                 YWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLI 
               
               
                   
               
               
                 KDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI   A   RGERHLAYYTLVD 
               
               
                   
               
               
                 GKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEI 
               
               
                   
               
               
                 AKLVIEYNAIVVF   E   DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRA 
               
               
                   
               
               
                 YQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKIC 
               
               
                   
               
               
                 YNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLL 
               
               
                   
               
               
                 KDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNF 
               
               
                   
               
               
                 FDSRQAPKNMPQDA   D   ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN 
               
               
                   
               
               
                   Francisella novicida  Cpf1 E1006A (D917, A1006, and D1255 are  
               
               
                 bolded and underlined) 
               
               
                 (SEQ ID NO: 109) 
                   
               
               
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFI 
                   
               
               
                   
               
               
                 EEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFN 
               
               
                   
               
               
                 QNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENR 
               
               
                   
               
               
                 KNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDY 
               
               
                   
               
               
                 KTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQ 
               
               
                   
               
               
                 INDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKE 
               
               
                   
               
               
                 TLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPS 
               
               
                   
               
               
                 KKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNK 
               
               
                   
               
               
                 DNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKD 
               
               
                   
               
               
                 EHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI 
               
               
                   
               
               
                 LFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFY 
               
               
                   
               
               
                 NPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDT 
               
               
                   
               
               
                 QRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTL 
               
               
                   
               
               
                 YWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLI 
               
               
                   
               
               
                 KDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI   D   RGERHLAYYTLVD 
               
               
                   
               
               
                 GKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEI 
               
               
                   
               
               
                 AKLVIEYNAIVVF   A   DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRA 
               
               
                   
               
               
                 YQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKIC 
               
               
                   
               
               
                 YNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLL 
               
               
                   
               
               
                 KDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNF 
               
               
                   
               
               
                 FDSRQAPKNMPQDA   D   ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN 
               
               
                   
               
               
                   Francisella novicida  Cpf1 D1255A (D917, E1006, and A1255 are  
               
               
                 bolded and underlined) 
               
               
                 (SEQ ID NO: 110) 
                   
               
               
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFI 
                   
               
               
                   
               
               
                 EEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFN 
               
               
                   
               
               
                 QNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENR 
               
               
                   
               
               
                 KNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDY 
               
               
                   
               
               
                 KTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQ 
               
               
                   
               
               
                 INDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKE 
               
               
                   
               
               
                 TLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPS 
               
               
                   
               
               
                 KKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNK 
               
               
                   
               
               
                 DNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKD 
               
               
                   
               
               
                 EHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI 
               
               
                   
               
               
                 LFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFY 
               
               
                   
               
               
                 NPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDT 
               
               
                   
               
               
                 QRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTL 
               
               
                   
               
               
                 YWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLI 
               
               
                   
               
               
                 KDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI   D   RGERHLAYYTLVD 
               
               
                   
               
               
                 GKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEI 
               
               
                   
               
               
                 AKLVIEYNAIVVF   E   DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRA 
               
               
                   
               
               
                 YQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKIC 
               
               
                   
               
               
                 YNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLL 
               
               
                   
               
               
                 KDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNF 
               
               
                   
               
               
                 FDSRQAPKNMPQDA   A   ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN 
               
               
                   
               
               
                   Francisella novicida  Cpf1 D917A/E1006A (A917, A1006, and D1255 
               
               
                 are bolded and underlined) 
               
               
                 (SEQ ID NO: 111) 
                   
               
               
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFI 
                   
               
               
                   
               
               
                 EEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFN 
               
               
                   
               
               
                 QNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENR 
               
               
                   
               
               
                 KNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDY 
               
               
                   
               
               
                 KTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQ 
               
               
                   
               
               
                 INDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKE 
               
               
                   
               
               
                 TLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPS 
               
               
                   
               
               
                 KKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNK 
               
               
                   
               
               
                 DNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKD 
               
               
                   
               
               
                 EHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI 
               
               
                   
               
               
                 LFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFY 
               
               
                   
               
               
                 NPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDT 
               
               
                   
               
               
                 QRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTL 
               
               
                   
               
               
                 YWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLI 
               
               
                   
               
               
                 KDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI   A   RGERHLAYYTLVD 
               
               
                   
               
               
                 GKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEI 
               
               
                   
               
               
                 AKLVIEYNAIVVF   A   DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRA 
               
               
                   
               
               
                 YQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKIC 
               
               
                   
               
               
                 YNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLL 
               
               
                   
               
               
                 KDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNF 
               
               
                   
               
               
                 FDSRQAPKNMPQDA   D   ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN 
               
               
                   
               
               
                   Francisella novicida  Cpf1 D917A/D1255A (A917, E1006, and A1255 
               
               
                 are bolded and underlined) 
               
               
                 (SEQ ID NO: 112) 
                   
               
               
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFI 
                   
               
               
                   
               
               
                 EEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFN 
               
               
                   
               
               
                 QNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENR 
               
               
                   
               
               
                 KNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDY 
               
               
                   
               
               
                 KTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQ 
               
               
                   
               
               
                 INDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKE 
               
               
                   
               
               
                 TLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPS 
               
               
                   
               
               
                 KKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNK 
               
               
                   
               
               
                 DNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKD 
               
               
                   
               
               
                 EHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI 
               
               
                   
               
               
                 LFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFY 
               
               
                   
               
               
                 NPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDT 
               
               
                   
               
               
                 QRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTL 
               
               
                   
               
               
                 YWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLI 
               
               
                   
               
               
                 KDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI   A   RGERHLAYYTLVD 
               
               
                   
               
               
                 GKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEI 
               
               
                   
               
               
                 AKLVIEYNAIVVF   E   DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRA 
               
               
                   
               
               
                 YQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKIC 
               
               
                   
               
               
                 YNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLL 
               
               
                   
               
               
                 KDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNF 
               
               
                   
               
               
                 FDSRQAPKNMPQDA   A   ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN 
               
               
                   
               
               
                   Francisella novicida  Cpf1 E1006A/D1255A (D917, A1006, and  
               
               
                 A1255 are bolded and underlined) 
               
               
                 (SEQ ID NO: 113) 
                   
               
               
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFI 
                   
               
               
                   
               
               
                 EEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFN 
               
               
                   
               
               
                 QNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENR 
               
               
                   
               
               
                 KNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDY 
               
               
                   
               
               
                 KTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQ 
               
               
                   
               
               
                 INDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKE 
               
               
                   
               
               
                 TLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPS 
               
               
                   
               
               
                 KKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNK 
               
               
                   
               
               
                 DNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKD 
               
               
                   
               
               
                 EHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI 
               
               
                   
               
               
                 LFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFY 
               
               
                   
               
               
                 NPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDT 
               
               
                   
               
               
                 QRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTL 
               
               
                   
               
               
                 YWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLI 
               
               
                   
               
               
                 KDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI   D   RGERHLAYYTLVD 
               
               
                   
               
               
                 GKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEI 
               
               
                   
               
               
                 AKLVIEYNAIVVF   A   DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRA 
               
               
                   
               
               
                 YQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKIC 
               
               
                   
               
               
                 YNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLL 
               
               
                   
               
               
                 KDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNF 
               
               
                   
               
               
                 FDSRQAPKNMPQDA   A   ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN 
               
               
                   
               
               
                   Francisella novicida  Cpf1 D917A/E1006A/D1255A (A917, A1006, 
               
               
                 and A1255 are bolded and underlined) 
               
               
                 (SEQ ID NO: 114) 
                   
               
               
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFI 
                   
               
               
                   
               
               
                 EEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFN 
               
               
                   
               
               
                 QNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENR 
               
               
                   
               
               
                 KNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDY 
               
               
                   
               
               
                 KTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQ 
               
               
                   
               
               
                 INDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKE 
               
               
                   
               
               
                 TLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPS 
               
               
                   
               
               
                 KKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNK 
               
               
                   
               
               
                 DNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKD 
               
               
                   
               
               
                 EHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI 
               
               
                   
               
               
                 LFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFY 
               
               
                   
               
               
                 NPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDT 
               
               
                   
               
               
                 QRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTL 
               
               
                   
               
               
                 YWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLI 
               
               
                   
               
               
                 KDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSI   A   RGERHLAYYTLVD 
               
               
                   
               
               
                 GKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEI 
               
               
                   
               
               
                 AKLVIEYNAIVVF   A   DLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRA 
               
               
                   
               
               
                 YQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKIC 
               
               
                   
               
               
                 YNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLL 
               
               
                   
               
               
                 KDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNF 
               
               
                   
               
               
                 FDSRQAPKNMPQDA   A   ANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN 
               
            
           
         
       
     
     In some embodiments, one of the Cas9 domains present in the fusion protein may be replaced with a guide nucleotide sequence-programmable DNA-binding protein domain that has no requirements for a PAM sequence. 
     In some embodiments, the Cas9 domain is a Cas9 domain from  Staphylococcus aureus  (SaCas9). In some embodiments, the SaCas9 domain is a nuclease active SaCas9, a nuclease inactive SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, the SaCas9 comprises a N579A mutation, or a corresponding mutation in any of the amino acid sequences provided herein. 
     In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having a NNGRRT or a NNGRRT PAM sequence. In some embodiments, the SaCas9 domain comprises one or more of a E781X, a N967X, and a R1014X mutation, or a corresponding mutation in any of the amino acid sequences provided herein, wherein X is any amino acid. In some embodiments, the SaCas9 domain comprises one or more of a E781K, a N967K, and a R1014H mutation, or one or more corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the SaCas9 domain comprises a E781K, a N967K, or a R1014H mutation, or corresponding mutations in any of the amino acid sequences provided herein. 
     
       
         
           
               
            
               
                 Exemplary SaCas9 sequence 
               
               
                 (SEQ ID NO: 115) 
               
               
                 KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR 
               
               
                   
               
               
                 GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLS 
               
               
                   
               
               
                 EEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVA 
               
               
                   
               
               
                 ELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTY 
               
               
                   
               
               
                 IDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY 
               
               
                   
               
               
                 NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK 
               
               
                   
               
               
                 EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQI 
               
               
                   
               
               
                 AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN 
               
               
                   
               
               
                 LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVK 
               
               
                   
               
               
                 RSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQT 
               
               
                   
               
               
                 NERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF 
               
               
                   
               
               
                 NYEVDHIIPRSVSFDNSFNNKVLVKQEE   N   SKKGNRTPFQYLSSSDSKISY 
               
               
                   
               
               
                 ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRY 
               
               
                   
               
               
                 ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH 
               
               
                   
               
               
                 AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYK 
               
               
                   
               
               
                 EIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLI 
               
               
                   
               
               
                 VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEK 
               
               
                   
               
               
                 NPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSR 
               
               
                   
               
               
                 NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK 
               
               
                   
               
               
                 KLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITY 
               
               
                   
               
               
                 REYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK 
               
               
                   
               
               
                 KG 
               
            
           
         
       
     
     Residue N579 above, which is underlined and in bold, may be mutated (e.g., to a A579) to yield a SaCas9 nickase. 
     
       
         
           
               
            
               
                 Exemplary SaCas9n sequence 
               
               
                 (SEQ ID NO: 116) 
               
               
                 KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR 
               
               
                   
               
               
                 GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLS 
               
               
                   
               
               
                 EEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVA 
               
               
                   
               
               
                 ELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTY 
               
               
                   
               
               
                 IDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY 
               
               
                   
               
               
                 NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK 
               
               
                   
               
               
                 EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQI 
               
               
                   
               
               
                 AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN 
               
               
                   
               
               
                 LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVK 
               
               
                   
               
               
                 RSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQT 
               
               
                   
               
               
                 NERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF 
               
               
                   
               
               
                 NYEVDHIIPRSVSFDNSFNNKVLVKQEE   A   SKKGNRTPFQYLSSSDSKISY 
               
               
                   
               
               
                 ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRY 
               
               
                   
               
               
                 ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH 
               
               
                   
               
               
                 AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYK 
               
               
                   
               
               
                 EIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLI 
               
               
                   
               
               
                 VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEK 
               
               
                   
               
               
                 NPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSR 
               
               
                   
               
               
                 NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK 
               
               
                   
               
               
                 KLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITY 
               
               
                   
               
               
                 REYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK 
               
               
                   
               
               
                 KG 
               
            
           
         
       
     
     Residue A579 above, which can be mutated from N579 to yield a SaCas9 nickase, is underlined and in bold. 
     
       
         
           
               
            
               
                 Exemplary SaKKH Cas9 
               
               
                 (SEQ ID NO: 117) 
               
               
                 KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR 
               
               
                   
               
               
                 GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLS 
               
               
                   
               
               
                 EEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVA 
               
               
                   
               
               
                 ELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTY 
               
               
                   
               
               
                 IDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY 
               
               
                   
               
               
                 NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK 
               
               
                   
               
               
                 EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQI 
               
               
                   
               
               
                 AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN 
               
               
                   
               
               
                 LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVK 
               
               
                   
               
               
                 RSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQT 
               
               
                   
               
               
                 NERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF 
               
               
                   
               
               
                 NYEVDHIIPRSVSFDNSFNNKVLVKQEE   A   SKKGNRTPFQYLSSSDSKISY 
               
               
                   
               
               
                 ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRY 
               
               
                   
               
               
                 ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH 
               
               
                   
               
               
                 AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYK 
               
               
                   
               
               
                 EIFITPHQIKHIKDFKDYKYSHRVDKKPNR   K   LINDTLYSTRKDDKGNTLI 
               
               
                   
               
               
                 VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEK 
               
               
                   
               
               
                 NPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSR 
               
               
                   
               
               
                 NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK 
               
               
                   
               
               
                 KLKKISNQAEFIASFY   K   NDLIKINGELYRVIGVNNDLLNRIEVNMIDITY 
               
               
                   
               
               
                 REYLENMNDKRPP   H   IIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK 
               
               
                   
               
               
                 KG. 
               
            
           
         
       
     
     Residue A579 above, which can be mutated from N579 to yield a SaCas9 nickase, is underlined and in bold. Residues K781, K967, and H1014 above, which can be mutated from E781, N967, and R1014 to yield a SaKKH Cas9 are underlined and in italics. 
     In some embodiments, the napDNAbp is a circular permutant. In the following sequences, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italics sequence denotes a linker sequence, and the underlined sequence denotes a bipartite nuclear localization sequence. 
     
       
         
           
               
            
               
                 CP5 (with MSP“NGC” PID and “D10A” nickase): 
               
               
                 (SEQ ID NO: 38) 
               
               
                 
                   EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG 
                 
               
               
                   
               
               
                 
                   RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD 
                 
               
               
                   
               
               
                 
                   PKKYGGFMQPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN 
                 
               
               
                   
               
               
                 
                   PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKFLQKGNELA 
                 
               
               
                   
               
               
                 
                   LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS 
                 
               
               
                   
               
               
                 
                   KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYF 
                 
               
               
                   
               
               
                 
                   DTTIARKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
                   GGSGGSGGS 
                 
               
               
                   
               
               
                 
                   GGSGGSGGSGGM 
                   DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTD 
                 
               
               
                   
               
               
                 
                   RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE 
                 
               
               
                   
               
               
                 
                   MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLR 
                 
               
               
                   
               
               
                 
                   KKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLV 
                 
               
               
                   
               
               
                 
                   QTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFG 
                 
               
               
                   
               
               
                 
                   NLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL 
                 
               
               
                   
               
               
                 
                   FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALV 
                 
               
               
                   
               
               
                 
                   RQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEEL 
                 
               
               
                   
               
               
                 LVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREK 
               
               
                   
               
               
                 
                   IEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQ 
                 
               
               
                   
               
               
                 
                   SFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAF 
                 
               
               
                   
               
               
                 
                   LSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNA 
                 
               
               
                   
               
               
                 
                   SLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY 
                 
               
               
                   
               
               
                 
                   AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFA 
                 
               
               
                   
               
               
                 
                   NRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQ 
                 
               
               
                   
               
               
                 
                   TVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIK 
                 
               
               
                   
               
               
                 
                   ELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVD 
                 
               
               
                   
               
               
                 
                   HIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNA 
                 
               
               
                   
               
               
                 
                   KLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRM 
                 
               
               
                   
               
               
                 
                   NTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL 
                 
               
               
                   
               
               
                 
                   NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ 
                   EGADKRTADGSE 
                 
               
               
                   
               
               
                   FESPKKKRKV * 
               
            
           
         
       
     
     In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) is a single effector of a microbial CRISPR-Cas system. Single effectors of microbial CRISPR-Cas systems include, without limitation, Cas9, Cpf1, Cas12b/C2c1, and Cas12c/C2c3. Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multisubunit effector complexes, while Class 2 systems have a single protein effector. For example, Cas9 and Cpf1 are Class 2 effectors. In addition to Cas9 and Cpf1, three distinct Class 2 CRISPR-Cas systems (Cas12b/C2c1, and Cas12c/C2c3) have been described by Shmakov et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems”, Mol. Cell, 2015 Nov. 5; 60(3): 385-397, the entire contents of which is hereby incorporated by reference. Effectors of two of the systems, Cas12b/C2c1, and Cas12c/C2c3, contain RuvC-like endonuclease domains related to Cpf1. A third system, contains an effector with two predicated HEPN RNase domains. Production of mature CRISPR RNA is tracrRNA-independent, unlike production of CRISPR RNA by Cas12b/C2c1. Cas12b/C2c1 depends on both CRISPR RNA and tracrRNA for DNA cleavage. 
     The crystal structure of  Alicyclobaccillus acidoterrastris  Cas12b/C2c1 (AacC2c1) has been reported in complex with a chimeric single-molecule guide RNA (sgRNA). See e.g., Liu et al., “C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism”,  Mol. Cell,  2017 Jan. 19; 65(2):310-322, the entire contents of which are hereby incorporated by reference. The crystal structure has also been reported in  Alicyclobacillus acidoterrestris  C2c1 bound to target DNAs as ternary complexes. See e.g., Yang et al., “PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease”,  Cell,  2016 Dec. 15; 167(7):1814-1828, the entire contents of which are hereby incorporated by reference. Catalytically competent conformations of AacC2c1, both with target and non-target DNA strands, have been captured independently positioned within a single RuvC catalytic pocket, with Cas12b/C2c1-mediated cleavage resulting in a staggered seven-nucleotide break of target DNA. Structural comparisons between Cas12b/C2c1 ternary complexes and previously identified Cas9 and Cpf1 counterparts demonstrate the diversity of mechanisms used by CRISPR-Cas9 systems. 
     In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) of any of the fusion proteins provided herein may be a Cas12b/C2c1, or a Cas12c/C2c3 protein. In some embodiments, the napDNAbp is a Cas12b/C2c1 protein. In some embodiments, the napDNAbp is a Cas12c/C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to a naturally-occurring Cas12b/C2c1 or Cas12c/C2c3 protein. In some embodiments, the napDNAbp is a naturally-occurring Cas12b/C2c1 or Cas12c/C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to any one of the napDNAbp sequences provided herein. It should be appreciated that Cas12b/C2c1 or Cas12c/C2c3 from other bacterial species may also be used in accordance with the present disclosure. 
     
       
         
           
               
               
            
               
                 ACas12b/C2c1 ((uniprot.org/uniprot/T0D7A2#2) sp|T0D7A2|C2C1_ALIAG 
                   
               
               
                 CRISPR-associated endonuclease C2c1 OS =  Alicyclobacillus acido - terrestris  (strain ATCC 
               
               
                 49025 / DSM 3922/ CIP106132 /NCIMB 13137/GD3B) GN = c2c1 PE = 1 SV = 1) amino acid 
               
               
                 sequence is as follows: 
               
               
                 (SEQ ID NO: 50) 
                   
               
               
                 MAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECD 
                   
               
               
                   
               
               
                 KTAEECKAELLERLRARQVENGHRGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKF 
               
               
                   
               
               
                 LSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKEKAETRKSADRTADVLRALADFG 
               
               
                   
               
               
                 LKPLMRVYTDSEMSSVEWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGQEYAKLVEQ 
               
               
                   
               
               
                 KNRFEQKNFVGQEHLVHLVNQLQQDMKEASPGLESKEQTAHYVTGRALRGSDKVFEKWGKLA 
               
               
                   
               
               
                 PDAPFDLYDAEIKNVQRRNTRRFGSHDLFAKLAEPEYQALWREDASFLTRYAVYNSILRKLN 
               
               
                   
               
               
                 HAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGERRHAIRFHKLLKVENGVAREV 
               
               
                   
               
               
                 DDVTVPISMSEQLDNLLPRDPNEPIALYFRDYGAEQHFTGEFGGAKIQCRRDQLAHMHRRRG 
               
               
                   
               
               
                 ARDVYLNVSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSE 
               
               
                   
               
               
                 GLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSKGRVPFFFPIKGNDNLVAVHERSQLL 
               
               
                   
               
               
                 KLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPVDAAN 
               
               
                   
               
               
                 HMTPDWREAFENELQKLKSLHGICSDKEWMDAVYESVRRVWRHMGKQVRDWRKDVRSGERPK 
               
               
                   
               
               
                 IRGYAKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKE 
               
               
                   
               
               
                 DRLKKLADRIIMEALGYVYALDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLM 
               
               
                   
               
               
                 QWSHRGVFQELINQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCTQEHNPEPFPW 
               
               
                   
               
               
                 WLNKFVVEHTLDACPLRADDLIPTGEGEIFVSPFSAEEGDFHQIHADLNAAQNLQQRLWSDF 
               
               
                   
               
               
                 DISQIRLRCDWGEVDGELVLIPRLTGKRTADSYSNKVFYTNTGVTYYERERGKKRRKVFAQE 
               
               
                   
               
               
                 KLSEEEAELLVEADEAREKSVVLMRDPSGIINRGNWTRQKEFWSMV NQRIEGYLVKQIRSR 
               
               
                   
               
               
                 VPLQDSACENTGDI. 
               
               
                   
               
               
                 AacCas12b ( Alicyclobacillus   acidiphilus ) - WP_067623834 
               
               
                 (SEQ ID NO: 118) 
                   
               
               
                 MAVKSMKVKLRLDNMPEIRAGLWKLHTEVNAGVRYYTEWLSLLRQENLYRRSPNGDGEQECY 
                   
               
               
                   
               
               
                 KTAEECKAELLERLRARQVENGHCGPAGSDDELLQLARQLYELLVPQAIGAKGDAQQIARKF 
               
               
                   
               
               
                 LSPLADKDAVGGLGIAKAGNKPRWVRMREAGEPGWEEEKAKAEARKSTDRTADVLRALADFG 
               
               
                   
               
               
                 LKPLMRVYTDSDMSSVQWKPLRKGQAVRTWDRDMFQQAIERMMSWESWNQRVGEAYAKLVEQ 
               
               
                   
               
               
                 KSRFEQKNFVGQEHLVQLVNQLQQDMKEASHGLESKEQTAHYLTGRALRGSDKVFEKWEKLD 
               
               
                   
               
               
                 PDAPFDLYDTEIKNVQRRNTRRFGSHDLFAKLAEPKYQALWREDASFLTRYAVYNSIVRKLN 
               
               
                   
               
               
                 HAKMFATFTLPDATAHPIWTRFDKLGGNLHQYTFLFNEFGEGRHAIRFQKLLTVEDGVAKEV 
               
               
                   
               
               
                 DDVTVPISMSAQLDDLLPRDPHELVALYFQDYGAEQHLAGEFGGAKIQYRRDQLNHLHARRG 
               
               
                   
               
               
                 ARDVYLNLSVRVQSQSEARGERRPPYAAVFRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSE 
               
               
                   
               
               
                 GLLSGLRVMSVDLGLRTSASISVFRVARKDELKPNSEGRVPFCFPIEGNENLVAVHERSQLL 
               
               
                   
               
               
                 KLPGETESKDLRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGRRERSWAKLIEQPMDANQ 
               
               
                   
               
               
                 MTPDWREAFEDELQKLKSLYGICGDREWTEAVYESVRRVWRHMGKQVRDWRKDVRSGERPKI 
               
               
                   
               
               
                 RGYQKDVVGGNSIEQIEYLERQYKFLKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAKED 
               
               
                   
               
               
                 RLKKLADRIIMEALGYVYALDDERGKGKWVAKYPPCQLILLEELSEYQFNNDRPPSENNQLM 
               
               
                   
               
               
                 QWSHRGVFQELLNQAQVHDLLVGTMYAAFSSRFDARTGAPGIRCRRVPARCAREQNPEPFPW 
               
               
                   
               
               
                 WLNKFVAEHKLDGCPLRADDLIPTGEGEFFVSPFSAEEGDFHQIHADLNAAQNLQRRLWSDF 
               
               
                   
               
               
                 DISQIRLRCDWGEVDGEPVLIPRTTGKRTADSYGNKVFYTKTGVTYYERERGKKRRKVFAQE 
               
               
                   
               
               
                 ELSEEEAELLVEADEAREKSVVLMRDPSGIINRGDWTRQKEFWSMVNQRIEGYLVKQIRSRV 
               
               
                   
               
               
                 RLQESACENTGDI 
               
               
                   
               
               
                 BhCas12b ( Bacillus   hisashii ) NCBI Reference Sequence: WP_095142515 
               
               
                 (SEQ ID NO: 55) 
                   
               
               
                 MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAI 
                   
               
               
                   
               
               
                 YEHHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVE 
               
               
                   
               
               
                 KKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDP 
               
               
                   
               
               
                 LAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWES 
               
               
                   
               
               
                 WNLKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLR 
               
               
                   
               
               
                 GWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPY 
               
               
                   
               
               
                 LYATFCEIDKKKKDAKQQATETLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKL 
               
               
                   
               
               
                 TVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGT 
               
               
                   
               
               
                 LGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKP 
               
               
                   
               
               
                 KELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIK 
               
               
                   
               
               
                 GTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITERE 
               
               
                   
               
               
                 KRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKS 
               
               
                   
               
               
                 LSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKED 
               
               
                   
               
               
                 RLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSR 
               
               
                   
               
               
                 REIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGR 
               
               
                   
               
               
                 LTLDKIAVLKEGDLYPDKGGEKFISLSKDRKCVTTHADINAAQNLQKRFWTRTHGFYKVYCK 
               
               
                   
               
               
                 AYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIKKGSSKQSSSELVDS 
               
               
                   
               
               
                 DILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQYSISTIE 
               
               
                   
               
               
                 DDSSKQSMKRPAATKKAGQAKKKK 
               
               
                   
               
               
                 BvCas12b V4 (S893R/K846R/E837G changes rel. to wild type) is expressed as 
               
               
                 follows: 5′ mRNA Cap-5′UTR-bhCas12b-STOP sequence-3′UTR - 120polyA tail 
               
               
                 5′UTR (“120polyA tail” disclosed as SEQ ID NO: 425): 
               
               
                 (SEQ ID NO: 119) 
                   
               
               
                 GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC 
                   
               
               
                   
               
               
                 3′ UTR (TriLink standard UTR) 
               
               
                 (SEQ ID NO: 120) 
                   
               
               
                 GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTT 
                   
               
               
                   
               
               
                 CCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGA 
               
               
                   
               
               
                 Nucleic acid sequence of bhCas12b (V4) 
               
               
                 (SEQ ID NO: 121) 
                   
               
               
                 ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGCCACCAGATC 
                   
               
               
                   
               
               
                 CTTCATCCTGAAGATCGAGCCCAACGAGGAAGTGAAGAAAGGCCTCTGGAAAACCCACGAGG 
               
               
                   
               
               
                 TGCTGAACCACGGAATCGCCTACTACATGAATATCCTGAAGCTGATCCGGCAAGAGGCCATC 
               
               
                   
               
               
                 TACGAGCACCACGAGCAGGACCCCAAGAATCCCAAGAAGGTGTCCAAGGCCGAGATCCAGGC 
               
               
                   
               
               
                 CGAGCTGTGGGATTTCGTGCTGAAGATGCAGAAGTGCAACAGCTTCACACACGAGGTGGACA 
               
               
                   
               
               
                 AGGACGAGGTGTTCAACATCCTGAGAGAGCTGTACGAGGAACTGGTGCCCAGCAGCGTGGAA 
               
               
                   
               
               
                 AAGAAGGGCGAAGCCAACCAGCTGAGCAACAAGTTTCTGTACCCTCTGGTGGACCCCAACAG 
               
               
                   
               
               
                 CCAGTCTGGAAAGGGAACAGCCAGCAGCGGCAGAAAGCCCAGATGGTACAACCTGAAGATTG 
               
               
                   
               
               
                 CCGGCGATCCCTCCTGGGAAGAAGAGAAGAAGAAGTGGGAAGAAGATAAGAAAAAGGACCCG 
               
               
                   
               
               
                 CTGGCCAAGATCCTGGGCAAGCTGGCTGAGTACGGACTGATCCCTCTGTTCATCCCCTACAC 
               
               
                   
               
               
                 CGACAGCAACGAGCCCATCGTGAAAGAAATCAAGTGGATGGAAAAGTCCCGGAACCAGAGCG 
               
               
                   
               
               
                 TGCGGCGGCTGGATAAGGACATGTTCATTCAGGCCCTGGAACGGTTCCTGAGCTGGGAGAGC 
               
               
                   
               
               
                 TGGAACCTGAAAGTGAAAGAGGAATACGAGAAGGTCGAGAAAGAGTACAAGACCCTGGAAGA 
               
               
                   
               
               
                 GAGGATCAAAGAGGACATCCAGGCTCTGAAGGCTCTGGAACAGTATGAGAAAGAGCGGCAAG 
               
               
                   
               
               
                 AACAGCTGCTGCGGGACACCCTGAACACCAACGAGTACCGGCTGAGCAAGAGAGGCCTTAGA 
               
               
                   
               
               
                 GGCTGGCGGGAAATCATCCAGAAATGGCTGAAAATGGACGAGAACGAGCCCTCCGAGAAGTA 
               
               
                   
               
               
                 CCTGGAAGTGTTCAAGGACTACCAGCGGAAGCACCCTAGAGAGGCCGGCGATTACAGCGTGT 
               
               
                   
               
               
                 ACGAGTTCCTGTCCAAGAAAGAGAACCACTTCATCTGGCGGAATCACCCTGAGTACCCCTAC 
               
               
                   
               
               
                 CTGTACGCCACCTTCTGCGAGATCGACAAGAAAAAGAAGGACGCCAAGCAGCAGGCCACCTT 
               
               
                   
               
               
                 CACACTGGCCGATCCTATCAATCACCCTCTGTGGGTCCGATTCGAGGAAAGAAGCGGCAGCA 
               
               
                   
               
               
                 ACCTGAACAAGTACAGAATCCTGACCGAGCAGCTGCACACCGAGAAGCTGAAGAAAAAGCTG 
               
               
                   
               
               
                 ACAGTGCAGCTGGACCGGCTGATCTACCCTACAGAATCTGGCGGCTGGGAAGAGAAGGGCAA 
               
               
                   
               
               
                 AGTGGACATTGTGCTGCTGCCCAGCCGGCAGTTCTACAACCAGATCTTCCTGGACATCGAGG 
               
               
                   
               
               
                 AAAAGGGCAAGCACGCCTTCACCTACAAGGATGAGAGCATCAAGTTCCCTCTGAAGGGCACA 
               
               
                   
               
               
                 CTCGGCGGAGCCAGAGTGCAGTTCGACAGAGATCACCTGAGAAGATACCCTCACAAGGTGGA 
               
               
                   
               
               
                 AAGCGGCAACGTGGGCAGAATCTACTTCAACATGACCGTGAACATCGAGCCTACAGAGTCCC 
               
               
                   
               
               
                 CAGTGTCCAAGTCTCTGAAGATCCACCGGGACGACTTCCCCAAGGTGGTCAACTTCAAGCCC 
               
               
                   
               
               
                 AAAGAACTGACCGAGTGGATCAAGGACAGCAAGGGCAAGAAACTGAAGTCCGGCATCGAGTC 
               
               
                   
               
               
                 CCTGGAAATCGGCCTGAGAGTGATGAGCATCGACCTGGGACAGAGACAGGCCGCTGCCGCCT 
               
               
                   
               
               
                 CTATTTTCGAGGTGGTGGATCAGAAGCCCGACATCGAAGGCAAGCTGTTTTTCCCAATCAAG 
               
               
                   
               
               
                 GGCACCGAGCTGTATGCCGTGCACAGAGCCAGCTTCAACATCAAGCTGCCCGGCGAGACACT 
               
               
                   
               
               
                 GGTCAAGAGCAGAGAAGTGCTGCGGAAGGCCAGAGAGGACAATCTGAAACTGATGAACCAGA 
               
               
                   
               
               
                 AGCTCAACTTCCTGCGGAACGTGCTGCACTTCCAGCAGTTCGAGGACATCACCGAGAGAGAG 
               
               
                   
               
               
                 AAGCGGGTCACCAAGTGGATCAGCAGACAAGAGAACAGCGACGTGCCCCTGGTGTACCAGGA 
               
               
                   
               
               
                 TGAGCTGATCCAGATCCGCGAGCTGATGTACAAGCCTTACAAGGACTGGGTCGCCTTCCTGA 
               
               
                   
               
               
                 AGCAGCTCCACAAGAGACTGGAAGTCGAGATCGGCAAAGAAGTGAAGCACTGGCGGAAGTCC 
               
               
                   
               
               
                 CTGAGCGACGGAAGAAAGGGCCTGTACGGCATCTCCCTGAAGAACATCGACGAGATCGATCG 
               
               
                   
               
               
                 GACCCGGAAGTTCCTGCTGAGATGGTCCCTGAGGCCTACCGAACCTGGCGAAGTGCGTAGAC 
               
               
                   
               
               
                 TGGAACCCGGCCAGAGATTCGCCATCGACCAGCTGAATCACCTGAACGCCCTGAAAGAAGAT 
               
               
                   
               
               
                 CGGCTGAAGAAGATGGCCAACACCATCATCATGCACGCCCTGGGCTACTGCTACGACGTGCG 
               
               
                   
               
               
                 GAAGAAGAAATGGCAGGCTAAGAACCCCGCCTGCCAGATCATCCTGTTCGAGGATCTGAGCA 
               
               
                   
               
               
                 ACTACAACCCCTACGAGGAAAGGTCCCGCTTCGAGAACAGCAAGCTCATGAAGTGGTCCAGA 
               
               
                   
               
               
                 CGCGAGATCCCCAGACAGGTTGCACTGCAGGGCGAGATCTATGGCCTGCAAGTGGGAGAAGT 
               
               
                   
               
               
                 GGGCGCTCAGTTCAGCAGCAGATTCCACGCCAAGACAGGCAGCCCTGGCATCAGATGTAGCG 
               
               
                   
               
               
                 TCGTGACCAAAGAGAAGCTGCAGGACAATCGGTTCTTCAAGAATCTGCAGAGAGAGGGCAGA 
               
               
                   
               
               
                 CTGACCCTGGACAAAATCGCCGTGCTGAAAGAGGGCGATCTGTACCCAGACAAAGGCGGCGA 
               
               
                   
               
               
                 GAAGTTCATCAGCCTGAGCAAGGATCGGAAGTGCGTGACCACACACGCCGACATCAACGCCG 
               
               
                   
               
               
                 CTCAGAACCTGCAGAAGCGGTTCTGGACAAGAACCCACGGCTTCTACAAGGTGTACTGCAAG 
               
               
                   
               
               
                 GCCTACCAGGTGGACGGCCAGACCGTGTACATCCCTGAGAGCAAGGACCAGAAGCAGAAGAT 
               
               
                   
               
               
                 CATCGAAGAGTTCGGCGAGGGCTACTTCATTCTGAAGGACGGGGTGTACGAATGGGTCAACG 
               
               
                   
               
               
                 CCGGCAAGCTGAAAATCAAGAAGGGCAGCTCCAAGCAGAGCAGCAGCGAGCTGGTGGATAGC 
               
               
                   
               
               
                 GACATCCTGAAAGACAGCTTCGACCTGGCCTCCGAGCTGAAAGGCGAAAAGCTGATGCTGTA 
               
               
                   
               
               
                 CAGGGACCCCAGCGGCAATGTGTTCCCCAGCGACAAATGGATGGCCGCTGGCGTGTTCTTCG 
               
               
                   
               
               
                 GAAAGCTGGAACGCATCCTGATCAGCAAGCTGACCAACCAGTACTCCATCAGCACCATCGAG 
               
               
                   
               
               
                 GACGACAGCAGCAAGCAGTCTATGAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAA 
               
               
                   
               
               
                 AAAGAAAAAG 
               
               
                   
               
               
                 In some embodiments, the Cas12b is BvCas12B, which is a variant of BhCas12b and 
               
               
                 comprises the following changes relative to BhCas12B: S893R, K846R, and E837G. 
               
               
                 BvCas12b ( Bacillus  sp. V3-13) NCBI Reference Sequence: WP_101661451.1 
               
               
                 (SEQ ID NO: 122) 
                   
               
               
                 MAIRSIKLKMKTNSGTDSIYLRKALWRTHQLINEGIAYYMNLLTLYRQEAIGDKTKEAYQAE 
                   
               
               
                   
               
               
                 LINIIRNQQRNNGSSEEHGSDQEILALLRQLYELIIPSSIGESGDANQLGNKFLYPLVDPNS 
               
               
                   
               
               
                 QSGKGTSNAGRKPRWKRLKEEGNPDWELEKKKDEERKAKDPTVKIFDNLNKYGLLPLFPLFT 
               
               
                   
               
               
                 NIQKDIEWLPLGKRQSVRKWDKDMFIQAIERLLSWESWNRRVADEYKQLKEKTESYYKEHLT 
               
               
                   
               
               
                 GGEEWIEKIRKFEKERNMELEKNAFAPNDGYFITSRQIRGWDRVYEKWSKLPESASPEELWK 
               
               
                   
               
               
                 VVAEQQNKMSEGFGDPKVFSFLANRENRDIWRGHSERIYHIAAYNGLQKKLSRTKEQATFTL 
               
               
                   
               
               
                 PDAIEHPLWIRYESPGGTNLNLFKLEEKQKKNYYVTLSKIIWPSEEKWIEKENIEIPLAPSI 
               
               
                   
               
               
                 QFNRQIKLKQHVKGKQEISFSDYSSRISLDGVLGGSRIQFNRKYIKNHKELLGEGDIGPVFF 
               
               
                   
               
               
                 NLVVDVAPLQETRNGRLQSPIGKALKVISSDFSKVIDYKPKELMDWMNTGSASNSFGVASLL 
               
               
                   
               
               
                 EGMRVMSIDMGQRTSASVSIFEVVKELPKDQEQKLFYSINDTELFAIHKRSFLLNLPGEVVT 
               
               
                   
               
               
                 KNNKQQRQERRKKRQFVRSQIRMLANVLRLETKKTPDERKKAIHKLMEIVQSYDSWTASQKE 
               
               
                   
               
               
                 VWEKELNLLTNMAAFNDEIWKESLVELHHRIEPYVGQIVSKWRKGLSEGRKNLAGISMWNID 
               
               
                   
               
               
                 ELEDTRRLLISWSKRSRTPGEANRIETDEPFGSSLLQHIQNVKDDRLKQMANLIIMTALGFK 
               
               
                   
               
               
                 YDKEEKDRYKRWKETYPACQIILFENLNRYLFNLDRSRRENSRLMKWAHRSIPRTVSMQGEM 
               
               
                   
               
               
                 FGLQVGDVRSEYSSRFHAKTGAPGIRCHALTEEDLKAGSNTLKRLIEDGFINESELAYLKKG 
               
               
                   
               
               
                 DIIPSQGGELFVTLSKRYKKDSDNNELTVIHADINAAQNLQKRFWQQNSEVYRVPCQLARMG 
               
               
                   
               
               
                 EDKLYIPKSQTETIKKYFGKGSFVKNNTEQEVYKWEKSEKMKIKTDTTFDLQDLDGFEDISK 
               
               
                   
               
               
                 TIELAQEQQKKYLTMFRDPSGYFFNNETWRPQKEYWSIVNNIIKSCLKKKILSNKVEL 
               
               
                   
               
               
                 In some embodiments, the Cas12b is BTCas12b.BTCas12b ( Bacillus   
               
               
                   thermoamylovorans ) NCBI Reference Sequence: WP_041902512 
               
               
                 (SEQ ID NO: 123) 
                   
               
               
                 MATRSEILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDPKNPKKV 
                   
               
               
                   
               
               
                 SKAEIQAELWDFVLKMQKCNSFTHEVDKDVVFNILRELYEELVPSSVEKKGEANQLSNKF 
               
               
                   
               
               
                 LYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDPLAKILGKLAE 
               
               
                   
               
               
                 YGLIPLFIPFTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWESWNLKVKEE 
               
               
                   
               
               
                 YEKVEKEHKTLEERIKEDIQAFKSLEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREII 
               
               
                   
               
               
                 QKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPYLYAT 
               
               
                   
               
               
                 FCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKLTV 
               
               
                   
               
               
                 QLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGT 
               
               
                   
               
               
                 LGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKFVNF 
               
               
                   
               
               
                 KPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLF 
               
               
                   
               
               
                 FPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFE 
               
               
                   
               
               
                 DITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGK 
               
               
                   
               
               
                 EVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQ 
               
               
                   
               
               
                 LNHLNALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERS 
               
               
                   
               
               
                 RFENSKLMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKL 
               
               
                   
               
               
                 QDNRFFKNLQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDRKLVTTHADINAAQNLQ 
               
               
                   
               
               
                 KRFWTRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWGNAGK 
               
               
                   
               
               
                 LKIKKGSSKQSSSELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFG 
               
               
                   
               
               
                 KLERILISKLTNQYSISTIEDDSSKQSM 
               
            
           
         
       
     
     In some embodiments, a napDNAbp refers to Cas12c. In some embodiments, the Cas12c protein is a Cas12c1 or a variant of Cas12c1. In some embodiments, the Cas12 protein is a Cas12c2 or a variant of Cas12c2. In some embodiments, the Cas12 protein is a Cas12c protein from Oleiphilus sp. HI0009 (i.e., OspCas12c) or a variant of OspCas12c. These Cas12c molecules have been described in Yan et al., “Functionally Diverse Type V CRISPR-Cas Systems,” Science, 2019 Jan. 4; 363: 88-91; the entire contents of which is hereby incorporated by reference. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring Cas12c1, Cas12c2, or OspCas12c protein. In some embodiments, the napDNAbp is a naturally-occurring Cas12c1, Cas12c2, or OspCas12c protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to any Cas12c1, Cas12c2, or OspCas12c protein described herein. It should be appreciated that Cas12c1, Cas12c2, or OspCas12c from other bacterial species may also be used in accordance with the present disclosure. 
     
       
         
           
               
               
            
               
                 Cas12c1 
                   
               
               
                 (SEQ ID NO: 124) 
                   
               
               
                 MQTKKTHLHLISAKASRKYRRTIACLSDTAKKDLERRKQSGAADPAQELSCLKTIKFKLEVP 
                   
               
               
                   
               
               
                 EGSKLPSFDRISQIYNALETIEKGSLSYLLFALILSGFRIFPNSSAAKTFASSSCYKNDQFA 
               
               
                   
               
               
                 SQIKEIFGEMVKNFIPSELESILKKGRRKNNKDWTEENIKRVLNSEFGRKNSEGSSALFDSF 
               
               
                   
               
               
                 LSKFSQELFRKFDSWNEVNKKYLEAAELLDSMLASYGPFDSVCKMIGDSDSRNSLPDKSTIA 
               
               
                   
               
               
                 FTNNAEITVDIESSVMPYMAIAALLREYRQSKSKAAPVAYVQSHLTTTNGNGLSWFFKFGLD 
               
               
                   
               
               
                 LIRKAPVSSKQSTSDGSKSLQELFSVPDDKLDGLKFIKEACEALPEASLLCGEKGELLGYQD 
               
               
                   
               
               
                 FRTSFAGHIDSWVANYVNRLFELIELVNQLPESIKLPSILTQKNHNLVASLGLQEAEVSHSL 
               
               
                   
               
               
                 ELFEGLVKNVRQTLKKLAGIDISSSPNEQDIKEFYAFSDVLNRLGSIRNQIENAVQTAKKDK 
               
               
                   
               
               
                 IDLESAIEWKEWKKLKKLPKLNGLGGGVPKQQELLDKALESVKQIRHYQRIDFERVIQWAVN 
               
               
                   
               
               
                 EHCLETVPKFLVDAEKKKINKESSTDFAAKENAVRFLLEGIGAAARGKTDSVSKAAYNWFVV 
               
               
                   
               
               
                 NNFLAKKDLNRYFINCQGCIYKPPYSKRRSLAFALRSDNKDTIEVVWEKFETFYKEISKEIE 
               
               
                   
               
               
                 KFNIFSQEFQTFLHLENLRMKLLLRRIQKPIPAEIAFFSLPQEYYDSLPPNVAFLALNQEIT 
               
               
                   
               
               
                 PSEYITQFNLYSSFLNGNLILLRRSRSYLRAKFSWVGNSKLIYAAKEARLWKIPNAYWKSDE 
               
               
                   
               
               
                 WKMILDSNVLVFDKAGNVLPAPTLKKVCEREGDLRLFYPLLRQLPHDWCYRNPFVKSVGREK 
               
               
                   
               
               
                 NVIEVNKEGEPKVASALPGSLFRLIGPAPFKSLLDDCFFNPLDKDLRECMLIVDQEISQKVE 
               
               
                   
               
               
                 AQKVEASLESCTYSIAVPIRYHLEEPKVSNQFENVLAIDQGEAGLAYAVFSLKSIGEAETKP 
               
               
                   
               
               
                 IAVGTIRIPSIRRLIHSVSTYRKKKQRLQNFKQNYDSTAFIMRENVTGDVCAKIVGLMKEFN 
               
               
                   
               
               
                 AFPVLEYDVKNLESGSRQLSAVYKAVNSHFLYFKEPGRDALRKQLWYGGDSWTIDGIEIVTR 
               
               
                   
               
               
                 ERKEDGKEGVEKIVPLKVFPGRSVSARFTSKTCSCCGRNVFDWLFTEKKAKTNKKFNVNSKG 
               
               
                   
               
               
                 ELTTADGVIQLFEADRSKGPKFYARRKERTPLTKPIAKGSYSLEEIERRVRTNLRRAPKSKQ 
               
               
                   
               
               
                 SRDTSQSQYFCVYKDCALHFSGMQADENAAINIGRRFLTALRKNRRSDFPSNVKISDRLLDN 
               
               
                   
               
               
                 Cas12c2 
               
               
                 (SEQ ID NO: 125) 
                   
               
               
                 MTKHSIPLHAFRNSGADARKWKGRIALLAKRGKETMRTLQFPLEMSEPEAAAINTTPFAVAY 
                   
               
               
                   
               
               
                 NAIEGTGKGTLFDYWAKLHLAGFRFFPSGGAATIFRQQAVFEDASWNAAFCQQSGKDWPWLV 
               
               
                   
               
               
                 PSKLYERFTKAPREVAKKDGSKKSIEFTQENVANESHVSLVGASITDKTPEDQKEFFLKMAG 
               
               
                   
               
               
                 ALAEKFDSWKSANEDRIVAMKVIDEFLKSEGLHLPSLENIAVKCSVETKPDNATVAWHDAPM 
               
               
                   
               
               
                 SGVQNLAIGVFATCASRIDNIYDLNGGKLSKLIQESATTPNVTALSWLFGKGLEYFRTTDID 
               
               
                   
               
               
                 TIMQDFNIPASAKESIKPLVESAQAIPTMTVLGKKNYAPFRPNFGGKIDSWIANYASRLMLL 
               
               
                   
               
               
                 NDILEQIEPGFELPQALLDNETLMSGIDMTGDELKELIEAVYAWVDAAKQGLATLLGRGGNV 
               
               
                   
               
               
                 DDAVQTFEQFSAMMDTLNGTLNTISARYVRAVEMAGKDEARLEKLIECKFDIPKWCKSVPKL 
               
               
                   
               
               
                 VGISGGLPKVEEEIKVMNAAFKDVRARMFVRFEEIAAYVASKGAGMDVYDALEKRELEQIKK 
               
               
                   
               
               
                 LKSAVPERAHIQAYRAVLHRIGRAVONCSEKTKQLFSSKVIEMGVFKNPSHLNNFIFNQKGA 
               
               
                   
               
               
                 IYRSPFDRSRHAPYQLHADKLLKNDWLELLAEISATLMASESTEQMEDALRLERTRLQLQLS 
               
               
                   
               
               
                 GLPDWEYPASLAKPDIEVEIQTALKMQLAKDTVTSDVLQRAFNLYSSVLSGLTFKLLRRSFS 
               
               
                   
               
               
                 LKMRFSVADTTQLIYVPKVCDWAIPKQYLQAEGEIGIAARVVTESSPAKMVTEVEMKEPKAL 
               
               
                   
               
               
                 GHFMQQAPHDWYFDASLGGTQVAGRIVEKGKEVGKERKLVGYRMRGNSAYKTVLDKSLVGNT 
               
               
                   
               
               
                 ELSQCSMIIEIPYTQTVDADFRAQVQAGLPKVSINLPVKETITASNKDEQMLFDRFVAIDLG 
               
               
                   
               
               
                 ERGLGYAVFDAKTLELQESGHRPIKAITNLLNRTHHYEQRPNQRQKFQAKFNVNLSELRENT 
               
               
                   
               
               
                 VGDVCHQINRICAYYNAFPVLEYMVPDRLDKQLKSVYESVTNRYIWSSTDAHKSARVQFWLG 
               
               
                   
               
               
                 GETWEHPYLKSAKDKKPLVLSPGRGASGKGTSQTCSCCGRNPFDLIKDMKPRAKIAVVDGKA 
               
               
                   
               
               
                 KLENSELKLFERNLESKDDMLARRHRNERAGMEQPLTPGNYTVDEIKALLRANLRRAPKNRR 
               
               
                   
               
               
                 TKDTTVSEYHCVFSDCGKTMHADENAAVNIGGKFIADIEK 
               
               
                   
               
               
                 OspCas12c 
               
               
                 (SEQ ID NO: 126) 
                   
               
               
                 MTKLRHRQKKLTHDWAGSKKREVLGSNGKLQNPLLMPVKKGQVTEFRKAFSAYARATKGEMT 
                   
               
               
                   
               
               
                 DGRKNMFTHSFEPFKTKPSLHQCELADKAYQSLHSYLPGSLAHFLLSAHALGFRIFSKSGEA 
               
               
                   
               
               
                 TAFQASSKIEAYESKLASELACVDLSIQNLTISTLFNALTTSVRGKGEETSADPLIARFYTL 
               
               
                   
               
               
                 LTGKPLSRDTQGPERDLAEVISRKIASSFGTWKEMTANPLQSLQFFEEELHALDANVSLSPA 
               
               
                   
               
               
                 FDVLIKMNDLQGDLKNRTIVFDPDAPVFEYNAEDPADIIIKLTARYAKEAVIKNQNVGNYVK 
               
               
                   
               
               
                 NAITTTNANGLGWLLNKGLSLLPVSTDDELLEFIGVERSHPSCHALIELIAQLEAPELFEKN 
               
               
                   
               
               
                 VFSDTRSEVQGMIDSAVSNHIARLSSSRNSLSMDSEELERLIKSFQIHTPHCSLFIGAQSLS 
               
               
                   
               
               
                 QQLESLPEALQSGVNSADILLGSTQYMLTNSLVEESIATYQRTLNRINYLSGVAGQINGAIK 
               
               
                   
               
               
                 RKAIDGEKIHLPAAWSELISLPFIGQPVIDVESDLAHLKNQYQTLSNEFDTLISALQKNFDL 
               
               
                   
               
               
                 NFNKALLNRTQHFEAMCRSTKKNALSKPEIVSYRDLLARLTSCLYRGSLVLRRAGIEVLKKH 
               
               
                   
               
               
                 KIFESNSELREHVHERKHFVFVSPLDRKAKKLLRLTDSRPDLLHVIDEILQHDNLENKDRES 
               
               
                   
               
               
                 LWLVRSGYLLAGLPDQLSSSFINLPIITQKGDRRLIDLIQYDQINRDAFVMLVTSAFKSNLS 
               
               
                   
               
               
                 GLQYRANKQSFVVTRTLSPYLGSKLVYVPKDKDWLVPSQMFEGRFADILQSDYMVWKDAGRL 
               
               
                   
               
               
                 CVIDTAKHLSNIKKSVFSSEEVLAFLRELPHRTFIQTEVRGLGVNVDGIAFNNGDIPSLKTF 
               
               
                   
               
               
                 SNCVQVKVSRTNTSLVQTLNRWFEGGKVSPPSIQFERAYYKKDDQIHEDAAKRKIRFQMPAT 
               
               
                   
               
               
                 ELVHASDDAGWTPSYLLGIDPGEYGMGLSLVSINNGEVLDSGFIHINSLINFASKKSNHQTK 
               
               
                   
               
               
                 VVPRQQYKSPYANYLEQSKDSAAGDIAHILDRLIYKLNALPVFEALSGNSQSAADQVWTKVL 
               
               
                   
               
               
                 SFYTWGDNDAQNSIRKQHWFGASHWDIKGMLRQPPTEKKPKPYIAFPGSQVSSYGNSQRCSC 
               
               
                   
               
               
                 CGRNPIEQLREMAKDTSIKELKIRNSEIQLEDGTIKLENPDPSTVIERRRHNLGPSRIPVAD 
               
               
                   
               
               
                 RTFKNISPSSLEFKELITIVSRSIRHSPEFIAKKRGIGSEYFCAYSDCNSSLNSEANAAANV 
               
               
                   
               
               
                 AQKFQKQLFFEL 
               
            
           
         
       
     
     In some embodiments, a napDNAbp refers to Cas12g, Cas12 h, or Cas12i, which have been described in, for example, Yan et al., “Functionally Diverse Type V CRISPR-Cas Systems,” Science, 2019 Jan. 4; 363: 88-91; the entire contents of each is hereby incorporated by reference. By aggregating more than 10 terabytes of sequence data, new classifications of Type V Cas proteins were identified that showed weak similarity to previously characterized Class V protein, including Cas12g, Cas12 h, and Cas12i. In some embodiments, the Cas12 protein is a Cas12g or a variant of Cas12g. In some embodiments, the Cas12 protein is a Cas12 h or a variant of Cas12 h. In some embodiments, the Cas12 protein is a Cas12i or a variant of Cas12i. It should be appreciated that other RNA-guided DNA binding proteins may be used as a napDNAbp, and are within the scope of this disclosure. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring Cas12g, Cas12 h, or Cas12i protein. In some embodiments, the napDNAbp is a naturally-occurring Cas12g, Cas12 h, or Cas12i protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at ease 99.5% identical to any Cas12g, Cas12 h, or Cas12i protein described herein. It should be appreciated that Cas12g, Cas12 h, or Cas12i from other bacterial species may also be used in accordance with the present disclosure. In some embodiments, the Cas12i is a Cas12i1 or a Cas12i2. 
     
       
         
           
               
               
            
               
                 Cas12g1 
                   
               
               
                 (SEQ ID NO: 127) 
                   
               
               
                 MAQASSTPAVSPRPRPRYREERTLVRKLLPRPGQSKQEFRENVKKLRKAFLQFNADVSGVCQ 
                   
               
               
                   
               
               
                 WAIQFRPRYGKPAEPTETFWKFFLEPETSLPPNDSRSPEFRRLQAFEAAAGINGAAALDDPA 
               
               
                   
               
               
                 FTNELRDSILAVASRPKTKEAQRLFSRLKDYQPAHRMILAKVAAEWIESRYRRAHQNWERNY 
               
               
                   
               
               
                 EEWKKEKQEWEQNHPELTPEIREAFNQIFQQLEVKEKRVRICPAARLLQNKDNCQYAGKNKH 
               
               
                   
               
               
                 SVLCNQFNEFKKNHLQGKAIKFFYKDAEKYLRCGLQSLKPNVQGPFREDWNKYLRYMNLKEE 
               
               
                   
               
               
                 TLRGKNGGRLPHCKNLGQECEFNPHTALCKQYQQQLSSRPDLVQHDELYRKWRREYWREPRK 
               
               
                   
               
               
                 PVFRYPSVKRHSIAKIFGENYFQADFKNSVVGLRLDSMPAGQYLEFAFAPWPRNYRPQPGET 
               
               
                   
               
               
                 EISSVHLHFVGTRPRIGFRFRVPHKRSRFDCTQEELDELRSRTFPRKAQDQKFLEAARKRLL 
               
               
                   
               
               
                 ETFPGNAEQELRLLAVDLGTDSARAAFFIGKTFQQAFPLKIVKIEKLYEQWPNQKQAGDRRD 
               
               
                   
               
               
                 ASSKQPRPGLSRDHVGRHLQKMRAQASEIAQKRQELTGTPAPETTTDQAAKKATLQPFDLRG 
               
               
                   
               
               
                 LTVHTARMIRDWARLNARQIIQLAEENQVDLIVLESLRGFRPPGYENLDQEKKRRVAFFAHG 
               
               
                   
               
               
                 RIRRKVTEKAVERGMRVVTVPYLASSKVCAECRKKQKDNKQWEKNKKRGLFKCEGCGSQAQV 
               
               
                   
               
               
                 DENAARVLGRVFWGEIELPTAIP 
               
               
                   
               
               
                 Cas12h1 
               
               
                 (SEQ ID NO: 128) 
                   
               
               
                 MKVHEIPRSQLLKIKQYEGSFVEWYRDLQEDRKKFASLLFRWAAFGYAAREDDGATYISPSQ 
                   
               
               
                   
               
               
                 ALLERRLLLGDAEDVAIKFLDVLFKGGAPSSSCYSLFYEDFALRDKAKYSGAKREFIEGLAT 
               
               
                   
               
               
                 MPLDKIIERIRQDEQLSKIPAEEWLILGAEYSPEEIWEQVAPRIVNVDRSLGKQLRERLGIK 
               
               
                   
               
               
                 CRRPHDAGYCKILMEVVARQLRSHNETYHEYLNQTHEMKTKVANNLTNEFDLVCEFAEVLEE 
               
               
                   
               
               
                 KNYGLGWYVLWQGVKQALKEQKKPTKIQIAVDQLRQPKFAGLLTAKWRALKGAYDTWKLKKR 
               
               
                   
               
               
                 LEKRKAFPYMPNWDNDYQIPVGLTGLGVFTLEVKRTEVVVDLKEHGKLFCSHSHYFGDLTAE 
               
               
                   
               
               
                 KHPSRYHLKFRHKLKLRKRDSRVEPTIGPWIEAALREITIQKKPNGVFYLGLPYALSHGIDN 
               
               
                   
               
               
                 FQIAKRFFSAAKPDKEVINGLPSEMVVGAADLNLSNIVAPVKARIGKGLEGPLHALDYGYGE 
               
               
                   
               
               
                 LIDGPKILTPDGPRCGELISLKRDIVEIKSAIKEFKACQREGLTMSEETTTWLSEVESPSDS 
               
               
                   
               
               
                 PRCMIQSRIADTSRRLNSFKYQMNKEGYQDLAEALRLLDAMDSYNSLLESYQRMHLSPGEQS 
               
               
                   
               
               
                 PKEAKFDTKRASFRDLLRRRVAHTIVEYFDDCDIVFFEDLDGPSDSDSRNNALVKLLSPRTL 
               
               
                   
               
               
                 LLYIRQALEKRGIGMVEVAKDGTSQNNPISGHVGWRNKQNKSEIYFYEDKELLVMDADEVGA 
               
               
                   
               
               
                 MNILCRGLNHSVCPYSFVTKAPEKKNDEKKEGDYGKRVKRFLKDRYGSSNVRFLVASMGFVT 
               
               
                   
               
               
                 VTTKRPKDALVGKRLYYHGGELVTHDLHNRMKDEIKYLVEKEVLARRVSLSDSTIKSYKSFA 
               
               
                   
               
               
                 HV 
               
               
                   
               
               
                 Cas12i1 
               
               
                 (SEQ ID NO: 129) 
                   
               
               
                 MSNKEKNASETRKAYTTKMIPRSHDRMKLLGNFMDYLMDGTPIFFELWNQFGGGIDRDIISG 
                   
               
               
                   
               
               
                 TANKDKISDDLLLAVNWFKVMPINSKPQGVSPSNLANLFQQYSGSEPDIQAQEYFASNFDTE 
               
               
                   
               
               
                 KHQWKDMRVEYERLLAELQLSRSDMHHDLKLMYKEKCIGLSLSTAHYITSVMFGTGAKNNRQ 
               
               
                   
               
               
                 TKHQFYSKVIQLLEESTQINSVEQLASIILKAGDCDSYRKLRIRCSRKGATPSILKIVQDYE 
               
               
                   
               
               
                 LGTNHDDEVNVPSLIANLKEKLGRFEYECEWKCMEKIKAFLASKVGPYYLGSYSAMLENALS 
               
               
                   
               
               
                 PIKGMTTKNCKFVLKQIDAKNDIKYENEPFGKIVEGFFDSPYFESDTNVKWVLHPHHIGESN 
               
               
                   
               
               
                 IKTLWEDLNAIHSKYEEDIASLSEDKKEKRIKVYQGDVCQTINTYCEEVGKEAKTPLVQLLR 
               
               
                   
               
               
                 YLYSRKDDIAVDKIIDGITFLSKKHKVEKQKINPVIQKYPSFNFGNNSKLLGKIISPKDKLK 
               
               
                   
               
               
                 HNLKCNRNQVDNYIWIEIKVLNTKTMRWEKHHYALSSTRFLEEVYYPATSENPPDALAARFR 
               
               
                   
               
               
                 TKTNGYEGKPALSAEQIEQIRSAPVGLRKVKKRQMRLEAARQQNLLPRYTWGKDFNINICKR 
               
               
                   
               
               
                 GNNFEVTLATKVKKKKEKNYKVVLGYDANIVRKNTYAAIEAHANGDGVIDYNDLPVKPIESG 
               
               
                   
               
               
                 FVTVESQVRDKSYDQLSYNGVKLLYCKPHVESRRSFLEKYRNGTMKDNRGNNIQIDFMKDFE 
               
               
                   
               
               
                 AIADDETSLYYFNMKYCKLLQSSIRNHSSQAKEYREEIFELLRDGKLSVLKLSSLSNLSFVM 
               
               
                   
               
               
                 FKVAKSLIGTYFGHLLKKPKNSKSDVKAPPITDEDKQKADPEMFALRLALEEKRLNKVKSKK 
               
               
                   
               
               
                 EVIANKIVAKALELRDKYGPVLIKGENISDTTKKGKKSSTNSFLMDWLARGVANKVKEMVMM 
               
               
                   
               
               
                 HQGLEFVEVNPNFTSHQDPFVHKNPENTFRARYSRCTPSELTEKNRKEILSFLSDKPSKRPT 
               
               
                   
               
               
                 NAYYNEGAMAFLATYGLKKNDVLGVSLEKFKQIMANILHQRSEDQLLFPSRGGMFYLATYKL 
               
               
                   
               
               
                 DADATSVNWNGKQFWVCNADLVAAYNVGLVDIQKDFKKK 
               
               
                   
               
               
                 Cas12i2 
               
               
                 (SEQ ID NO: 130) 
                   
               
               
                 MSSAIKSYKSVLRPNERKNQLLKSTIQCLEDGSAFFFKMLQGLFGGITPEIVRFSTEQEKQQ 
                   
               
               
                   
               
               
                 QDIALWCAVNWFRPVSQDSLTHTIASDNLVEKFEEYYGGTASDAIKQYFSASIGESYYWNDC 
               
               
                   
               
               
                 RQQYYDLCRELGVEVSDLTHDLEILCREKCLAVATESNQNNSIISVLFGTGEKEDRSVKLRI 
               
               
                   
               
               
                 TKKILEAISNLKEIPKNVAPIQEIILNVAKATKETFRQVYAGNLGAPSTLEKFIAKDGQKEF 
               
               
                   
               
               
                 DLKKLQTDLKKVIRGKSKERDWCCQEELRSYVEQNTIQYDLWAWGEMFNKAHTALKIKSTRN 
               
               
                   
               
               
                 YNFAKQRLEQFKEIQSLNNLLVVKKLNDFFDSEFFSGEETYTICVHHLGGKDLSKLYKAWED 
               
               
                   
               
               
                 DPADPENAIVVLCDDLKNNFKKEPIRNILRYIFTIRQECSAQDILAAAKYNQQLDRYKSQKA 
               
               
                   
               
               
                 NPSVLGNQGFTWTNAVILPEKAQRNDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQ 
               
               
                   
               
               
                 EIYAAGNSPVDTCQFRTPRFGYHLPKLTDQTAIRVNKKHVKAAKTEARIRLAIQQGTLPVSN 
               
               
                   
               
               
                 LKITEISATINSKGQVRIPVKFDVGRQKGTLQIGDRFCGYDQNQTASHAYSLWEVVKEGQYH 
               
               
                   
               
               
                 KELGCFVRFISSGDIVSITENRGNQFDQLSYEGLAYPQYADWRKKASKFVSLWQITKKNKKK 
               
               
                   
               
               
                 EIVTVEAKEKFDAICKYQPRLYKFNKEYAYLLRDIVRGKSLVELQQIRQEIFRFIEQDCGVT 
               
               
                   
               
               
                 RLGSLSLSTLETVKAVKGIIYSYFSTALNASKNNPISDEQRKEFDPELFALLEKLELIRTRK 
               
               
                   
               
               
                 KKQKVERIANSLIQTCLENNIKFIRGEGDLSTTNNATKKKANSRSMDWLARGVFNKIRQLAP 
               
               
                   
               
               
                 MHNITLFGCGSLYTSHQDPLVHRNPDKAMKCRWAAIPVKDIGDWVLRKLSQNLRAKNIGTGE 
               
               
                   
               
               
                 YYHQGVKEFLSHYELQDLEEELLKWRSDRKSNIPCWVLQNRLAEKLGNKEAVVYIPVRGGRI 
               
               
                   
               
               
                 YFATHKVATGAVSIVFDQKQVWVCNADHVAAANIALTVKGIGEQSSDEENPDGSRIKLQLTS 
               
            
           
         
       
     
     Representative nucleic acid and protein sequences of the base editors follow: 
     
       
         
           
               
               
            
               
                 BhCas12b GGSGGS-ABE8-Xten20 at P153 
                   
               
               
                 (SEQ ID NO: 131) 
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 CAGATCCTTCATCCTGAAGATCGAGCCCAACGAGGAAGTGAAGAAAGGCCTCTGGAAAACCC 
               
               
                   
               
               
                 ACGAGGTGCTGAACCACGGAATCGCCTACTACATGAATATCCTGAAGCTGATCCGGCAAGAG 
               
               
                   
               
               
                 GCCATCTACGAGCACCACGAGCAGGACCCCAAGAATCCCAAGAAGGTGTCCAAGGCCGAGAT 
               
               
                   
               
               
                 CCAGGCCGAGCTGTGGGATTTCGTGCTGAAGATGCAGAAGTGCAACAGCTTCACACACGAGG 
               
               
                   
               
               
                 TGGACAAGGACGAGGTGTTCAACATCCTGAGAGAGCTGTACGAGGAACTGGTGCCCAGCAGC 
               
               
                   
               
               
                 GTGGAAAAGAAGGGCGAAGCCAACCAGCTGAGCAACAAGTTTCTGTACCCTCTGGTGGACCC 
               
               
                   
               
               
                 CAACAGCCAGTCTGGAAAGGGAACAGCCAGCAGCGGCAGAAAGCCCAGATGGTACAACCTGA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                   GCACAAGCGAGAGCGCCACCCCTGAGAGCTCTGGC TCCTGGGAAGAAGAGAAGAAGAAGTGG 
               
               
                   
               
               
                 GAAGAAGATAAGAAAAAGGACCCGCTGGCCAAGATCCTGGGCAAGCTGGCTGAGTACGGACT 
               
               
                   
               
               
                 GATCCCTCTGTTCATCCCCTACACCGACAGCAACGAGCCCATCGTGAAAGAAATCAAGTGGA 
               
               
                   
               
               
                 TGGAAAAGTCCCGGAACCAGAGCGTGCGGCGGCTGGATAAGGACATGTTCATTCAGGCCCTG 
               
               
                   
               
               
                 GAACGGTTCCTGAGCTGGGAGAGCTGGAACCTGAAAGTGAAAGAGGAATACGAGAAGGTCGA 
               
               
                   
               
               
                 GAAAGAGTACAAGACCCTGGAAGAGAGGATCAAAGAGGACATCCAGGCTCTGAAGGCTCTGG 
               
               
                   
               
               
                 AACAGTATGAGAAAGAGCGGCAAGAACAGCTGCTGCGGGACACCCTGAACACCAACGAGTAC 
               
               
                   
               
               
                 CGGCTGAGCAAGAGAGGCCTTAGAGGCTGGCGGGAAATCATCCAGAAATGGCTGAAAATGGA 
               
               
                   
               
               
                 CGAGAACGAGCCCTCCGAGAAGTACCTGGAAGTGTTCAAGGACTACCAGCGGAAGCACCCTA 
               
               
                   
               
               
                 GAGAGGCCGGCGATTACAGCGTGTACGAGTTCCTGTCCAAGAAAGAGAACCACTTCATCTGG 
               
               
                   
               
               
                 CGGAATCACCCTGAGTACCCCTACCTGTACGCCACCTTCTGCGAGATCGACAAGAAAAAGAA 
               
               
                   
               
               
                 GGACGCCAAGCAGCAGGCCACCTTCACACTGGCCGATCCTATCAATCACCCTCTGTGGGTCC 
               
               
                   
               
               
                 GATTCGAGGAAAGAAGCGGCAGCAACCTGAACAAGTACAGAATCCTGACCGAGCAGCTGCAC 
               
               
                   
               
               
                 ACCGAGAAGCTGAAGAAAAAGCTGACAGTGCAGCTGGACCGGCTGATCTACCCTACAGAATC 
               
               
                   
               
               
                 TGGCGGCTGGGAAGAGAAGGGCAAAGTGGACATTGTGCTGCTGCCCAGCCGGCAGTTCTACA 
               
               
                   
               
               
                 ACCAGATCTTCCTGGACATCGAGGAAAAGGGCAAGCACGCCTTCACCTACAAGGATGAGAGC 
               
               
                   
               
               
                 ATCAAGTTCCCTCTGAAGGGCACACTCGGCGGAGCCAGAGTGCAGTTCGACAGAGATCACCT 
               
               
                   
               
               
                 GAGAAGATACCCTCACAAGGTGGAAAGCGGCAACGTGGGCAGAATCTACTTCAACATGACCG 
               
               
                   
               
               
                 TGAACATCGAGCCTACAGAGTCCCCAGTGTCCAAGTCTCTGAAGATCCACCGGGACGACTTC 
               
               
                   
               
               
                 CCCAAGGTGGTCAACTTCAAGCCCAAAGAACTGACCGAGTGGATCAAGGACAGCAAGGGCAA 
               
               
                   
               
               
                 GAAACTGAAGTCCGGCATCGAGTCCCTGGAAATCGGCCTGAGAGTGATGAGCATCGACCTGG 
               
               
                   
               
               
                 GACAGAGACAGGCCGCTGCCGCCTCTATTTTCGAGGTGGTGGATCAGAAGCCCGACATCGAA 
               
               
                   
               
               
                 GGCAAGCTGTTTTTCCCAATCAAGGGCACCGAGCTGTATGCCGTGCACAGAGCCAGCTTCAA 
               
               
                   
               
               
                 CATCAAGCTGCCCGGCGAGACACTGGTCAAGAGCAGAGAAGTGCTGCGGAAGGCCAGAGAGG 
               
               
                   
               
               
                 ACAATCTGAAACTGATGAACCAGAAGCTCAACTTCCTGCGGAACGTGCTGCACTTCCAGCAG 
               
               
                   
               
               
                 TTCGAGGACATCACCGAGAGAGAGAAGCGGGTCACCAAGTGGATCAGCAGACAAGAGAACAG 
               
               
                   
               
               
                 CGACGTGCCCCTGGTGTACCAGGATGAGCTGATCCAGATCCGCGAGCTGATGTACAAGCCTT 
               
               
                   
               
               
                 ACAAGGACTGGGTCGCCTTCCTGAAGCAGCTCCACAAGAGACTGGAAGTCGAGATCGGCAAA 
               
               
                   
               
               
                 GAAGTGAAGCACTGGCGGAAGTCCCTGAGCGACGGAAGAAAGGGCCTGTACGGCATCTCCCT 
               
               
                   
               
               
                 GAAGAACATCGACGAGATCGATCGGACCCGGAAGTTCCTGCTGAGATGGTCCCTGAGGCCTA 
               
               
                   
               
               
                 CCGAACCTGGCGAAGTGCGTAGACTGGAACCCGGCCAGAGATTCGCCATCGACCAGCTGAAT 
               
               
                   
               
               
                 CACCTGAACGCCCTGAAAGAAGATCGGCTGAAGAAGATGGCCAACACCATCATCATGCACGC 
               
               
                   
               
               
                 CCTGGGCTACTGCTACGACGTGCGGAAGAAGAAATGGCAGGCTAAGAACCCCGCCTGCCAGA 
               
               
                   
               
               
                 TCATCCTGTTCGAGGATCTGAGCAACTACAACCCCTACGAGGAAAGGTCCCGCTTCGAGAAC 
               
               
                   
               
               
                 AGCAAGCTCATGAAGTGGTCCAGACGCGAGATCCCCAGACAGGTTGCACTGCAGGGCGAGAT 
               
               
                   
               
               
                 CTATGGCCTGCAAGTGGGAGAAGTGGGCGCTCAGTTCAGCAGCAGATTCCACGCCAAGACAG 
               
               
                   
               
               
                 GCAGCCCTGGCATCAGATGTAGCGTCGTGACCAAAGAGAAGCTGCAGGACAATCGGTTCTTC 
               
               
                   
               
               
                 AAGAATCTGCAGAGAGAGGGCAGACTGACCCTGGACAAAATCGCCGTGCTGAAAGAGGGCGA 
               
               
                   
               
               
                 TCTGTACCCAGACAAAGGCGGCGAGAAGTTCATCAGCCTGAGCAAGGATCGGAAGTGCGTGA 
               
               
                   
               
               
                 CCACACACGCCGACATCAACGCCGCTCAGAACCTGCAGAAGCGGTTCTGGACAAGAACCCAC 
               
               
                   
               
               
                 GGCTTCTACAAGGTGTACTGCAAGGCCTACCAGGTGGAC GGCCAGACCGTGTACATCC CT 
               
               
                   
               
               
                 GAGAGCAAGGACCAGAAGCAGAAGATCATCGAAGAGTTCGGCGAGGGCTACTTCATTCTGAA 
               
               
                   
               
               
                 GGACGGGGTGTACGAATGGGTCAACGCCGGCAAGCTGAAAATCAAGAAGGGCAGCTCCAAGC 
               
               
                   
               
               
                 AGAGCAGCAGCGAGCTGGTGGATAGCGACATCCTGAAAGACAGCTTCGACCTGGCCTCCGAG 
               
               
                   
               
               
                 CTGAAAGGCGAAAAGCTGATGCTGTACAGGGACCCCAGCGGCAATGTGTTCCCCAGCGACAA 
               
               
                   
               
               
                 ATGGATGGCCGCTGGCGTGTTCTTCGGAAAGCTGGAACGCATCCTGATCAGCAAGCTGACCA 
               
               
                   
               
               
                 ACCAGTACTCCATCAGCACCATCGAGGACGACAGCAGCAAGCAGTCTATG AAAAGGCCGGCG   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                   TTACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCC T 
               
               
                   
               
               
                 AA  
               
               
                 (SEQ ID NO: 132) 
                   
               
               
                 MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAI 
                   
               
               
                   
               
               
                 YEHHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVE 
               
               
                   
               
               
                 KKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPGGSGGSSEVEFSHEYWM 
               
               
                   
               
               
                 RHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYR 
               
               
                   
               
               
                 LYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGI 
               
               
                   
               
               
                 LADECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSGSWEEEKKKWEE 
               
               
                   
               
               
                 DKKKDPLAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALER 
               
               
                   
               
               
                 FLSWESWNLKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRL 
               
               
                   
               
               
                 SKRGLRGWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRN 
               
               
                   
               
               
                 HPEYPYLYATFCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTE 
               
               
                   
               
               
                 KLKKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIK 
               
               
                   
               
               
                 FPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPK 
               
               
                   
               
               
                 VVNFKPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGK 
               
               
                   
               
               
                 LFFPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFE 
               
               
                   
               
               
                 DITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEV 
               
               
                   
               
               
                 KHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHL 
               
               
                   
               
               
                 NALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSK 
               
               
                   
               
               
                 LMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKN 
               
               
                   
               
               
                 LQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDRKCVTTHADINAAQNLQKRFWTRTHGF 
               
               
                   
               
               
                 YKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIKKGSSKQSS 
               
               
                   
               
               
                 SELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQY 
               
               
                   
               
               
                 SISTIEDDSSKQSMKRPAATKKAGQAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA 
               
               
                   
               
               
                 BhCas12b GGSGGS-ABE8-Xten20 at K255 
               
               
                 (SEQ ID NO: 133) 
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 CAGATCCTTCATCCTGAAGATCGAGCCCAACGAGGAAGTGAAGAAAGGCCTCTGGAAAACCC 
               
               
                   
               
               
                 ACGAGGTGCTGAACCACGGAATCGCCTACTACATGAATATCCTGAAGCTGATCCGGCAAGAG 
               
               
                   
               
               
                 GCCATCTACGAGCACCACGAGCAGGACCCCAAGAATCCCAAGAAGGTGTCCAAGGCCGAGAT 
               
               
                   
               
               
                 CCAGGCCGAGCTGTGGGATTTCGTGCTGAAGATGCAGAAGTGCAACAGCTTCACACACGAGG 
               
               
                   
               
               
                 TGGACAAGGACGAGGTGTTCAACATCCTGAGAGAGCTGTACGAGGAACTGGTGCCCAGCAGC 
               
               
                   
               
               
                 GTGGAAAAGAAGGGCGAAGCCAACCAGCTGAGCAACAAGTTTCTGTACCCTCTGGTGGACCC 
               
               
                   
               
               
                 CAACAGCCAGTCTGGAAAGGGAACAGCCAGCAGCGGCAGAAAGCCCAGATGGTACAACCTGA 
               
               
                   
               
               
                 AGATTGCCGGCGATCCCTCCTGGGAAGAAGAGAAGAAGAAGTGGGAAGAAGATAAGAAAAAG 
               
               
                   
               
               
                 GACCCGCTGGCCAAGATCCTGGGCAAGCTGGCTGAGTACGGACTGATCCCTCTGTTCATCCC 
               
               
                   
               
               
                 CTACACCGACAGCAACGAGCCCATCGTGAAAGAAATCAAGTGGATGGAAAAGTCCCGGAACC 
               
               
                   
               
               
                 AGAGCGTGCGGCGGCTGGATAAGGACATGTTCATTCAGGCCCTGGAACGGTTCCTGAGCTGG 
               
               
                   
               
               
                 GAGAGCTGGAACCTGAAAGTGAAAGAGGAATACGAGAAGGTCGAGAAAGAGTACAAGACCCT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                   CACAAGCGAGAGCGCCACCCCTGAGAGCTCTGGC GAGGACATCCAGGCTCTGAAGGCTCTGG 
               
               
                   
               
               
                 AACAGTATGAGAAAGAGCGGCAAGAACAGCTGCTGCGGGACACCCTGAACACCAACGAGTAC 
               
               
                   
               
               
                 CGGCTGAGCAAGAGAGGCCTTAGAGGCTGGCGGGAAATCATCCAGAAATGGCTGAAAATGGA 
               
               
                   
               
               
                 CGAGAACGAGCCCTCCGAGAAGTACCTGGAAGTGTTCAAGGACTACCAGCGGAAGCACCCTA 
               
               
                   
               
               
                 GAGAGGCCGGCGATTACAGCGTGTACGAGTTCCTGTCCAAGAAAGAGAACCACTTCATCTGG 
               
               
                   
               
               
                 CGGAATCACCCTGAGTACCCCTACCTGTACGCCACCTTCTGCGAGATCGACAAGAAAAAGAA 
               
               
                   
               
               
                 GGACGCCAAGCAGCAGGCCACCTTCACACTGGCCGATCCTATCAATCACCCTCTGTGGGTCC 
               
               
                   
               
               
                 GATTCGAGGAAAGAAGCGGCAGCAACCTGAACAAGTACAGAATCCTGACCGAGCAGCTGCAC 
               
               
                   
               
               
                 ACCGAGAAGCTGAAGAAAAAGCTGACAGTGCAGCTGGACCGGCTGATCTACCCTACAGAATC 
               
               
                   
               
               
                 TGGCGGCTGGGAAGAGAAGGGCAAAGTGGACATTGTGCTGCTGCCCAGCCGGCAGTTCTACA 
               
               
                   
               
               
                 ACCAGATCTTCCTGGACATCGAGGAAAAGGGCAAGCACGCCTTCACCTACAAGGATGAGAGC 
               
               
                   
               
               
                 ATCAAGTTCCCTCTGAAGGGCACACTCGGCGGAGCCAGAGTGCAGTTCGACAGAGATCACCT 
               
               
                   
               
               
                 GAGAAGATACCCTCACAAGGTGGAAAGCGGCAACGTGGGCAGAATCTACTTCAACATGACCG 
               
               
                   
               
               
                 TGAACATCGAGCCTACAGAGTCCCCAGTGTCCAAGTCTCTGAAGATCCACCGGGACGACTTC 
               
               
                   
               
               
                 CCCAAGGTGGTCAACTTCAAGCCCAAAGAACTGACCGAGTGGATCAAGGACAGCAAGGGCAA 
               
               
                   
               
               
                 GAAACTGAAGTCCGGCATCGAGTCCCTGGAAATCGGCCTGAGAGTGATGAGCATCGACCTGG 
               
               
                   
               
               
                 GACAGAGACAGGCCGCTGCCGCCTCTATTTTCGAGGTGGTGGATCAGAAGCCCGACATCGAA 
               
               
                   
               
               
                 GGCAAGCTGTTTTTCCCAATCAAGGGCACCGAGCTGTATGCCGTGCACAGAGCCAGCTTCAA 
               
               
                   
               
               
                 CATCAAGCTGCCCGGCGAGACACTGGTCAAGAGCAGAGAAGTGCTGCGGAAGGCCAGAGAGG 
               
               
                   
               
               
                 ACAATCTGAAACTGATGAACCAGAAGCTCAACTTCCTGCGGAACGTGCTGCACTTCCAGCAG 
               
               
                   
               
               
                 TTCGAGGACATCACCGAGAGAGAGAAGCGGGTCACCAAGTGGATCAGCAGACAAGAGAACAG 
               
               
                   
               
               
                 CGACGTGCCCCTGGTGTACCAGGATGAGCTGATCCAGATCCGCGAGCTGATGTACAAGCCTT 
               
               
                   
               
               
                 ACAAGGACTGGGTCGCCTTCCTGAAGCAGCTCCACAAGAGACTGGAAGTCGAGATCGGCAAA 
               
               
                   
               
               
                 GAAGTGAAGCACTGGCGGAAGTCCCTGAGCGACGGAAGAAAGGGCCTGTACGGCATCTCCCT 
               
               
                   
               
               
                 GAAGAACATCGACGAGATCGATCGGACCCGGAAGTTCCTGCTGAGATGGTCCCTGAGGCCTA 
               
               
                   
               
               
                 CCGAACCTGGCGAAGTGCGTAGACTGGAACCCGGCCAGAGATTCGCCATCGACCAGCTGAAT 
               
               
                   
               
               
                 CACCTGAACGCCCTGAAAGAAGATCGGCTGAAGAAGATGGCCAACACCATCATCATGCACGC 
               
               
                   
               
               
                 CCTGGGCTACTGCTACGACGTGCGGAAGAAGAAATGGCAGGCTAAGAACCCCGCCTGCCAGA 
               
               
                   
               
               
                 TCATCCTGTTCGAGGATCTGAGCAACTACAACCCCTACGAGGAAAGGTCCCGCTTCGAGAAC 
               
               
                   
               
               
                 AGCAAGCTCATGAAGTGGTCCAGACGCGAGATCCCCAGACAGGTTGCACTGCAGGGCGAGAT 
               
               
                   
               
               
                 CTATGGCCTGCAAGTGGGAGAAGTGGGCGCTCAGTTCAGCAGCAGATTCCACGCCAAGACAG 
               
               
                   
               
               
                 GCAGCCCTGGCATCAGATGTAGCGTCGTGACCAAAGAGAAGCTGCAGGACAATCGGTTCTTC 
               
               
                   
               
               
                 AAGAATCTGCAGAGAGAGGGCAGACTGACCCTGGACAAAATCGCCGTGCTGAAAGAGGGCGA 
               
               
                   
               
               
                 TCTGTACCCAGACAAAGGCGGCGAGAAGTTCATCAGCCTGAGCAAGGATCGGAAGTGCGTGA 
               
               
                   
               
               
                 CCACACACGCCGACATCAACGCCGCTCAGAACCTGCAGAAGCGGTTCTGGACAAGAACCCAC 
               
               
                   
               
               
                 GGCTTCTACAAGGTGTACTGCAAGGCCTACCAGGTGGACGGCCAGACCGTGTACATCCCTGA 
               
               
                   
               
               
                 GAGCAAGGACCAGAAGCAGAAGATCATCGAAGAGTTCGGCGAGGGCTACTTCATTCTGAAGG 
               
               
                   
               
               
                 ACGGGGTGTACGAATGGGTCAACGCCGGCAAGCTGAAAATCAAGAAGGGCAGCTCCAAGCAG 
               
               
                   
               
               
                 AGCAGCAGCGAGCTGGTGGATAGCGACATCCTGAAAGACAGCTTCGACCTGGCCTCCGAGCT 
               
               
                   
               
               
                 GAAAGGCGAAAAGCTGATGCTGTACAGGGACCCCAGCGGCAATGTGTTCCCCAGCGACAAAT 
               
               
                   
               
               
                 GGATGGCCGCTGGCGTGTTCTTCGGAAAGCTGGAACGCATCCTGATCAGCAAGCTGACCAAC 
               
               
                   
               
               
                 CAGTACTCCATCAGCACCATCGAGGACGACAGCAGCAAGCAGTCTATG AAAAGGCCGGCGGC   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                   ACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCC TAA 
               
               
                   
               
               
                 (SEQ ID NO: 134) 
                   
               
               
                 MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAI 
                   
               
               
                   
               
               
                 YEHHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVE 
               
               
                   
               
               
                 KKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDP 
               
               
                   
               
               
                 LAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWES 
               
               
                   
               
               
                 WNLKVKEEYEKVEKEYKTLEERIKGGSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAV 
               
               
                   
               
               
                 LVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMI 
               
               
                   
               
               
                 HSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMPRRVFN 
               
               
                   
               
               
                 AQKKAQSSTDGSSGSETPGTSESATPESSGEDIQALKALEQYEKERQEQLLRDTLNTNEYRL 
               
               
                   
               
               
                 SKRGLRGWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRN 
               
               
                   
               
               
                 HPEYPYLYATFCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTE 
               
               
                   
               
               
                 KLKKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIK 
               
               
                   
               
               
                 FPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPK 
               
               
                   
               
               
                 VVNEKPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIEEVVDQKPDIEGK 
               
               
                   
               
               
                 LFFPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFE 
               
               
                   
               
               
                 DITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEV 
               
               
                   
               
               
                 KHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHL 
               
               
                   
               
               
                 NALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSK 
               
               
                   
               
               
                 LMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKN 
               
               
                   
               
               
                 LQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDRKCVTTHADINAAQNLQKRFWTRTHGF 
               
               
                   
               
               
                 YKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIKKGSSKQSS 
               
               
                   
               
               
                 SELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQY 
               
               
                   
               
               
                 SISTIEDDSSKQSMKRPAATKKAGQAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA 
               
               
                   
               
               
                 BhCas12b GGSGGS-ABE8-Xten20 at D306 
               
               
                 (SEQ ID NO: 135) 
                   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 CAGATCCTTCATCCTGAAGATCGAGCCCAACGAGGAAGTGAAGAAAGGCCTCTGGAAAACCC 
               
               
                   
               
               
                 ACGAGGTGCTGAACCACGGAATCGCCTACTACATGAATATCCTGAAGCTGATCCGGCAAGAG 
               
               
                   
               
               
                 GCCATCTACGAGCACCACGAGCAGGACCCCAAGAATCCCAAGAAGGTGTCCAAGGCCGAGAT 
               
               
                   
               
               
                 CCAGGCCGAGCTGTGGGATTTCGTGCTGAAGATGCAGAAGTGCAACAGCTTCACACACGAGG 
               
               
                   
               
               
                 TGGACAAGGACGAGGTGTTCAACATCCTGAGAGAGCTGTACGAGGAACTGGTGCCCAGCAGC 
               
               
                   
               
               
                 GTGGAAAAGAAGGGCGAAGCCAACCAGCTGAGCAACAAGTTTCTGTACCCTCTGGTGGACCC 
               
               
                   
               
               
                 CAACAGCCAGTCTGGAAAGGGAACAGCCAGCAGCGGCAGAAAGCCCAGATGGTACAACCTGA 
               
               
                   
               
               
                 AGATTGCCGGCGATCCCTCCTGGGAAGAAGAGAAGAAGAAGTGGGAAGAAGATAAGAAAAAG 
               
               
                   
               
               
                 GACCCGCTGGCCAAGATCCTGGGCAAGCTGGCTGAGTACGGACTGATCCCTCTGTTCATCCC 
               
               
                   
               
               
                 CTACACCGACAGCAACGAGCCCATCGTGAAAGAAATCAAGTGGATGGAAAAGTCCCGGAACC 
               
               
                   
               
               
                 AGAGCGTGCGGCGGCTGGATAAGGACATGTTCATTCAGGCCCTGGAACGGTTCCTGAGCTGG 
               
               
                   
               
               
                 GAGAGCTGGAACCTGAAAGTGAAAGAGGAATACGAGAAGGTCGAGAAAGAGTACAAGACCCT 
               
               
                   
               
               
                 GGAAGAGAGGATCAAAGAGGACATCCAGGCTCTGAAGGCTCTGGAACAGTATGAGAAAGAGC 
               
               
                   
               
               
                 GGCAAGAACAGCTGCTGCGGGACACCCTGAACACCAACGAGTACCGGCTGAGCAAGAGAGGC 
               
               
                   
               
               
                 CTTAGAGGCTGGCGGGAAATCATCCAGAAATGGCTGAAAATGGACggaggctctggaggaag 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                   C AGAACGAGCCCTCCGAGAAGTACCTGGAAGTGTTCAAGGACTACCAGCGGAAGCACCCTA 
               
               
                   
               
               
                 GAGAGGCCGGCGATTACAGCGTGTACGAGTTCCTGTCCAAGAAAGAGAACCACTTCATCTGG 
               
               
                   
               
               
                 CGGAATCACCCTGAGTACCCCTACCTGTACGCCACCTTCTGCGAGATCGACAAGAAAAAGAA 
               
               
                   
               
               
                 GGACGCCAAGCAGCAGGCCACCTTCACACTGGCCGATCCTATCAATCACCCTCTGTGGGTCC 
               
               
                   
               
               
                 GATTCGAGGAAAGAAGCGGCAGCAACCTGAACAAGTACAGAATCCTGACCGAGCAGCTGCAC 
               
               
                   
               
               
                 ACCGAGAAGCTGAAGAAAAAGCTGACAGTGCAGCTGGACCGGCTGATCTACCCTACAGAATC 
               
               
                   
               
               
                 TGGCGGCTGGGAAGAGAAGGGCAAAGTGGACATTGTGCTGCTGCCCAGCCGGCAGTTCTACA 
               
               
                   
               
               
                 ACCAGATCTTCCTGGACATCGAGGAAAAGGGCAAGCACGCCTTCACCTACAAGGATGAGAGC 
               
               
                   
               
               
                 ATCAAGTTCCCTCTGAAGGGCACACTCGGCGGAGCCAGAGTGCAGTTCGACAGAGATCACCT 
               
               
                   
               
               
                 GAGAAGATACCCTCACAAGGTGGAAAGCGGCAACGTGGGCAGAATCTACTTCAACATGACCG 
               
               
                   
               
               
                 TGAACATCGAGCCTACAGAGTCCCCAGTGTCCAAGTCTCTGAAGATCCACCGGGACGACTTC 
               
               
                   
               
               
                 CCCAAGGTGGTCAACTTCAAGCCCAAAGAACTGACCGAGTGGATCAAGGACAGCAAGGGCAA 
               
               
                   
               
               
                 GAAACTGAAGTCCGGCATCGAGTCCCTGGAAATCGGCCTGAGAGTGATGAGCATCGACCTGG 
               
               
                   
               
               
                 GACAGAGACAGGCCGCTGCCGCCTCTATTTTCGAGGTGGTGGATCAGAAGCCCGACATCGAA 
               
               
                   
               
               
                 GGCAAGCTGTTTTTCCCAATCAAGGGCACCGAGCTGTATGCCGTGCACAGAGCCAGCTTCAA 
               
               
                   
               
               
                 CATCAAGCTGCCCGGCGAGACACTGGTCAAGAGCAGAGAAGTGCTGCGGAAGGCCAGAGAGG 
               
               
                   
               
               
                 ACAATCTGAAACTGATGAACCAGAAGCTCAACTTCCTGCGGAACGTGCTGCACTTCCAGCAG 
               
               
                   
               
               
                 TTCGAGGACATCACCGAGAGAGAGAAGCGGGTCACCAAGTGGATCAGCAGACAAGAGAACAG 
               
               
                   
               
               
                 CGACGTGCCCCTGGTGTACCAGGATGAGCTGATCCAGATCCGCGAGCTGATGTACAAGCCTT 
               
               
                   
               
               
                 ACAAGGACTGGGTCGCCTTCCTGAAGCAGCTCCACAAGAGACTGGAAGTCGAGATCGGCAAA 
               
               
                   
               
               
                 GAAGTGAAGCACTGGCGGAAGTCCCTGAGCGACGGAAGAAAGGGCCTGTACGGCATCTCCCT 
               
               
                   
               
               
                 GAAGAACATCGACGAGATCGATCGGACCCGGAAGTTCCTGCTGAGATGGTCCCTGAGGCCTA 
               
               
                   
               
               
                 CCGAACCTGGCGAAGTGCGTAGACTGGAACCCGGCCAGAGATTCGCCATCGACCAGCTGAAT 
               
               
                   
               
               
                 CACCTGAACGCCCTGAAAGAAGATCGGCTGAAGAAGATGGCCAACACCATCATCATGCACGC 
               
               
                   
               
               
                 CCTGGGCTACTGCTACGACGTGCGGAAGAAGAAATGGCAGGCTAAGAACCCCGCCTGCCAGA 
               
               
                   
               
               
                 TCATCCTGTTCGAGGATCTGAGCAACTACAACCCCTACGAGGAAAGGTCCCGCTTCGAGAAC 
               
               
                   
               
               
                 AGCAAGCTCATGAAGTGGTCCAGACGCGAGATCCCCAGACAGGTTGCACTGCAGGGCGAGAT 
               
               
                   
               
               
                 CTATGGCCTGCAAGTGGGAGAAGTGGGCGCTCAGTTCAGCAGCAGATTCCACGCCAAGACAG 
               
               
                   
               
               
                 GCAGCCCTGGCATCAGATGTAGCGTCGTGACCAAAGAGAAGCTGCAGGACAATCGGTTCTTC 
               
               
                   
               
               
                 AAGAATCTGCAGAGAGAGGGCAGACTGACCCTGGACAAAATCGCCGTGCTGAAAGAGGGCGA 
               
               
                   
               
               
                 TCTGTACCCAGACAAAGGCGGCGAGAAGTTCATCAGCCTGAGCAAGGATCGGAAGTGCGTGA 
               
               
                   
               
               
                 CCACACACGCCGACATCAACGCCGCTCAGAACCTGCAGAAGCGGTTCTGGACAAGAACCCAC 
               
               
                   
               
               
                 GGCTTCTACAAGGTGTACTGCAAGGCCTACCAGGTGGACGGCCAGACCGTGTACATCCCTGA 
               
               
                   
               
               
                 GAGCAAGGACCAGAAGCAGAAGATCATCGAAGAGTTCGGCGAGGGCTACTTCATTCTGAAGG 
               
               
                   
               
               
                 ACGGGGTGTACGAATGGGTCAACGCCGGCAAGCTGAAAATCAAGAAGGGCAGCTCCAAGCAG 
               
               
                   
               
               
                 AGCAGCAGCGAGCTGGTGGATAGCGACATCCTGAAAGACAGCTTCGACCTGGCCTCCGAGCT 
               
               
                   
               
               
                 GAAAGGCGAAAAGCTGATGCTGTACAGGGACCCCAGCGGCAATGTGTTCCCCAGCGACAAAT 
               
               
                   
               
               
                 GGATGGCCGCTGGCGTGTTCTTCGGAAAGCTGGAACGCATCCTGATCAGCAAGCTGACCAAC 
               
               
                   
               
               
                 CAGTACTCCATCAGCACCATCGAGGACGACAGCAGCAAGCAGTCTATG AAAAGGCCGGCGGC   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                   ACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCC TAA 
               
               
                   
               
               
                 (SEQ ID NO: 136) 
                   
               
               
                 MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAI 
                   
               
               
                   
               
               
                 YEHHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVE 
               
               
                   
               
               
                 KKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDP 
               
               
                   
               
               
                 LAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWES 
               
               
                   
               
               
                 WNLKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLR 
               
               
                   
               
               
                 GWREIIQKWLKMDGGSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEG 
               
               
                   
               
               
                 WNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGV 
               
               
                   
               
               
                 RNAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDG 
               
               
                   
               
               
                 SSGSETPGTSESATPESSGENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRN 
               
               
                   
               
               
                 HPEYPYLYATFCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTE 
               
               
                   
               
               
                 KLKKKLTVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIK 
               
               
                   
               
               
                 FPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPK 
               
               
                   
               
               
                 VVNFKPKELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGK 
               
               
                   
               
               
                 LFFPIKGTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFE 
               
               
                   
               
               
                 DITEREKRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEV 
               
               
                   
               
               
                 KHWRKSLSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHL 
               
               
                   
               
               
                 NALKEDRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSK 
               
               
                   
               
               
                 LMKWSRREIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKN 
               
               
                   
               
               
                 LQREGRLTLDKIAVLKEGDLYPDKGGEKFISLSKDRKCVTTHADINAAQNLQKRFWTRTHGF 
               
               
                   
               
               
                 YKVYCKAYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIKKGSSKQSS 
               
               
                   
               
               
                 SELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQY 
               
               
                   
               
               
                 SISTIEDDSSKQSMKRPAATKKAGQAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA 
               
               
                   
               
               
                 BhCas12b GGSGGS-ABE8-Xten20 at D980 
               
               
                 (SEQ ID NO: 137) 
                   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 CAGATCCTTCATCCTGAAGATCGAGCCCAACGAGGAAGTGAAGAAAGGCCTCTGGAAAACCC 
               
               
                   
               
               
                 ACGAGGTGCTGAACCACGGAATCGCCTACTACATGAATATCCTGAAGCTGATCCGGCAAGAG 
               
               
                   
               
               
                 GCCATCTACGAGCACCACGAGCAGGACCCCAAGAATCCCAAGAAGGTGTCCAAGGCCGAGAT 
               
               
                   
               
               
                 CCAGGCCGAGCTGTGGGATTTCGTGCTGAAGATGCAGAAGTGCAACAGCTTCACACACGAGG 
               
               
                   
               
               
                 TGGACAAGGACGAGGTGTTCAACATCCTGAGAGAGCTGTACGAGGAACTGGTGCCCAGCAGC 
               
               
                   
               
               
                 GTGGAAAAGAAGGGCGAAGCCAACCAGCTGAGCAACAAGTTTCTGTACCCTCTGGTGGACCC 
               
               
                   
               
               
                 CAACAGCCAGTCTGGAAAGGGAACAGCCAGCAGCGGCAGAAAGCCCAGATGGTACAACCTGA 
               
               
                   
               
               
                 AGATTGCCGGCGATCCCTCCTGGGAAGAAGAGAAGAAGAAGTGGGAAGAAGATAAGAAAAAG 
               
               
                   
               
               
                 GACCCGCTGGCCAAGATCCTGGGCAAGCTGGCTGAGTACGGACTGATCCCTCTGTTCATCCC 
               
               
                   
               
               
                 CTACACCGACAGCAACGAGCCCATCGTGAAAGAAATCAAGTGGATGGAAAAGTCCCGGAACC 
               
               
                   
               
               
                 AGAGCGTGCGGCGGCTGGATAAGGACATGTTCATTCAGGCCCTGGAACGGTTCCTGAGCTGG 
               
               
                   
               
               
                 GAGAGCTGGAACCTGAAAGTGAAAGAGGAATACGAGAAGGTCGAGAAAGAGTACAAGACCCT 
               
               
                   
               
               
                 GGAAGAGAGGATCAAAGAGGACATCCAGGCTCTGAAGGCTCTGGAACAGTATGAGAAAGAGC 
               
               
                   
               
               
                 GGCAAGAACAGCTGCTGCGGGACACCCTGAACACCAACGAGTACCGGCTGAGCAAGAGAGGC 
               
               
                   
               
               
                 CTTAGAGGCTGGCGGGAAATCATCCAGAAATGGCTGAAAATGGACGAGAACGAGCCCTCCGA 
               
               
                   
               
               
                 GAAGTACCTGGAAGTGTTCAAGGACTACCAGCGGAAGCACCCTAGAGAGGCCGGCGATTACA 
               
               
                   
               
               
                 GCGTGTACGAGTTCCTGTCCAAGAAAGAGAACCACTTCATCTGGCGGAATCACCCTGAGTAC 
               
               
                   
               
               
                 CCCTACCTGTACGCCACCTTCTGCGAGATCGACAAGAAAAAGAAGGACGCCAAGCAGCAGGC 
               
               
                   
               
               
                 CACCTTCACACTGGCCGATCCTATCAATCACCCTCTGTGGGTCCGATTCGAGGAAAGAAGCG 
               
               
                   
               
               
                 GCAGCAACCTGAACAAGTACAGAATCCTGACCGAGCAGCTGCACACCGAGAAGCTGAAGAAA 
               
               
                   
               
               
                 AAGCTGACAGTGCAGCTGGACCGGCTGATCTACCCTACAGAATCTGGCGGCTGGGAAGAGAA 
               
               
                   
               
               
                 GGGCAAAGTGGACATTGTGCTGCTGCCCAGCCGGCAGTTCTACAACCAGATCTTCCTGGACA 
               
               
                   
               
               
                 TCGAGGAAAAGGGCAAGCACGCCTTCACCTACAAGGATGAGAGCATCAAGTTCCCTCTGAAG 
               
               
                   
               
               
                 GGCACACTCGGCGGAGCCAGAGTGCAGTTCGACAGAGATCACCTGAGAAGATACCCTCACAA 
               
               
                   
               
               
                 GGTGGAAAGCGGCAACGTGGGCAGAATCTACTTCAACATGACCGTGAACATCGAGCCTACAG 
               
               
                   
               
               
                 AGTCCCCAGTGTCCAAGTCTCTGAAGATCCACCGGGACGACTTCCCCAAGGTGGTCAACTTC 
               
               
                   
               
               
                 AAGCCCAAAGAACTGACCGAGTGGATCAAGGACAGCAAGGGCAAGAAACTGAAGTCCGGCAT 
               
               
                   
               
               
                 CGAGTCCCTGGAAATCGGCCTGAGAGTGATGAGCATCGACCTGGGACAGAGACAGGCCGCTG 
               
               
                   
               
               
                 CCGCCTCTATTTTCGAGGTGGTGGATCAGAAGCCCGACATCGAAGGCAAGCTGTTTTTCCCA 
               
               
                   
               
               
                 ATCAAGGGCACCGAGCTGTATGCCGTGCACAGAGCCAGCTTCAACATCAAGCTGCCCGGCGA 
               
               
                   
               
               
                 GACACTGGTCAAGAGCAGAGAAGTGCTGCGGAAGGCCAGAGAGGACAATCTGAAACTGATGA 
               
               
                   
               
               
                 ACCAGAAGCTCAACTTCCTGCGGAACGTGCTGCACTTCCAGCAGTTCGAGGACATCACCGAG 
               
               
                   
               
               
                 AGAGAGAAGCGGGTCACCAAGTGGATCAGCAGACAAGAGAACAGCGACGTGCCCCTGGTGTA 
               
               
                   
               
               
                 CCAGGATGAGCTGATCCAGATCCGCGAGCTGATGTACAAGCCTTACAAGGACTGGGTCGCCT 
               
               
                   
               
               
                 TCCTGAAGCAGCTCCACAAGAGACTGGAAGTCGAGATCGGCAAAGAAGTGAAGCACTGGCGG 
               
               
                   
               
               
                 AAGTCCCTGAGCGACGGAAGAAAGGGCCTGTACGGCATCTCCCTGAAGAACATCGACGAGAT 
               
               
                   
               
               
                 CGATCGGACCCGGAAGTTCCTGCTGAGATGGTCCCTGAGGCCTACCGAACCTGGCGAAGTGC 
               
               
                   
               
               
                 GTAGACTGGAACCCGGCCAGAGATTCGCCATCGACCAGCTGAATCACCTGAACGCCCTGAAA 
               
               
                   
               
               
                 GAAGATCGGCTGAAGAAGATGGCCAACACCATCATCATGCACGCCCTGGGCTACTGCTACGA 
               
               
                   
               
               
                 CGTGCGGAAGAAGAAATGGCAGGCTAAGAACCCCGCCTGCCAGATCATCCTGTTCGAGGATC 
               
               
                   
               
               
                 TGAGCAACTACAACCCCTACGAGGAAAGGTCCCGCTTCGAGAACAGCAAGCTCATGAAGTGG 
               
               
                   
               
               
                 TCCAGACGCGAGATCCCCAGACAGGTTGCACTGCAGGGCGAGATCTATGGCCTGCAAGTGGG 
               
               
                   
               
               
                 AGAAGTGGGCGCTCAGTTCAGCAGCAGATTCCACGCCAAGACAGGCAGCCCTGGCATCAGAT 
               
               
                   
               
               
                 GTAGCGTCGTGACCAAAGAGAAGCTGCAGGACAATCGGTTCTTCAAGAATCTGCAGAGAGAG 
               
               
                   
               
               
                 GGCAGACTGACCCTGGACAAAATCGCCGTGCTGAAAGAGGGCGATCTGTACCCAGACAAAGG 
               
               
                   
               
               
                 CGGCGAGAAGTTCATCAGCCTGAGCAAGGATCGGAAGTGCGTGACCACACACGCCGACATCA 
               
               
                   
               
               
                 ACGCCGCTCAGAACCTGCAGAAGCGGTTCTGGACAAGAACCCACGGCTTCTACAAGGTGTAC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                   CCTGGCACAAGCGAGAGCGCCACCCCTGAGAGCTCTGGC GGCCAGACCGTGTACATCCCTGA 
               
               
                   
               
               
                 GAGCAAGGACCAGAAGCAGAAGATCATCGAAGAGTTCGGCGAGGGCTACTTCATTCTGAAGG 
               
               
                   
               
               
                 ACGGGGTGTACGAATGGGTCAACGCCGGCAAGCTGAAAATCAAGAAGGGCAGCTCCAAGCAG 
               
               
                   
               
               
                 AGCAGCAGCGAGCTGGTGGATAGCGACATCCTGAAAGACAGCTTCGACCTGGCCTCCGAGCT 
               
               
                   
               
               
                 GAAAGGCGAAAAGCTGATGCTGTACAGGGACCCCAGCGGCAATGTGTTCCCCAGCGACAAAT 
               
               
                   
               
               
                 GGATGGCCGCTGGCGTGTTCTTCGGAAAGCTGGAACGCATCCTGATCAGCAAGCTGACCAAC 
               
               
                   
               
               
                 CAGTACTCCATCAGCACCATCGAGGACGACAGCAGCAAGCAGTCTATGAAAAGGCCGGCGGC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                   ACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCC TAA 
               
               
                   
               
               
                 (SEQ ID NO: 138) 
                   
               
               
                 MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAI 
                   
               
               
                   
               
               
                 YEHHEQDPKNPKKVSKAEIQAELWDEVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVE 
               
               
                   
               
               
                 KKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDP 
               
               
                   
               
               
                 LAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWES 
               
               
                   
               
               
                 WNLKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLR 
               
               
                   
               
               
                 GWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPY 
               
               
                   
               
               
                 LYATFCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKL 
               
               
                   
               
               
                 TVQLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGT 
               
               
                   
               
               
                 LGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKP 
               
               
                   
               
               
                 KELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIK 
               
               
                   
               
               
                 GTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITERE 
               
               
                   
               
               
                 KRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKS 
               
               
                   
               
               
                 LSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKED 
               
               
                   
               
               
                 RLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSR 
               
               
                   
               
               
                 REIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGR 
               
               
                   
               
               
                 LTLDKIAVLKEGDLYPDKGGEKFISLSKDRKCVTTHADINAAQNLQKRFWTRTHGFYKVYCK 
               
               
                   
               
               
                 AYQVDGGSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLH 
               
               
                   
               
               
                 DPTAHAEIMALRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAA 
               
               
                   
               
               
                 GSLMDVLHHPGMNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPG 
               
               
                   
               
               
                 TSESATPESSGGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKLKIKKGSSKQSS 
               
               
                   
               
               
                 SELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQY 
               
               
                   
               
               
                 SISTIEDDSSKQSMKRPAATKKAGQAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA 
               
               
                   
               
               
                 BhCas12b GGSGGS-ABE8-Xten20 at K1019 
               
               
                 (SEQ ID NO: 139) 
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 CAGATCCTTCATCCTGAAGATCGAGCCCAACGAGGAAGTGAAGAAAGGCCTCTGGAAAACCC 
               
               
                   
               
               
                 ACGAGGTGCTGAACCACGGAATCGCCTACTACATGAATATCCTGAAGCTGATCCGGCAAGAG 
               
               
                   
               
               
                 GCCATCTACGAGCACCACGAGCAGGACCCCAAGAATCCCAAGAAGGTGTCCAAGGCCGAGAT 
               
               
                   
               
               
                 CCAGGCCGAGCTGTGGGATTTCGTGCTGAAGATGCAGAAGTGCAACAGCTTCACACACGAGG 
               
               
                   
               
               
                 TGGACAAGGACGAGGTGTTCAACATCCTGAGAGAGCTGTACGAGGAACTGGTGCCCAGCAGC 
               
               
                   
               
               
                 GTGGAAAAGAAGGGCGAAGCCAACCAGCTGAGCAACAAGTTTCTGTACCCTCTGGTGGACCC 
               
               
                   
               
               
                 CAACAGCCAGTCTGGAAAGGGAACAGCCAGCAGCGGCAGAAAGCCCAGATGGTACAACCTGA 
               
               
                   
               
               
                 AGATTGCCGGCGATCCCTCCTGGGAAGAAGAGAAGAAGAAGTGGGAAGAAGATAAGAAAAAG 
               
               
                   
               
               
                 GACCCGCTGGCCAAGATCCTGGGCAAGCTGGCTGAGTACGGACTGATCCCTCTGTTCATCCC 
               
               
                   
               
               
                 CTACACCGACAGCAACGAGCCCATCGTGAAAGAAATCAAGTGGATGGAAAAGTCCCGGAACC 
               
               
                   
               
               
                 AGAGCGTGCGGCGGCTGGATAAGGACATGTTCATTCAGGCCCTGGAACGGTTCCTGAGCTGG 
               
               
                   
               
               
                 GAGAGCTGGAACCTGAAAGTGAAAGAGGAATACGAGAAGGTCGAGAAAGAGTACAAGACCCT 
               
               
                   
               
               
                 GGAAGAGAGGATCAAAGAGGACATCCAGGCTCTGAAGGCTCTGGAACAGTATGAGAAAGAGC 
               
               
                   
               
               
                 GGCAAGAACAGCTGCTGCGGGACACCCTGAACACCAACGAGTACCGGCTGAGCAAGAGAGGC 
               
               
                   
               
               
                 CTTAGAGGCTGGCGGGAAATCATCCAGAAATGGCTGAAAATGGACGAGAACGAGCCCTCCGA 
               
               
                   
               
               
                 GAAGTACCTGGAAGTGTTCAAGGACTACCAGCGGAAGCACCCTAGAGAGGCCGGCGATTACA 
               
               
                   
               
               
                 GCGTGTACGAGTTCCTGTCCAAGAAAGAGAACCACTTCATCTGGCGGAATCACCCTGAGTAC 
               
               
                   
               
               
                 CCCTACCTGTACGCCACCTTCTGCGAGATCGACAAGAAAAAGAAGGACGCCAAGCAGCAGGC 
               
               
                   
               
               
                 CACCTTCACACTGGCCGATCCTATCAATCACCCTCTGTGGGTCCGATTCGAGGAAAGAAGCG 
               
               
                   
               
               
                 GCAGCAACCTGAACAAGTACAGAATCCTGACCGAGCAGCTGCACACCGAGAAGCTGAAGAAA 
               
               
                   
               
               
                 AAGCTGACAGTGCAGCTGGACCGGCTGATCTACCCTACAGAATCTGGCGGCTGGGAAGAGAA 
               
               
                   
               
               
                 GGGCAAAGTGGACATTGTGCTGCTGCCCAGCCGGCAGTTCTACAACCAGATCTTCCTGGACA 
               
               
                   
               
               
                 TCGAGGAAAAGGGCAAGCACGCCTTCACCTACAAGGATGAGAGCATCAAGTTCCCTCTGAAG 
               
               
                   
               
               
                 GGCACACTCGGCGGAGCCAGAGTGCAGTTCGACAGAGATCACCTGAGAAGATACCCTCACAA 
               
               
                   
               
               
                 GGTGGAAAGCGGCAACGTGGGCAGAATCTACTTCAACATGACCGTGAACATCGAGCCTACAG 
               
               
                   
               
               
                 AGTCCCCAGTGTCCAAGTCTCTGAAGATCCACCGGGACGACTTCCCCAAGGTGGTCAACTTC 
               
               
                   
               
               
                 AAGCCCAAAGAACTGACCGAGTGGATCAAGGACAGCAAGGGCAAGAAACTGAAGTCCGGCAT 
               
               
                   
               
               
                 CGAGTCCCTGGAAATCGGCCTGAGAGTGATGAGCATCGACCTGGGACAGAGACAGGCCGCTG 
               
               
                   
               
               
                 CCGCCTCTATTTTCGAGGTGGTGGATCAGAAGCCCGACATCGAAGGCAAGCTGTTTTTCCCA 
               
               
                   
               
               
                 ATCAAGGGCACCGAGCTGTATGCCGTGCACAGAGCCAGCTTCAACATCAAGCTGCCCGGCGA 
               
               
                   
               
               
                 GACACTGGTCAAGAGCAGAGAAGTGCTGCGGAAGGCCAGAGAGGACAATCTGAAACTGATGA 
               
               
                   
               
               
                 ACCAGAAGCTCAACTTCCTGCGGAACGTGCTGCACTTCCAGCAGTTCGAGGACATCACCGAG 
               
               
                   
               
               
                 AGAGAGAAGCGGGTCACCAAGTGGATCAGCAGACAAGAGAACAGCGACGTGCCCCTGGTGTA 
               
               
                   
               
               
                 CCAGGATGAGCTGATCCAGATCCGCGAGCTGATGTACAAGCCTTACAAGGACTGGGTCGCCT 
               
               
                   
               
               
                 TCCTGAAGCAGCTCCACAAGAGACTGGAAGTCGAGATCGGCAAAGAAGTGAAGCACTGGCGG 
               
               
                   
               
               
                 AAGTCCCTGAGCGACGGAAGAAAGGGCCTGTACGGCATCTCCCTGAAGAACATCGACGAGAT 
               
               
                   
               
               
                 CGATCGGACCCGGAAGTTCCTGCTGAGATGGTCCCTGAGGCCTACCGAACCTGGCGAAGTGC 
               
               
                   
               
               
                 GTAGACTGGAACCCGGCCAGAGATTCGCCATCGACCAGCTGAATCACCTGAACGCCCTGAAA 
               
               
                   
               
               
                 GAAGATCGGCTGAAGAAGATGGCCAACACCATCATCATGCACGCCCTGGGCTACTGCTACGA 
               
               
                   
               
               
                 CGTGCGGAAGAAGAAATGGCAGGCTAAGAACCCCGCCTGCCAGATCATCCTGTTCGAGGATC 
               
               
                   
               
               
                 TGAGCAACTACAACCCCTACGAGGAAAGGTCCCGCTTCGAGAACAGCAAGCTCATGAAGTGG 
               
               
                   
               
               
                 TCCAGACGCGAGATCCCCAGACAGGTTGCACTGCAGGGCGAGATCTATGGCCTGCAAGTGGG 
               
               
                   
               
               
                 AGAAGTGGGCGCTCAGTTCAGCAGCAGATTCCACGCCAAGACAGGCAGCCCTGGCATCAGAT 
               
               
                   
               
               
                 GTAGCGTCGTGACCAAAGAGAAGCTGCAGGACAATCGGTTCTTCAAGAATCTGCAGAGAGAG 
               
               
                   
               
               
                 GGCAGACTGACCCTGGACAAAATCGCCGTGCTGAAAGAGGGCGATCTGTACCCAGACAAAGG 
               
               
                   
               
               
                 CGGCGAGAAGTTCATCAGCCTGAGCAAGGATCGGAAGTGCGTGACCACACACGCCGACATCA 
               
               
                   
               
               
                 ACGCCGCTCAGAACCTGCAGAAGCGGTTCTGGACAAGAACCCACGGCTTCTACAAGGTGTAC 
               
               
                   
               
               
                 TGCAAGGCCTACCAGGTGGACGGCCAGACCGTGTACATCCCTGAGAGCAAGGACCAGAAGCA 
               
               
                   
               
               
                 GAAGATCATCGAAGAGTTCGGCGAGGGCTACTTCATTCTGAAGGACGGGGTGTACGAATGGG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                   CAAGCGAGAGCGCCACCCCTGAGAGCTCTGGC CTGAAAATCAAGAAGGGCAGCTCCAAGCAG 
               
               
                   
               
               
                 AGCAGCAGCGAGCTGGTGGATAGCGACATCCTGAAAGACAGCTTCGACCTGGCCTCCGAGCT 
               
               
                   
               
               
                 GAAAGGCGAAAAGCTGATGCTGTACAGGGACCCCAGCGGCAATGTGTTCCCCAGCGACAAAT 
               
               
                   
               
               
                 GGATGGCCGCTGGCGTGTTCTTCGGAAAGCTGGAACGCATCCTGATCAGCAAGCTGACCAAC 
               
               
                   
               
               
                 CAGTACTCCATCAGCACCATCGAGGACGACAGCAGCAAGCAGTCTATG AAAAGGCCGGCGGC   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                   ACGCTTATCCCTACGACGTGCCTGATTATGCATACCCATATGATGTCCCCGACTATGCC TAA 
               
               
                   
               
               
                 (SEQ ID NO: 140) 
                   
               
               
                 MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKGLWKTHEVLNHGIAYYMNILKLIRQEAI 
                   
               
               
                   
               
               
                 YEHHEQDPKNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKDEVFNILRELYEELVPSSVE 
               
               
                   
               
               
                 KKGEANQLSNKFLYPLVDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEKKKWEEDKKKDP 
               
               
                   
               
               
                 LAKILGKLAEYGLIPLFIPYTDSNEPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLSWES 
               
               
                   
               
               
                 WNLKVKEEYEKVEKEYKTLEERIKEDIQALKALEQYEKERQEQLLRDTLNTNEYRLSKRGLR 
               
               
                   
               
               
                 GWREIIQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYSVYEFLSKKENHFIWRNHPEYPY 
               
               
                   
               
               
                 LYATFCEIDKKKKDAKQQATFTLADPINHPLWVRFEERSGSNLNKYRILTEQLHTEKLKKKL 
               
               
                   
               
               
                 TVOLDRLIYPTESGGWEEKGKVDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIKFPLKGT 
               
               
                   
               
               
                 LGGARVQFDRDHLRRYPHKVESGNVGRIYFNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKP 
               
               
                   
               
               
                 KELTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQAAAASIFEVVDQKPDIEGKLFFPIK 
               
               
                   
               
               
                 GTELYAVHRASFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNFLRNVLHFQQFEDITERE 
               
               
                   
               
               
                 KRVTKWISRQENSDVPLVYQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIGKEVKHWRKS 
               
               
                   
               
               
                 LSDGRKGLYGISLKNIDEIDRTRKFLLRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKED 
               
               
                   
               
               
                 RLKKMANTIIMHALGYCYDVRKKKWQAKNPACQIILFEDLSNYNPYEERSRFENSKLMKWSR 
               
               
                   
               
               
                 REIPRQVALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSVVTKEKLQDNRFFKNLQREGR 
               
               
                   
               
               
                 LTLDKIAVLKEGDLYPDKGGEKFISLSKDRKCVTTHADINAAQNLQKRFWTRTHGFYKVYCK 
               
               
                   
               
               
                 AYQVDGQTVYIPESKDQKQKIIEEFGEGYFILKDGVYEWVNAGKGGSGGSSEVEFSHEYWMR 
               
               
                   
               
               
                 HALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRL 
               
               
                   
               
               
                 YDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGIL 
               
               
                   
               
               
                 ADECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSGLKIKKGSSKQSS 
               
               
                   
               
               
                 SELVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPSDKWMAAGVFFGKLERILISKLTNQY 
               
               
                   
               
               
                 SISTIEDDSSKQSMKRPAATKKAGQAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA 
               
            
           
         
       
     
     For the sequences above, the Kozak sequence is bolded and underlined; marks the N-terminal nuclear localization signal (NLS); lower case characters denote the GGGSGGS linker (SEQ ID NO: 141);   marks the sequence encoding ABE8, unmodified sequence encodes BhCas12b; double underling denotes the Xten20 linker; single underlining denotes the C-terminal NLS;   denotes the GS linker; and italicized characters represent the coding sequence of the 3× hemagglutinin (HA) tag. 
     Guide Polynucleotides 
     In an embodiment, the guide polynucleotide is a guide RNA. An RNA/Cas complex can assist in “guiding” Cas protein to a target DNA. Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA,” or simply “gNRA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M. et al., Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (see e.g., “Complete genome sequence of an M1 strain of  Streptococcus pyogenes .” Ferretti, J. J. et al., Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E. et al., Nature 471:602-607(2011); and “Programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M. et al, Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to,  S. pyogenes  and  S. thermophilus . Additional suitable Cas9 nucleases and sequences can be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase. 
     In some embodiments, the guide polynucleotide is at least one single guide RNA (“sgRNA” or “gNRA”). In some embodiments, the guide polynucleotide is at least one tracrRNA. In some embodiments, the guide polynucleotide does not require PAM sequence to guide the polynucleotide-programmable DNA-binding domain (e.g., Cas9 or Cpf1) to the target nucleotide sequence. 
     The polynucleotide programmable nucleotide binding domain (e.g., a CRISPR-derived domain) of the base editors disclosed herein can recognize a target polynucleotide sequence by associating with a guide polynucleotide. A guide polynucleotide (e.g., gRNA) is typically single-stranded and can be programmed to site-specifically bind (i.e., via complementary base pairing) to a target sequence of a polynucleotide, thereby directing a base editor that is in conjunction with the guide nucleic acid to the target sequence. A guide polynucleotide can be DNA. A guide polynucleotide can be RNA. In some embodiments, the guide polynucleotide comprises natural nucleotides (e.g., adenosine). In some embodiments, the guide polynucleotide comprises non-natural (or unnatural) nucleotides (e.g., peptide nucleic acid or nucleotide analogs). In some embodiments, the targeting region of a guide nucleic acid sequence can be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. A targeting region of a guide nucleic acid can be between 10-30 nucleotides in length, or between 15-25 nucleotides in length, or between 15-20 nucleotides in length. 
     In some embodiments, a guide polynucleotide comprises two or more individual polynucleotides, which can interact with one another via for example complementary base pairing (e.g., a dual guide polynucleotide). For example, a guide polynucleotide can comprise a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). For example, a guide polynucleotide can comprise one or more trans-activating CRISPR RNA (tracrRNA). 
     In type II CRISPR systems, targeting of a nucleic acid by a CRISPR protein (e.g., Cas9) typically requires complementary base pairing between a first RNA molecule (crRNA) comprising a sequence that recognizes the target sequence and a second RNA molecule (trRNA) comprising repeat sequences which forms a scaffold region that stabilizes the guide RNA-CRISPR protein complex. Such dual guide RNA systems can be employed as a guide polynucleotide to direct the base editors disclosed herein to a target polynucleotide sequence. 
     In some embodiments, the base editor provided herein utilizes a single guide polynucleotide (e.g., gRNA). In some embodiments, the base editor provided herein utilizes a dual guide polynucleotide (e.g., dual gRNAs). In some embodiments, the base editor provided herein utilizes one or more guide polynucleotide (e.g., multiple gRNA). In some embodiments, a single guide polynucleotide is utilized for different base editors described herein. For example, a single guide polynucleotide can be utilized for a cytidine base editor and an adenosine base editor. 
     In other embodiments, a guide polynucleotide can comprise both the polynucleotide targeting portion of the nucleic acid and the scaffold portion of the nucleic acid in a single molecule (i.e., a single-molecule guide nucleic acid). For example, a single-molecule guide polynucleotide can be a single guide RNA (sgRNA or gRNA). Herein the term guide polynucleotide sequence contemplates any single, dual or multi-molecule nucleic acid capable of interacting with and directing a base editor to a target polynucleotide sequence. 
     Typically, a guide polynucleotide (e.g., crRNA/trRNA complex or a gRNA) comprises a “polynucleotide-targeting segment” that includes a sequence capable of recognizing and binding to a target polynucleotide sequence, and a “protein-binding segment” that stabilizes the guide polynucleotide within a polynucleotide programmable nucleotide binding domain component of a base editor. In some embodiments, the polynucleotide targeting segment of the guide polynucleotide recognizes and binds to a DNA polynucleotide, thereby facilitating the editing of a base in DNA. In other embodiments, the polynucleotide targeting segment of the guide polynucleotide recognizes and binds to an RNA polynucleotide, thereby facilitating the editing of a base in RNA. Herein a “segment” refers to a section or region of a molecule, e.g., a contiguous stretch of nucleotides in the guide polynucleotide. A segment can also refer to a region/section of a complex such that a segment can comprise regions of more than one molecule. For example, where a guide polynucleotide comprises multiple nucleic acid molecules, the protein-binding segment of can include all or a portion of multiple separate molecules that are for instance hybridized along a region of complementarity. In some embodiments, a protein-binding segment of a DNA-targeting RNA that comprises two separate molecules can comprise (i) base pairs 40-75 of a first RNA molecule that is 100 base pairs in length; and (ii) base pairs 10-25 of a second RNA molecule that is 50 base pairs in length. The definition of “segment,” unless otherwise specifically defined in a particular context, is not limited to a specific number of total base pairs, is not limited to any particular number of base pairs from a given RNA molecule, is not limited to a particular number of separate molecules within a complex, and can include regions of RNA molecules that are of any total length and can include regions with complementarity to other molecules. 
     A guide RNA or a guide polynucleotide can comprise two or more RNAs, e.g., CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA). A guide RNA or a guide polynucleotide can sometimes comprise a single-chain RNA, or single guide RNA (sgRNA) formed by fusion of a portion (e.g., a functional portion) of crRNA and tracrRNA. A guide RNA or a guide polynucleotide can also be a dual RNA comprising a crRNA and a tracrRNA. Furthermore, a crRNA can hybridize with a target DNA. 
     As discussed above, a guide RNA or a guide polynucleotide can be an expression product. For example, a DNA that encodes a guide RNA can be a vector comprising a sequence coding for the guide RNA. A guide RNA or a guide polynucleotide can be transferred into a cell by transfecting the cell with an isolated guide RNA or plasmid DNA comprising a sequence coding for the guide RNA and a promoter. A guide RNA or a guide polynucleotide can also be transferred into a cell in other way, such as using virus-mediated gene delivery. 
     A guide RNA or a guide polynucleotide can be isolated. For example, a guide RNA can be transfected in the form of an isolated RNA into a cell or organism. A guide RNA can be prepared by in vitro transcription using any in vitro transcription system known in the art. A guide RNA can be transferred to a cell in the form of isolated RNA rather than in the form of plasmid comprising encoding sequence for a guide RNA. 
     A guide RNA or a guide polynucleotide can comprise three regions: a first region at the 5′ end that can be complementary to a target site in a chromosomal sequence, a second internal region that can form a stem loop structure, and a third 3′ region that can be single-stranded. A first region of each guide RNA can also be different such that each guide RNA guides a fusion protein to a specific target site. Further, second and third regions of each guide RNA can be identical in all guide RNAs. 
     A first region of a guide RNA or a guide polynucleotide can be complementary to sequence at a target site in a chromosomal sequence such that the first region of the guide RNA can base pair with the target site. In some embodiments, a first region of a guide RNA can comprise from or from about 10 nucleotides to 25 nucleotides (i.e., from 10 nucleotides to nucleotides; or from about 10 nucleotides to about 25 nucleotides; or from 10 nucleotides to about 25 nucleotides; or from about 10 nucleotides to 25 nucleotides) or more. For example, a region of base pairing between a first region of a guide RNA and a target site in a chromosomal sequence can be or can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more nucleotides in length. Sometimes, a first region of a guide RNA can be or can be about 19, 20, or 21 nucleotides in length. 
     A guide RNA or a guide polynucleotide can also comprise a second region that forms a secondary structure. For example, a secondary structure formed by a guide RNA can comprise a stem (or hairpin) and a loop. A length of a loop and a stem can vary. For example, a loop can range from or from about 3 to 10 nucleotides in length, and a stem can range from or from about 6 to 20 base pairs in length. A stem can comprise one or more bulges of 1 to 10 or about 10 nucleotides. The overall length of a second region can range from or from about 16 to 60 nucleotides in length. For example, a loop can be or can be about 4 nucleotides in length and a stem can be or can be about 12 base pairs. 
     A guide RNA or a guide polynucleotide can also comprise a third region at the 3′ end that can be essentially single-stranded. For example, a third region is sometimes not complementarity to any chromosomal sequence in a cell of interest and is sometimes not complementarity to the rest of a guide RNA. Further, the length of a third region can vary. A third region can be more than or more than about 4 nucleotides in length. For example, the length of a third region can range from or from about 5 to 60 nucleotides in length. 
     A guide RNA or a guide polynucleotide can target any exon or intron of a gene target. In some embodiments, a guide can target exon 1 or 2 of a gene; in other embodiments, a guide can target exon 3 or 4 of a gene. A composition can comprise multiple guide RNAs that all target the same exon or in some embodiments, multiple guide RNAs that can target different exons. An exon and an intron of a gene can be targeted. 
     A guide RNA or a guide polynucleotide can target a nucleic acid sequence of or of about 20 nucleotides. A target nucleic acid can be less than or less than about 20 nucleotides. A target nucleic acid can be at least or at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or anywhere between 1-100 nucleotides in length. A target nucleic acid can be at most or at most about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, or anywhere between 1-100 nucleotides in length. A target nucleic acid sequence can be or can be about 20 bases immediately 5′ of the first nucleotide of the PAM. A guide RNA can target a nucleic acid sequence. A target nucleic acid can be at least or at least about 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, or 1-100 nucleotides. 
     A guide polynucleotide, for example, a guide RNA, can refer to a nucleic acid that can hybridize to another nucleic acid, for example, the target nucleic acid or protospacer in a genome of a cell. A guide polynucleotide can be RNA. A guide polynucleotide can be DNA. The guide polynucleotide can be programmed or designed to bind to a sequence of nucleic acid site-specifically. A guide polynucleotide can comprise a polynucleotide chain and can be called a single guide polynucleotide. A guide polynucleotide can comprise two polynucleotide chains and can be called a double guide polynucleotide. A guide RNA can be introduced into a cell or embryo as an RNA molecule. For example, an RNA molecule can be transcribed in vitro and/or can be chemically synthesized. An RNA can be transcribed from a synthetic DNA molecule, e.g., a gBlocks® gene fragment. A guide RNA can then be introduced into a cell or embryo as an RNA molecule. A guide RNA can also be introduced into a cell or embryo in the form of a non-RNA nucleic acid molecule, e.g., DNA molecule. For example, a DNA encoding a guide RNA can be operably linked to promoter control sequence for expression of the guide RNA in a cell or embryo of interest. A RNA coding sequence can be operably linked to a promoter sequence that is recognized by RNA polymerase III (Pol III). Plasmid vectors that can be used to express guide RNA include, but are not limited to, px330 vectors and px333 vectors. In some embodiments, a plasmid vector (e.g., px333 vector) can comprise at least two guide RNA-encoding DNA sequences. 
     Methods for selecting, designing, and validating guide polynucleotides, e.g., guide RNAs and targeting sequences are described herein and known to those skilled in the art. For example, to minimize the impact of potential substrate promiscuity of a deaminase domain in the nucleobase editor system (e.g., an AID domain), the number of residues that could unintentionally be targeted for deamination (e.g., off-target C residues that could potentially reside on ssDNA within the target nucleic acid locus) may be minimized. In addition, software tools can be used to optimize the gRNAs corresponding to a target nucleic acid sequence, e.g., to minimize total off-target activity across the genome. For example, for each possible targeting domain choice using  S. pyogenes  Cas9, all off-target sequences (preceding selected PAMs, e.g., NAG or NGG) may be identified across the genome that contain up to certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base-pairs. First regions of gRNAs complementary to a target site can be identified, and all first regions (e.g., crRNAs) can be ranked according to its total predicted off-target score; the top-ranked targeting domains represent those that are likely to have the greatest on-target and the least off-target activity. Candidate targeting gRNAs can be functionally evaluated by using methods known in the art and/or as set forth herein. 
     As a non-limiting example, target DNA hybridizing sequences in crRNAs of a guide RNA for use with Cas9s may be identified using a DNA sequence searching algorithm. gRNA design may be carried out using custom gRNA design software based on the public tool cas-offinder as described in Bae S., Park J., &amp; Kim J.-S. Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473-1475 (2014). This software scores guides after calculating their genome-wide off-target propensity. Typically matches ranging from perfect matches to 7 mismatches are considered for guides ranging in length from 17 to 24. Once the off-target sites are computationally-determined, an aggregate score is calculated for each guide and summarized in a tabular output using a web-interface. In addition to identifying potential target sites adjacent to PAM sequences, the software also identifies all PAM adjacent sequences that differ by 1, 2, 3 or more than 3 nucleotides from the selected target sites. Genomic DNA sequences for a target nucleic acid sequence, e.g., a target gene may be obtained and repeat elements may be screened using publicly available tools, for example, the RepeatMasker program. RepeatMasker searches input DNA sequences for repeated elements and regions of low complexity. The output is a detailed annotation of the repeats present in a given query sequence. 
     Following identification, first regions of guide RNAs, e.g., crRNAs, may be ranked into tiers based on their distance to the target site, their orthogonality and presence of 5′ nucleotides for close matches with relevant PAM sequences (for example, a 5′ G based on identification of close matches in the human genome containing a relevant PAM e.g., NGG PAM for  S. pyogenes , NNGRRT or NNGRRV PAM for  S. aureus ). As used herein, orthogonality refers to the number of sequences in the human genome that contain a minimum number of mismatches to the target sequence. A “high level of orthogonality” or “good orthogonality” may, for example, refer to 20-mer targeting domains that have no identical sequences in the human genome besides the intended target, nor any sequences that contain one or two mismatches in the target sequence. Targeting domains with good orthogonality may be selected to minimize off-target DNA cleavage. 
     In some embodiments, a reporter system may be used for detecting base-editing activity and testing candidate guide polynucleotides. In some embodiments, a reporter system may comprise a reporter gene-based assay where base editing activity leads to expression of the reporter gene. For example, a reporter system may include a reporter gene comprising a deactivated start codon, e.g., a mutation on the template strand from 3′-TAC-5′ to 3′-CAC-5′. Upon successful deamination of the target C, the corresponding mRNA will be transcribed as 5′-AUG-3′ instead of 5′-GUG-3′, enabling the translation of the reporter gene. Suitable reporter genes will be apparent to those of skill in the art. Non-limiting examples of reporter genes include gene encoding green fluorescence protein (GFP), red fluorescence protein (RFP), luciferase, secreted alkaline phosphatase (SEAP), or any other gene whose expression are detectable and apparent to those skilled in the art. The reporter system can be used to test many different gRNAs, e.g., in order to determine which residue(s) with respect to the target DNA sequence the respective deaminase will target. sgRNAs that target non-template strand can also be tested in order to assess off-target effects of a specific base editing protein, e.g., a Cas9 deaminase fusion protein. In some embodiments, such gRNAs can be designed such that the mutated start codon will not be base-paired with the gRNA. The guide polynucleotides can comprise standard ribonucleotides, modified ribonucleotides (e.g., pseudouridine), ribonucleotide isomers, and/or ribonucleotide analogs. In some embodiments, the guide polynucleotide can comprise at least one detectable label. The detectable label can be a fluorophore (e.g., FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluors, Halo tags, or suitable fluorescent dye), a detection tag (e.g., biotin, digoxigenin, and the like), quantum dots, or gold particles. 
     The guide polynucleotides can be synthesized chemically, synthesized enzymatically, or a combination thereof. For example, the guide RNA can be synthesized using standard phosphoramidite-based solid-phase synthesis methods. Alternatively, the guide RNA can be synthesized in vitro by operably linking DNA encoding the guide RNA to a promoter control sequence that is recognized by a phage RNA polymerase. Examples of suitable phage promoter sequences include T7, T3, SP6 promoter sequences, or variations thereof. In embodiments in which the guide RNA comprises two separate molecules (e.g., crRNA and tracrRNA), the crRNA can be chemically synthesized and the tracrRNA can be enzymatically synthesized. 
     In some embodiments, a base editor system may comprise multiple guide polynucleotides, e.g., gRNAs. For example, the gRNAs may target to one or more target loci (e.g., at least 1 gRNA, at least 2 gRNA, at least 5 gRNA, at least 10 gRNA, at least 20 gRNA, at least 30 g RNA, at least 50 gRNA) comprised in a base editor system. The multiple gRNA sequences can be tandemly arranged and are preferably separated by a direct repeat. 
     A DNA sequence encoding a guide RNA or a guide polynucleotide can also be part of a vector. Further, a vector can comprise additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences, etc.), selectable marker sequences (e.g., GFP or antibiotic resistance genes such as puromycin), origins of replication, and the like. A DNA molecule encoding a guide RNA can also be linear. A DNA molecule encoding a guide RNA or a guide polynucleotide can also be circular. 
     In some embodiments, one or more components of a base editor system may be encoded by DNA sequences. Such DNA sequences may be introduced into an expression system, e.g., a cell, together or separately. For example, DNA sequences encoding a polynucleotide programmable nucleotide binding domain and a guide RNA may be introduced into a cell, each DNA sequence can be part of a separate molecule (e.g., one vector containing the polynucleotide programmable nucleotide binding domain coding sequence and a second vector containing the guide RNA coding sequence) or both can be part of a same molecule (e.g., one vector containing coding (and regulatory) sequence for both the polynucleotide programmable nucleotide binding domain and the guide RNA). 
     A guide polynucleotide can comprise one or more modifications to provide a nucleic acid with a new or enhanced feature. A guide polynucleotide can comprise a nucleic acid affinity tag. A guide polynucleotide can comprise synthetic nucleotide, synthetic nucleotide analog, nucleotide derivatives, and/or modified nucleotides. 
     In some embodiments, a gRNA or a guide polynucleotide can comprise modifications. A modification can be made at any location of a gRNA or a guide polynucleotide. More than one modification can be made to a single gRNA or a guide polynucleotide. A gRNA or a guide polynucleotide can undergo quality control after a modification. In some embodiments, quality control can include PAGE, HPLC, MS, or any combination thereof. 
     A modification of a gRNA or a guide polynucleotide can be a substitution, insertion, deletion, chemical modification, physical modification, stabilization, purification, or any combination thereof. 
     A gRNA or a guide polynucleotide can also be modified by 5′adenylate, 5′ guanosine-triphosphate cap, 5′N7-Methylguanosine-triphosphate cap, 5′triphosphate cap, 3′phosphate, 3′thiophosphate, 5′phosphate, 5′thiophosphate, Cis-Syn thymidine dimer, trimers, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9,3′-3′ modifications, 5′-5′ modifications, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-Biotin, dual biotin, PC biotin, psoralen C2, psoralen C6, TINA, 3′DABCYL, black hole quencher 1, black hole quencer 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linkers, 2′-deoxyribonucleoside analog purine, 2′-deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2′-O-methyl ribonucleoside analog, sugar modified analogs, wobble/universal bases, fluorescent dye label, 2′-fluoro RNA, 2′-O-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5′-triphosphate, 5′-methylcytidine-5′-triphosphate, or any combination thereof. 
     In some embodiments, a modification is permanent. In other embodiments, a modification is transient. In some embodiments, multiple modifications are made to a gRNA or a guide polynucleotide. A gRNA or a guide polynucleotide modification can alter physiochemical properties of a nucleotide, such as their conformation, polarity, hydrophobicity, chemical reactivity, base-pairing interactions, or any combination thereof. 
     The PAM sequence can be any PAM sequence known in the art. Suitable PAM sequences include, but are not limited to, NGG, NGA, NGC, NGN, NGT, NGCG, NGAG, NGAN, NGNG, NGCN, NGCG, NGTN, NNGRRT, NNNRRT, NNGRR(N), TTTV, TYCV, TYCV, TATV, NNNNGATT, NNAGAAW, or NAAAAC. Y is a pyrimidine; N is any nucleotide base; W is A or T. 
     A modification can also be a phosphorothioate substitute. In some embodiments, a natural phosphodiester bond can be susceptible to rapid degradation by cellular nucleases and; a modification of internucleotide linkage using phosphorothioate (PS) bond substitutes can be more stable towards hydrolysis by cellular degradation. A modification can increase stability in a gRNA or a guide polynucleotide. A modification can also enhance biological activity. In some embodiments, a phosphorothioate enhanced RNA gRNA can inhibit RNase A, RNase T1, calf serum nucleases, or any combinations thereof. These properties can allow the use of PS-RNA gRNAs to be used in applications where exposure to nucleases is of high probability in vivo or in vitro. For example, phosphorothioate (PS) bonds can be introduced between the last 3-5 nucleotides at the 5′- or ″-end of a gRNA which can inhibit exonuclease degradation. In some embodiments, phosphorothioate bonds can be added throughout an entire gRNA to reduce attack by endonucleases. 
     Different Cas12b orthologs (e.g., BhCas12b, BvCas12b, and AaCas12b) use different scaffold sequences (also referred to as tracrRNA). In some embodiments, the scaffold sequence is optimized for use with a BhCas12b protein and has the following sequence: (where the T&#39;s are replaced by uridines (U&#39;s) in the actual gRNA). 
     BhCas12b sgRNA Scaffold (Underlined)+20 nt to 23 nt Guide Sequence (Denoted by Ns). 
     
       
         
           
               
            
               
                 (SEQ ID NO: 142) 
               
               
                 5′ GTTCTGTCTTTTGGTCAGGACAACCGTCTAGCTATAAGTGCTGCAGGG   
               
               
                   
               
               
                   TGTGAGAAACTCCTATTGCTGGACGATGTCTCTTACGAGGCATTAGCA CN 
               
               
                   
               
               
                 NNNNNNNNNNNNNNNNNNN-3′ 
               
            
           
         
       
     
     In some embodiments, the scaffold sequence is optimized for use with a BvCas12b protein and has the following sequence: (where the T&#39;s are replaced by uridines (U&#39;s) in the actual gRNA). 
     BvCas12b sgRNA Scaffold (Underlined)+20 nt to 23 nt Guide Sequence (Denoted by Ns) 
     
       
         
           
               
            
               
                 (SEQ ID NO: 143) 
               
               
                 5′ GACCTATAGGGTCAATGAATCTGTGCGTGTGCCATAAGTAATTAAAAA   
               
               
                   
               
               
                   TTACCCACCACAGGAGCACCTGAAAACAGGTGCTTGGCAC NNNNNNNNNN 
               
               
                   
               
               
                 NNNNNNNNNN-3′ 
               
            
           
         
       
     
     In some embodiments, the scaffold sequence is optimized for use with a AaCas12b protein and has the following sequence: (where the T&#39;s are replaced by uridines (U&#39;s) in the actual gRNA). 
     AaCas12b sgRNA Scaffold (Underlined)+20 nt to 23 nt Guide Sequence (Denoted by Ns) 
     
       
         
           
               
            
               
                 (SEQ ID NO: 144) 
               
               
                 5′ GTCTAAAGGACAGAATTTTTCAACGGGTGTGCCAATGGCCACTTTCCA   
               
               
                   
               
               
                 
                   GGTGGCAAAGCCCGTTGAACTTCTCAAAAAGAACGATCTGAGAAGTGGCA 
                 
               
               
                   
               
               
                   C NNNNNNNNNNNNNNNNNNNN-3′ 
               
            
           
         
       
     
     Thus, a skilled artisan can change the genomic target of the Cas protein specificity is partially determined by how specific the gRNA targeting sequence is for the genomic target compared to the rest of the genome. 
     Protospacer Adjacent Motif 
     The term “protospacer adjacent motif (PAM)” or PAM-like motif refers to a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. In some embodiments, the PAM can be a 5′ PAM (i.e., located upstream of the 5′ end of the protospacer). In other embodiments, the PAM can be a 3′ PAM (i.e., located downstream of the 5′ end of the protospacer). 
     The PAM sequence is essential for target binding, but the exact sequence depends on a type of Cas protein. 
     A base editor provided herein can comprise a CRISPR protein-derived domain that is capable of binding a nucleotide sequence that contains a canonical or non-canonical protospacer adjacent motif (PAM) sequence. A PAM site is a nucleotide sequence in proximity to a target polynucleotide sequence. Some aspects of the disclosure provide for base editors comprising all or a portion of CRISPR proteins that have different PAM specificities. 
     For example, typically Cas9 proteins, such as Cas9 from  S. pyogenes  (spCas9), require a canonical NGG PAM sequence to bind a particular nucleic acid region, where the “N” in “NGG” is adenine (A), thymine (T), guanine (G), or cytosine (C), and the G is guanine. A PAM can be CRISPR protein-specific and can be different between different base editors comprising different CRISPR protein-derived domains. A PAM can be 5′ or 3′ of a target sequence. A PAM can be upstream or downstream of a target sequence. A PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length. Often, a PAM is between 2-6 nucleotides in length. Several PAM variants are described in Table 1 below. 
     
       
         
           
               
             
               
                 TABLE1 
               
             
            
               
                   
               
               
                 Cas9 proteins and corresponding PAM sequences 
               
            
           
           
               
               
               
            
               
                   
                 Variant 
                 PAM 
               
               
                   
                   
               
               
                   
                 spCas9 
                 NGG 
               
               
                   
                   
               
               
                   
                 spCas9-VRQR 
                 NGA 
               
               
                   
                   
               
               
                   
                 spCas9-VRER 
                 NGCG 
               
               
                   
                   
               
               
                   
                 xCas9 (sp) 
                 NGN 
               
               
                   
                   
               
               
                   
                 saCas9 
                 NNGRRT 
               
               
                   
                   
               
               
                   
                 saCas9-KKH 
                 NNNRRT 
               
               
                   
                   
               
               
                   
                 spCas9-MQKSER 
                 NGCG 
               
               
                   
                   
               
               
                   
                 spCas9-MQKSER 
                 NGCN 
               
               
                   
                   
               
               
                   
                 spCas9-LRKIQK 
                 NGTN 
               
               
                   
                   
               
               
                   
                 spCas9-LRVSQK 
                 NGTN 
               
               
                   
                   
               
               
                   
                 spCas9-LRVSQL 
                 NGTN 
               
               
                   
                   
               
               
                   
                 spCas9-MQKFRAER 
                 NGC 
               
               
                   
                   
               
               
                   
                 Cpf1 
                 5′ (TTTV) 
               
               
                   
                   
               
               
                   
                 SpyMac 
                 5′-NAA-3′ 
               
               
                   
                   
               
            
           
         
       
     
     In some embodiments, the PAM is NGC. In some embodiments, the NGC PAM is recognized by a Cas9 variant. In some embodiments, the NGC PAM variant includes one or more amino acid substitutions selected from D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (collectively termed “MQKFRAER”). 
     In some embodiments, the PAM is NGT. In some embodiments, the NGT PAM is recognized by a Cas9 variant. In some embodiments, the NGT PAM variant is generated through targeted mutations at one or more residues 1335, 1337, 1135, 1136, 1218, and/or 1219. In some embodiments, the NGT PAM variant is created through targeted mutations at one or more residues 1219, 1335, 1337, 1218. In some embodiments, the NGT PAM variant is created through targeted mutations at one or more residues 1135, 1136, 1218, 1219, and 1335. In some embodiments, the NGT PAM variant is selected from the set of targeted mutations provided in Table 2 and Table 3 below. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 NGT PAM Variant Mutations at residues 1219, 1335, 1337, 1218 
               
            
           
           
               
               
               
               
               
            
               
                 Variant 
                 E1219V 
                 R1335Q 
                 T1337 
                 G1218 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 F 
                 V 
                 T 
                   
               
               
                 2 
                 F 
                 V 
                 R 
               
               
                 3 
                 F 
                 V 
                 Q 
               
               
                 4 
                 F 
                 V 
                 L 
               
               
                 5 
                 F 
                 V 
                 T 
                 R 
               
               
                 6 
                 F 
                 V 
                 R 
                 R 
               
               
                 7 
                 F 
                 V 
                 Q 
                 R 
               
               
                 8 
                 F 
                 V 
                 L 
                 R 
               
               
                 9 
                 L 
                 L 
                 T 
               
               
                 10 
                 L 
                 L 
                 R 
               
               
                 11 
                 L 
                 L 
                 Q 
               
               
                 12 
                 L 
                 L 
                 L 
               
               
                 13 
                 F 
                 I 
                 T 
               
               
                 14 
                 F 
                 I 
                 R 
               
               
                 15 
                 F 
                 I 
                 Q 
               
               
                 16 
                 F 
                 I 
                 L 
               
               
                 17 
                 F 
                 G 
                 C 
               
               
                 18 
                 H 
                 L 
                 N 
               
               
                 19 
                 F 
                 G 
                 C 
                 A 
               
               
                 20 
                 H 
                 L 
                 N 
                 V 
               
               
                 21 
                 L 
                 A 
                 W 
               
               
                 22 
                 L 
                 A 
                 F 
               
               
                 23 
                 L 
                 A 
                 Y 
               
               
                 24 
                 I 
                 A 
                 W 
               
               
                 25 
                 I 
                 A 
                 F 
               
               
                 26 
                 I 
                 A 
                 Y 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 NGT PAM Variant Mutations at residues 
               
               
                 1135, 1136, 1218, 1219, and 1335 
               
            
           
           
               
               
               
               
               
               
            
               
                 Variant 
                 D1135L 
                 S1136R 
                 G1218S 
                 E1219V 
                 R1335Q 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 27 
                 G 
                   
                   
                   
                   
               
               
                 28 
                 V 
               
               
                 29 
                 I 
               
               
                 30 
                   
                 A 
               
               
                 31 
                   
                 W 
               
               
                 32 
                   
                 H 
               
               
                 33 
                   
                 K 
               
               
                 34 
                   
                   
                 K 
               
               
                 35 
                   
                   
                 R 
               
               
                 36 
                   
                   
                 Q 
               
               
                 37 
                   
                   
                 T 
               
               
                 38 
                   
                   
                 N 
               
               
                 39 
                   
                   
                   
                 I 
               
               
                 40 
                   
                   
                   
                 A 
               
               
                 41 
                   
                   
                   
                 N 
               
               
                 42 
                   
                   
                   
                 Q 
               
               
                 43 
                   
                   
                   
                 G 
               
               
                 44 
                   
                   
                   
                 L 
               
               
                 45 
                   
                   
                   
                 S 
               
               
                 46 
                   
                   
                   
                 T 
               
               
                 47 
                   
                   
                   
                   
                 L 
               
               
                 48 
                   
                   
                   
                   
                 I 
               
               
                 49 
                   
                   
                   
                   
                 V 
               
               
                 50 
                   
                   
                   
                   
                 N 
               
               
                 51 
                   
                   
                   
                   
                 S 
               
               
                 52 
                   
                   
                   
                   
                 T 
               
               
                 53 
                   
                   
                   
                   
                 F 
               
               
                 54 
                   
                   
                   
                   
                 Y 
               
               
                 55 
                 N1286Q 
                 I1331F 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the NGT PAM variant is selected from variant 5, 7, 28, 31, or 36 in Tables 2 and 3. In some embodiments, the variants have improved NGT PAM recognition. 
     In some embodiments, the NGT PAM variants have mutations at residues 1219, 1335, 1337, and/or 1218. In some embodiments, the NGT PAM variant is selected with mutations for improved recognition from the variants provided in Table 4 below. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 NGT PAM Variant Mutations at residues 
               
               
                 1219, 1335, 1337, and 1218 
               
            
           
           
               
               
               
               
               
            
               
                 Variant 
                 E1219V 
                 R1335Q 
                 T1337 
                 G1218 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 F 
                 V 
                 T 
                   
               
               
                 2 
                 F 
                 V 
                 R 
               
               
                 3 
                 F 
                 V 
                 Q 
               
               
                 4 
                 F 
                 V 
                 L 
               
               
                 5 
                 F 
                 V 
                 T 
                 R 
               
               
                 6 
                 F 
                 V 
                 R 
                 R 
               
               
                 7 
                 F 
                 V 
                 Q 
                 R 
               
               
                 8 
                 F 
                 V 
                 L 
                 R 
               
               
                   
               
            
           
         
       
     
     In some embodiments, base editors with specificity for NGT PAM may be generated as provided in Table 5 below. 
     
       
         
           
               
             
               
                 TABLE5A 
               
             
            
               
                   
               
               
                 NGT PAM variants 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 NGTN 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 variant 
                 D1135 
                 S1136 
                 G1218 
                 E1219 
                 A1322R 
                 R1335 
                 T1337 
               
               
                   
               
               
                 Variant 1 
                 LRKIQK 
                 L 
                 R 
                 K 
                 I 
                 — 
                 Q 
                 K 
               
               
                   
               
               
                 Variant 2 
                 LRSVQK 
                 L 
                 R 
                 S 
                 V 
                 — 
                 Q 
                 K 
               
               
                   
               
               
                 Variant 3 
                 LRSVQL 
                 L 
                 R 
                 S 
                 V 
                 — 
                 Q 
                 L 
               
               
                   
               
               
                 Variant 4 
                 LRKIRQK 
                 L 
                 R 
                 K 
                 I 
                 R 
                 Q 
                 K 
               
               
                   
               
               
                 Variant 5 
                 LRSVRQK 
                 L 
                 R 
                 S 
                 V 
                 R 
                 Q 
                 K 
               
               
                   
               
               
                 Variant 6 
                 LRSVRQL 
                 L 
                 R 
                 S 
                 V 
                 R 
                 Q 
                 L 
               
               
                   
               
            
           
         
       
     
     In some embodiments the NGTN variant is variant 1. In some embodiments, the NGTN variant is variant 2. In some embodiments, the NGTN variant is variant 3. In some embodiments, the NGTN variant is variant 4. In some embodiments, the NGTN variant is variant 5. In some embodiments, the NGTN variant is variant 6. 
     In some embodiments, the Cas9 domain is a Cas9 domain from  Streptococcus pyogenes  (SpCas9). In some embodiments, the SpCas9 domain is a nuclease active SpCas9, a nuclease inactive SpCas9 (SpCas9d), or a SpCas9 nickase (SpCas9n). In some embodiments, the SpCas9 comprises a D10X mutation, or a corresponding mutation in any of the amino acid sequences provided herein, as numbered in SEQ ID NO: 1, wherein Xis any amino acid except for D. In some embodiments, the SpCas9 comprises a D10A mutation, as numbered in SEQ ID NO: 1, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SpCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence having an NGG, a NGA, or a NGCG PAM sequence. In some embodiments, the SpCas9 domain comprises one or more of a D1135X, a R1335X, and a T1337X mutation, as numbered in SEQ ID NO: 1, or a corresponding mutation in any of the amino acid sequences provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1136E, R1335Q, and T1337R mutation, as numbered in SEQ ID NO: 1, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises a D1135E, a R1335Q, and a T1337R mutation, as numbered in SEQ ID NO: 1, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises one or more of a D1135X, a R1335X, and a T1337X mutation, as numbered in SEQ ID NO: 1, or a corresponding mutation in any of the amino acid sequences provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1135V, a R1335Q, and a T1337R mutation, as numbered in SEQ ID NO: 1, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises a D1135V, a R1335Q, and a T1337R mutation, as numbered in SEQ ID NO: 1, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises one or more of a D1135X, a G1218X, as numbered in SEQ ID NO: 1, a R1335X, and a T1337X mutation, or a corresponding mutation in any of the amino acid sequences provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1135V, a G1218R, a R1335Q, and a T1337R mutation, as numbered in SEQ ID NO: 1, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises a D1135V, a G1218R, a R1335Q, and a T1337R mutation, as numbered in SEQ ID NO: 1, or corresponding mutations in any of the amino acid sequences provided herein. 
     In some embodiments, the Cas9 is a Cas9 variant having specificity for an altered PAM sequence. In some embodiments, the Additional Cas9 variants and PAM sequences are described in Miller et al., Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat Biotechnol (2020). doi.org/10.1038/s41587-020-0412-8, the entirety of which is incorporated herein by reference. in some embodiments, a Cas9 variate have no specific PAM requirements. In some embodiments, a Cas9 variant, e.g. a SpCas9 variant has specificity for a NRNH PAM, wherein R is A or G and H is A, C, or T. In some embodiments, the SpCas9 variant has specificity for a PAM sequence AAA, TAA, CAA, GAA, TAT, GAT, or CAC. In some embodiments, the SpCas9 variant comprises an amino acid substitution at position 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1218, 1219, 1221, 1249, 1256, 1264, 1290, 1318, 1317, 1320, 1321, 1323, 1332, 1333, 1335, 1337, or 1339 as numbered in SEQ ID NO: 1 or a corresponding position thereof. In some embodiments, the SpCas9 variant comprises an amino acid substitution at position 1114, 1135, 1218, 1219, 1221, 1249, 1320, 1321, 1323, 1332, 1333, 1335, or 1337 as numbered in SEQ ID NO: 1 or a corresponding position thereof. In some embodiments, the SpCas9 variant comprises an amino acid substitution at position 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1219, 1221, 1256, 1264, 1290, 1318, 1317, 1320, 1323, 1333 as numbered in SEQ ID NO: 1 or a corresponding position thereof. In some embodiments, the SpCas9 variant comprises an amino acid substitution at position 1114, 1131, 1135, 1150, 1156, 1180, 1191, 1218, 1219, 1221, 1227, 1249, 1253, 1286, 1293, 1320, 1321, 1332, 1335, 1339 as numbered in SEQ ID NO: 1 or a corresponding position thereof. In some embodiments, the SpCas9 variant comprises an amino acid substitution at position 1114, 1127, 1135, 1180, 1207, 1219, 1234, 1286, 1301, 1332, 1335, 1337, 1338, 1349 as numbered in SEQ ID NO: 1 or a corresponding position thereof. Exemplary amino acid substitutions and PAM specificity of SpCas9 variants are shown in Tables 5B, 5C, 5D, and 5E below. 
     
       
         
           
               
             
               
                 TABLE 5B 
               
             
            
               
                   
               
               
                 Additional variant mutations and PAMs 
               
            
           
           
               
               
            
               
                   
                 SpCas9 amino acid position 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 SpCas9/ 
                 1114 
                 1135 
                 1218 
                 1219 
                 1221 
                 1249 
                 1320 
                 1321 
                 1323 
                 1332 
                 1333 
                 1335 
                 1337 
               
               
                 PAM 
                 R 
                 D 
                 G 
                 E 
                 Q 
                 P 
                 A 
                 P 
                 A 
                 D 
                 R 
                 R 
                 T 
               
               
                   
               
               
                 AAA 
                   
                 N 
                   
                 V 
                 H 
                   
                   
                   
                   
                   
                 G 
                   
                   
               
               
                 AAA 
                   
                 N 
                   
                 V 
                 H 
                   
                   
                   
                   
                   
                 G 
               
               
                 AAA 
                   
                   
                   
                 V 
                   
                   
                   
                   
                   
                   
                 G 
               
               
                 TAA 
                 G 
                 N 
                   
                 V 
                   
                   
                   
                   
                   
                   
                 I 
               
               
                 TAA 
                   
                 N 
                   
                 V 
                   
                   
                   
                   
                   
                   
                 I 
                   
                 A 
               
               
                 TAA 
                 G 
                 N 
                   
                 V 
                   
                   
                   
                   
                   
                   
                 I 
                   
                 A 
               
               
                 CAA 
                   
                   
                   
                 V 
                   
                   
                   
                   
                   
                   
                 K 
               
               
                 CAA 
                   
                 N 
                   
                 V 
                   
                   
                   
                   
                   
                   
                 K 
               
               
                 CAA 
                   
                 N 
                   
                 V 
                   
                   
                   
                   
                   
                   
                 K 
               
               
                 GAA 
                   
                   
                   
                 V 
                 H 
                   
                 V 
                   
                   
                   
                 K 
               
               
                 GAA 
                   
                 N 
                   
                 V 
                   
                   
                 V 
                   
                   
                   
                 K 
               
               
                 GAA 
                   
                   
                   
                 V 
                 H 
                   
                 V 
                   
                   
                   
                 K 
               
               
                 TAT 
                   
                   
                 S 
                 V 
                 H 
                 S 
                   
                 S 
                   
                   
                   
                 L 
               
               
                 TAT 
                   
                   
                 S 
                 V 
                 H 
                 S 
                   
                 S 
                   
                   
                   
                 L 
               
               
                 TAT 
                   
                   
                 S 
                 V 
                 H 
                 S 
                   
                 S 
                   
                   
                   
                 L 
               
               
                 GAT 
                   
                   
                   
                 V 
                   
                   
                   
                   
                   
                   
                 I 
               
               
                 GAT 
                   
                   
                   
                 V 
                   
                   
                   
                   
                 D 
                   
                   
                 Q 
               
               
                 GAT 
                   
                   
                   
                 V 
                   
                   
                   
                   
                 D 
                   
                   
                 Q 
               
               
                 CAC 
                   
                   
                   
                 V 
                   
                   
                   
                   
                   
                 N 
                   
                 Q 
                 N 
               
               
                 CAC 
                   
                 N 
                   
                 V 
                   
                   
                   
                   
                   
                   
                   
                 Q 
                 N 
               
               
                 CAC 
                   
                   
                   
                 V 
                   
                   
                   
                   
                   
                 N 
                   
                 Q 
                 N 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5C 
               
             
            
               
                   
               
               
                 Additional variant mutations and PAMs 
               
            
           
           
               
               
            
               
                   
                 SpCas9 amino acid position 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 SpCas9/ 
                 1114 
                 1134 
                 1135 
                 1137 
                 1139 
                 1151 
                 1180 
                 1188 
                 1211 
                 1219 
                 1221 
                 1256 
                 1264 
                 1290 
                 1318 
                 1317 
                 1320 
                 1323 
                 1333 
               
               
                 PAM 
                 R 
                 F 
                 D 
                 P 
                 V 
                 K 
                 D 
                 K 
                 K 
                 E 
                 Q 
                 Q 
                 H 
                 V 
                 L 
                 N 
                 A 
                 A 
                 R 
               
               
                   
               
               
                 GAA 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 V 
                 H 
                   
                   
                   
                   
                   
                 V 
                   
                 K 
               
               
                 GAA 
                   
                   
                 N 
                 S 
                   
                   
                   
                   
                   
                 V 
                   
                   
                   
                   
                   
                   
                 V 
                 D 
                 K 
               
               
                 GAA 
                   
                   
                 N 
                   
                   
                   
                   
                   
                   
                 V 
                 H 
                   
                 Y 
                   
                   
                   
                 V 
                   
                 K 
               
               
                 CAA 
                   
                   
                 N 
                   
                   
                   
                   
                   
                   
                 V 
                 H 
                   
                 Y 
                   
                   
                   
                 V 
                   
                 K 
               
               
                 CAA 
                 G 
                   
                 N 
                 S 
                   
                   
                   
                   
                   
                 V 
                 H 
                   
                 Y 
                   
                   
                   
                 V 
                   
                 K 
               
               
                 CAA 
                   
                   
                 N 
                   
                   
                   
                   
                 R 
                   
                 V 
                 H 
                   
                   
                   
                   
                   
                 V 
                   
                 K 
               
               
                 CAA 
                   
                   
                 N 
                   
                   
                   
                 G 
                   
                 R 
                 V 
                 H 
                   
                 Y 
                   
                   
                   
                 V 
                   
                 K 
               
               
                 CAA 
                   
                   
                 N 
                   
                   
                   
                   
                   
                   
                 V 
                 H 
                   
                 Y 
                   
                   
                   
                 V 
                   
                 K 
               
               
                 AAA 
                   
                   
                 N 
                   
                   
                   
                 G 
                   
                   
                 V 
                 H 
                 R 
                 Y 
                   
                   
                   
                 V 
                 D 
                 K 
               
               
                 CAA 
                 G 
                   
                 N 
                   
                   
                   
                 G 
                   
                   
                 V 
                 H 
                   
                 Y 
                   
                   
                   
                 V 
                 D 
                 K 
               
               
                 CAA 
                   
                 L 
                 N 
                   
                   
                   
                 G 
                   
                   
                 V 
                 H 
                   
                 Y 
                   
                   
                 T 
                 V 
                 D 
                 K 
               
               
                 TAA 
                 G 
                   
                 N 
                   
                   
                   
                 G 
                   
                   
                 V 
                 H 
                   
                 Y 
                 G 
                 S 
                   
                 V 
                 D 
                 K 
               
               
                 TAA 
                 G 
                   
                 N 
                   
                   
                 E 
                 G 
                   
                   
                 V 
                 H 
                   
                 Y 
                   
                 S 
                   
                 V 
                   
                 K 
               
               
                 TAA 
                 G 
                   
                 N 
                   
                   
                   
                 G 
                   
                   
                 V 
                 H 
                   
                 Y 
                   
                 S 
                   
                 V 
                 D 
                 K 
               
               
                 TAA 
                 G 
                   
                 N 
                   
                   
                   
                 G 
                   
                 R 
                 V 
                 H 
                   
                   
                   
                   
                   
                 V 
                   
                 K 
               
               
                 TAA 
                   
                   
                 N 
                   
                   
                   
                 G 
                   
                 R 
                 V 
                 H 
                   
                 Y 
                   
                   
                   
                 V 
                   
                 K 
               
               
                 TAA 
                 G 
                   
                 N 
                   
                 A 
                   
                 G 
                   
                   
                 V 
                 H 
                   
                   
                   
                   
                   
                 V 
                   
                 K 
               
               
                 TAA 
                 G 
                   
                 N 
                   
                   
                   
                   
                   
                   
                 V 
                 H 
                   
                   
                   
                   
                   
                 V 
                   
                 K 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5D 
               
               
                   
               
               
                 Additional variant mutations and PAMs 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 SpCas9 amino acid position 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 SpCas9/ 
                 1114 
                 1131 
                 1135 
                 1150 
                 1156 
                 1180 
                 1191 
                 1218 
                 1219 
                 1221 
               
               
                 PAM 
                 R 
                 Y 
                 D 
                 E 
                 K 
                 D 
                 K 
                 G 
                 E 
                 Q 
               
               
                   
               
               
                 SacB.TAT 
                   
                   
                 N 
                   
                   
                   
                 N 
                   
                 V 
                 H 
               
               
                 SacB.TAT 
                   
                   
                 N 
                   
                   
                   
                   
                 S 
                 V 
                 H 
               
               
                 AAT 
                   
                   
                 N 
                   
                   
                   
                   
                 S 
                 V 
                 H 
               
               
                 TAT 
                 G 
                   
                 N 
                   
                   
                 G 
                   
                 S 
                 V 
                 H 
               
               
                 TAT 
                 G 
                   
                 N 
                   
                   
                 G 
                   
                 S 
                 V 
                 H 
               
               
                 TAT 
                 G 
                 C 
                 N 
                   
                   
                 G 
                   
                 S 
                 V 
                 H 
               
               
                 TAT 
                 G 
                 C 
                 N 
                   
                   
                 G 
                   
                 S 
                 V 
                 H 
               
               
                 TAT 
                 G 
                 C 
                 N 
                   
                   
                 G 
                   
                 S 
                 V 
                 H 
               
               
                 TAT 
                 G 
                 C 
                 N 
                   
                 E 
                 G 
                   
                 S 
                 V 
                 H 
               
               
                 TAT 
                 G 
                 C 
                 N 
                 V 
                   
                 G 
                   
                 S 
                 V 
                 H 
               
               
                 TAT 
                   
                 C 
                 N 
                   
                   
                 G 
                   
                 S 
                 V 
                 H 
               
               
                 TAT 
                 G 
                 C 
                 N 
                   
                   
                 G 
                   
                 S 
                 V 
                 H 
               
               
                   
               
            
           
           
               
               
            
               
                   
                 SpCas9 amino acid position 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 SpCas9/ 
                 1227 
                 1249 
                 1253 
                 1286 
                 1293 
                 1320 
                 1321 
                 1332 
                 1335 
                 1339 
               
               
                   
                 PAM 
                 A 
                 P 
                 E 
                 N 
                 A 
                 A 
                 P 
                 D 
                 R 
                 T 
               
               
                   
                   
               
               
                   
                 SacB.TAT 
                   
                   
                   
                   
                   
                 V 
                 S 
                   
                 L 
               
               
                   
                 SacB.TAT 
                   
                 S 
                   
                   
                   
                   
                 S 
                 G 
                 L 
               
               
                   
                 AAT 
                 V 
                 S 
                   
                 K 
                 T 
                   
                 S 
                 G 
                 L 
                 I 
               
               
                   
                 TAT 
                   
                 S 
                 K 
                   
                   
                   
                 S 
                 G 
                 L 
               
               
                   
                 TAT 
                   
                 S 
                   
                   
                   
                   
                 S 
                 G 
                 L 
               
               
                   
                 TAT 
                   
                 S 
                   
                   
                   
                   
                 S 
                 G 
                 L 
               
               
                   
                 TAT 
                   
                 S 
                   
                   
                   
                   
                 S 
                 G 
                 L 
               
               
                   
                 TAT 
                   
                 S 
                   
                   
                   
                   
                 S 
                 G 
                 L 
               
               
                   
                 TAT 
                   
                 S 
                   
                   
                   
                   
                 S 
                 G 
                 L 
               
               
                   
                 TAT 
                   
                 S 
                   
                   
                   
                   
                 S 
                 G 
                 L 
               
               
                   
                 TAT 
                   
                 S 
                   
                   
                   
                   
                 S 
                 G 
                 L 
               
               
                   
                 TAT 
                   
                 S 
                   
                   
                   
                   
                 S 
                 G 
                 L 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5E 
               
             
            
               
                   
               
               
                 Additional variant mutations and PAM. 
               
            
           
           
               
               
            
               
                   
                 SpCas9 amino acid position 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 1114 
                 1127 
                 1135 
                 1180 
                 1207 
                 1219 
                 1234 
                 1286 
                 1301 
                 1332 
                 1335 
                 1337 
                 1338 
                 1349 
               
               
                 SpCas9 
                 R 
                 D 
                 D 
                 D 
                 E 
                 E 
                 N 
                 N 
                 P 
                 D 
                 R 
                 T 
                 S 
                 H 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 SacB.CAC 
                   
                   
                 N 
                   
                   
                 V 
                   
                   
                 N 
                 Q 
                 N 
                   
                   
               
               
                 AAC 
                 G 
                   
                 N 
                   
                   
                 V 
                   
                   
                 N 
                 Q 
                 N 
               
               
                 AAC 
                 G 
                   
                 N 
                   
                   
                 V 
                   
                   
                 N 
                 Q 
                 N 
               
               
                 TAC 
                 G 
                   
                 N 
                   
                   
                 V 
                   
                   
                 N 
                 Q 
                 N 
               
               
                 TAC 
                 G 
                   
                 N 
                   
                   
                 V 
                   
                 H 
                 N 
                 Q 
                 N 
               
               
                 TAC 
                 G 
                   
                 N 
                   
                 G 
                 V 
                 D 
                 H 
                 N 
                 Q 
                 N 
               
               
                 TAC 
                 G 
                   
                 N 
                   
                   
                 V 
                   
                   
                 N 
                 Q 
                 N 
               
               
                 TAC 
                 G 
                 G 
                 N 
                 E 
                   
                 V 
                   
                 H 
                 N 
                 Q 
                 N 
               
               
                 TAC 
                 G 
                   
                 N 
                   
                   
                 V 
                   
                 H 
                 N 
                 Q 
                 N 
               
               
                 TAC 
                 G 
                   
                 N 
                   
                   
                 V 
                   
                   
                 N 
                 Q 
                 N 
                 T 
                 R 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the Cas9 is a  Neisseria  menigitidis Cas9 (NmeCas9) or a variant thereof. In some embodiments, the NmeCas9 has specificity for a NNNNGAYW 
     PAM, wherein Y is C or T and W is A or T. In some embodiments, the NmeCas9 has specificity for a NNNNGYTT PAM, wherein Y is C or T. In some embodiments, the NmeCas9 has specificity for a NNNNGTCT PAM. In some embodiments, the NmeCas9 is a Nme1Cas9. In some embodiments, the NmeCas9 has specificity for a NNNNGATT PAM, a NNNNCCTA PAM, a NNNNCCTC PAM, a NNNNCCTT PAM, a NNNNCCTG PAM, a NNNNCCGT PAM, a NNNNCCGGPAM, a NNNNCCCA PAM, a NNNNCCCT PAM, a NNNNCCCC PAM, a NNNNCCAT PAM, a NNNNCCAG PAM, a NNNNCCAT PAM, or a NNNGATT PAM. In some embodiments, the Nme1Cas9 has specificity for a NNNNGATT PAM, a NNNNCCTA PAM, a NNNNCCTC PAM, a NNNNCCTT PAM, or a NNNNCCTG PAM. In some embodiments, the NmeCas9 has specificity for a CAA PAM, a CAAA PAM, or a CCA PAM. In some embodiments, the NmeCas9 is a Nme2 Cas9. In some embodiments, the NmeCas9 has specificity for a NNNNCC (N4CC) PAM, wherein N is any one of A, G, C, or T. in some embodiments, the NmeCas9 has specificity for a NNNNCCGT PAM, a NNNNCCGGPAM, a NNNNCCCA PAM, a NNNNCCCT PAM, a NNNNCCCC PAM, a NNNNCCAT PAM, a NNNNCCAG PAM, a NNNNCCAT PAM, or a NNNGATT PAM. In some embodiments, the NmeCas9 is a Nme3Cas9. In some embodiments, the NmeCas9 has specificity for a NNNNCAAA PAM, a NNNNCC PAM, or a NNNNCNNN PAM. Additional NmeCas9 features and PAM sequences as described in Edraki et al. Mol. Cell. (2019) 73(4): 714-726 is incorporated herein by reference in its entirety. 
     An exemplary amino acid sequence of a Nme1Cas9 is provided below: 
     
       
         
           
               
            
               
                 type II CRISPR RNA-guided endonuclease Cas9 
               
               
                 [ Neisseria   meningitidis ] WP_002235 162.1 
               
               
                 (SEQ ID NO: 104) 
               
            
           
           
               
               
            
               
                 1 
                 maafkpnpin yilgldigia svgwamveid edenpiclid 
               
               
                   
               
               
                   
                 lgvrvferae vpktgdslam 
               
               
                   
               
               
                 61 
                 arrlarsvrr ltrrrahrll rarrllkreg vlqaadfden 
               
               
                   
               
               
                   
                 glikslpntp wqlraaaldr 
               
               
                   
               
               
                 121 
                 kltplewsav llhlikhrgy lsqrkneget adkelgallk 
               
               
                   
               
               
                   
                 gvadnahalq tgdfrtpael 
               
               
                   
               
               
                 181 
                 alnkfekesg hirnqrgdys htfsrkdlqa elillfekqk 
               
               
                   
               
               
                   
                 efgnphvsgg lkegietllm 
               
               
                   
               
               
                 241 
                 tqrpalsgda vqkmlghctf epaepkaakn tytaerfiwl 
               
               
                   
               
               
                   
                 tklnnlrile qgserpltdt 
               
               
                   
               
               
                 301 
                 eratlmdepy rkskltyaqa rkllgledta ffkglrygkd 
               
               
                   
               
               
                   
                 naeastlmem kayhaisral 
               
               
                   
               
               
                 361 
                 ekeglkdkks plnlspelqd eigtafslfk tdeditgrlk 
               
               
                   
               
               
                   
                 driqpeilea llkhisfdkf 
               
               
                   
               
               
                 421 
                 vqislkalrr ivplmeqgkr ydeacaeiyg dhygkkntee 
               
               
                   
               
               
                   
                 kiylppipad eirnpvvlra 
               
               
                   
               
               
                 481 
                 lsqarkving vvrrygspar ihietarevg ksfkdrkeie 
               
               
                   
               
               
                   
                 krqeenrkdr ekaaakfrey 
               
               
                   
               
               
                 541 
                 fpnfvgepks kdilklrlye qqhgkclysg keinlgrlne 
               
               
                   
               
               
                   
                 kgyveidhal pfsrtwddsf 
               
               
                   
               
               
                 601 
                 nnkvlvlgse nqnkgnqtpy eyfngkdnsr ewqefkarve 
               
               
                   
               
               
                   
                 tsrfprskkq rillqkfded 
               
               
                   
               
               
                 661 
                 gfkernlndt ryvnrflcqf vadrmrltgk gkkrvfasng 
               
               
                   
               
               
                   
                 qitnllrgfw glrkvraend 
               
               
                   
               
               
                 721 
                 rhhaldavvv acstvamqqk itrfvrykem nafdgktidk 
               
               
                   
               
               
                   
                 etgevlhqkt hfpqpweffa 
               
               
                   
               
               
                 781 
                 qevmirvfgk pdgkpefeea dtpeklrtll aeklssrpea 
               
               
                   
               
               
                   
                 vheyvtplfv srapnrkmsg 
               
               
                   
               
               
                 841 
                 qghmetvksa krldegvsvl rvpltqlklk dlekmvnrer 
               
               
                   
               
               
                   
                 epklyealka rleahkddpa 
               
               
                   
               
               
                 901 
                 kafaepfyky dkagnrtqqv kavrveqvqk tgvwvrnhng 
               
               
                   
               
               
                   
                 iadnatmvrv dvfekgdkyy 
               
               
                   
               
               
                 961 
                 lvpiyswqva kgilpdravv qgkdeedwql iddsfnfkfs 
               
               
                   
               
               
                   
                 lhpndlvevi tkkarmfgyf 
               
               
                   
               
               
                 1021 
                 aschrgtgni nirihdldhk igkngilegi gvktalsfqk 
               
               
                   
               
               
                   
                 yqidelgkei rpcrlkkrpp 
               
               
                   
               
               
                 1081 
                 vr 
               
            
           
         
       
     
     An exemplary amino acid sequence of a Nme2Cas9 is provided below: 
     
       
         
           
               
            
               
                 type II CRISPR RNA-guided endonuclease Cas9 
               
               
                 [ Neisseria   meningitidis ] WP_002230835.1 
               
               
                 (SEQ ID NO: 105) 
               
            
           
           
               
               
            
               
                 1 
                 maafkpnpin yilgldigia svgwamveid eeenpirlid 
               
               
                   
               
               
                   
                 lgvrvferae vpktgdslam 
               
               
                   
               
               
                 61 
                 arrlarsvrr ltrrrahrll rarrllkreg vlqaadfden 
               
               
                   
               
               
                   
                 glikslpntp wqlraaaldr 
               
               
                   
               
               
                 121 
                 kltplewsav llhlikhrgy lsqrkneget adkelgallk 
               
               
                   
               
               
                   
                 gvannahalq tgdfrtpael 
               
               
                   
               
               
                 181 
                 alnkfekesg hirnqrgdys htfsrkdlqa elillfekqk 
               
               
                   
               
               
                   
                 efgnphvsgg lkegietllm 
               
               
                   
               
               
                 241 
                 tqrpalsgda vqkmlghctf epaepkaakn tytaerfiwl 
               
               
                   
               
               
                   
                 tklnnlrile qgserpltdt 
               
               
                   
               
               
                 301 
                 eratlmdepy rkskltyaqa rkllgledta ffkglrygkd 
               
               
                   
               
               
                   
                 naeastlmem kayhaisral 
               
               
                   
               
               
                 361 
                 ekeglkdkks plnlsselqd eigtafslfk tdeditgrlk 
               
               
                   
               
               
                   
                 drvqpeilea llkhisfdkf 
               
               
                   
               
               
                 421 
                 vqislkalrr ivplmeqgkr ydeacaeiyg dhygkkntee 
               
               
                   
               
               
                   
                 kiylppipad eirnpvvlra 
               
               
                   
               
               
                 481 
                 lsqarkving vvrrygspar ihietarevg ksfkdrkeie 
               
               
                   
               
               
                   
                 krqeenrkdr ekaaakfrey 
               
               
                   
               
               
                 541 
                 fpnfvgepks kdilklrlye qqhgkclysg keinlvrlne 
               
               
                   
               
               
                   
                 kgyveidhal pfsrtwddsf 
               
               
                   
               
               
                 601 
                 nnkvlvlgse nqnkgnqtpy eyfngkdnsr ewqefkarve 
               
               
                   
               
               
                   
                 tsrfprskkq rillqkfded 
               
               
                   
               
               
                 661 
                 gfkecnlndt ryvnrflcqf vadhilltgk gkrrvfasng 
               
               
                   
               
               
                   
                 qitnllrgfw glrkvraend 
               
               
                   
               
               
                 721 
                 rhhaldavvv acstvamqqk itrfvrykem nafdgktidk 
               
               
                   
               
               
                   
                 etgkvlhqkt hfpqpweffa 
               
               
                   
               
               
                 781 
                 qevmirvfgk pdgkpefeea dtpeklrtll aeklssrpea 
               
               
                   
               
               
                   
                 vheyvtplfv srapnrkmsg 
               
               
                   
               
               
                 841 
                 ahkdtlrsak rfvkhnekis vkrvwlteik ladlenmvny 
               
               
                   
               
               
                   
                 kngreielye alkarleayg 
               
               
                   
               
               
                 901 
                 gnakqafdpk dnpfykkggq lvkavrvekt qesgvllnkk 
               
               
                   
               
               
                   
                 naytiadngd mvrvdvfckv 
               
               
                   
               
               
                 961 
                 dkkgknqyfi vpiyawqvae nilpdidckg yriddsytfc 
               
               
                   
               
               
                   
                 fslhkydlia fqkdekskve 
               
               
                   
               
               
                 1021 
                 fayyincdss ngrfylawhd kgskeqqfri stqnlvliqk 
               
               
                   
               
               
                   
                 yqvnelgkei rpcrlkkrpp 
               
               
                   
               
               
                 1081 
                 vr 
               
            
           
         
       
     
     In some embodiments, the Cas9 domains of any of the fusion proteins provided herein comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a Cas9 polypeptide described herein. In some embodiments, the Cas9 domains of any of the fusion proteins provided herein comprises the amino acid sequence of any Cas9 polypeptide described herein. In some embodiments, the Cas9 domains of any of the fusion proteins provided herein consists of the amino acid sequence of any Cas9 polypeptide described herein. 
     In some examples, a PAM recognized by a CRISPR protein-derived domain of a base editor disclosed herein can be provided to a cell on a separate oligonucleotide to an insert (e.g., an AAV insert) encoding the base editor. In such embodiments, providing PAM on a separate oligonucleotide can allow cleavage of a target sequence that otherwise would not be able to be cleaved, because no adjacent PAM is present on the same polynucleotide as the target sequence. 
     In an embodiment,  S. pyogenes  Cas9 (SpCas9) can be used as a CRISPR endonuclease for genome engineering. However, others can be used. In some embodiments, a different endonuclease can be used to target certain genomic targets. In some embodiments, synthetic SpCas9-derived variants with non-NGG PAM sequences can be used. Additionally, other Cas9 orthologues from various species have been identified and these “non-SpCas9s” can bind a variety of PAM sequences that can also be useful for the present disclosure. For example, the relatively large size of SpCas9 (approximately 4 kb coding sequence) can lead to plasmids carrying the SpCas9 cDNA that cannot be efficiently expressed in a cell. Conversely, the coding sequence for  Staphylococcus aureus  Cas9 (SaCas9) is approximately 1 kilobase shorter than SpCas9, possibly allowing it to be efficiently expressed in a cell. Similar to SpCas9, the SaCas9 endonuclease is capable of modifying target genes in mammalian cells in vitro and in mice in vivo. In some embodiments, a Cas protein can target a different PAM sequence. In some embodiments, a target gene can be adjacent to a Cas9 PAM, 5′-NGG, for example. In other embodiments, other Cas9 orthologs can have different PAM requirements. For example, other PAMs such as those of  S. thermophilus  (5′-NNAGAA for CRISPR1 and 5′-NGGNG for CRISPR3) and  Neisseria meningiditis  (5′-NNNNGATT) can also be found adjacent to a target gene. 
     In some embodiments, for a  S. pyogenes  system, a target gene sequence can precede (i.e., be 5′ to) a 5′-NGG PAM, and a 20-nt guide RNA sequence can base pair with an opposite strand to mediate a Cas9 cleavage adjacent to a PAM. In some embodiments, an adjacent cut can be or can be about 3 base pairs upstream of a PAM. In some embodiments, an adjacent cut can be or can be about 10 base pairs upstream of a PAM. In some embodiments, an adjacent cut can be or can be about 0-20 base pairs upstream of a PAM. For example, an adjacent cut can be next to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs upstream of a PAM. An adjacent cut can also be downstream of a PAM by 1 to 30 base pairs. The sequences of exemplary SpCas9 proteins capable of binding a PAM sequence follow: 
     The amino acid sequence of an exemplary PAM-binding SpCas9 is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 1) 
               
               
                 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE 
               
               
                   
               
               
                 DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK 
               
               
                   
               
               
                 PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ 
               
               
                   
               
               
                 SITGLYETRIDLSQLGGD 
               
            
           
         
       
     
     The amino acid sequence of an exemplary PAM-binding SpCas9n is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 102) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE 
               
               
                   
               
               
                 DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK 
               
               
                   
               
               
                 PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ 
               
               
                   
               
               
                 SITGLYETRIDLSQLGGD 
               
            
           
         
       
     
     The amino acid sequence of an exemplary PAM-binding SpEQR Cas9 is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 145) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGF   E   SPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE 
               
               
                   
               
               
                 DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK 
               
               
                   
               
               
                 PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK   Q   Y   R   STKEVLDATLIHQ 
               
               
                   
               
               
                 SITGLYETRIDLSQLGGD 
               
            
           
         
       
     
     In the above sequence, residues E1134, Q1334, and R1336, which can be mutated from D1134, R1334, and T1336 to yield a SpEQR Cas9, are underlined and in bold. 
     The amino acid sequence of an exemplary PAM-binding SpVQR Cas9 is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 146) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNEMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGF   V   SPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE 
               
               
                   
               
               
                 DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK 
               
               
                   
               
               
                 PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK   Q   Y   R   STKEVLDATLIHQ 
               
               
                   
               
               
                 SITGLYETRIDLSQLGGD 
               
            
           
         
       
     
     In the above sequence, residues V1134, Q1334, and R1336, which can be mutated from D1134, R1334, and T1336 to yield a SpVQR Cas9, are underlined and in bold. 
     The amino acid sequence of an exemplary PAM-binding SpVRER Cas9 is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 147) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGF   V   SPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASA   R   ELQKGNELALPSKYVNELYLASHYEKLKGSPE 
               
               
                   
               
               
                 DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK 
               
               
                   
               
               
                 PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK   E   Y   R   STKEVLDATLIHQ 
               
               
                   
               
               
                 SITGLYETRIDLSQLGGD. 
               
            
           
         
       
     
     In the above sequence, residues V1134, R1217, Q1334, and R1336, which can be mutated from D1134, G1217, R1334, and T1336 to yield a SpVRER Cas9, are underlined and in bold. 
     In some embodiments, the Cas9 domain is a recombinant Cas9 domain. In some embodiments, the recombinant Cas9 domain is a SpyMacCas9 domain. In some embodiments, the SpyMacCas9 domain is a nuclease active SpyMacCas9, a nuclease inactive SpyMacCas9 (SpyMacCas9d), or a SpyMacCas9 nickase (SpyMacCas9n). In some embodiments, the SaCas9 domain, the SaCas9d domain, or the SaCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SpyMacCas9 domain, the SpCas9d domain, or the SpCas9n domain can bind to a nucleic acid sequence having a NAA PAM sequence. 
     The sequence of an exemplary Cas9 A homolog of Spy Cas9 in  Streptococcus macacae  with native 5′-NAAN-3′ PAM specificity is known in the art and described, for example, by Jakimo et al., (www.biorxiv.org/content/biorxiv/early/2018/09/27/429654.full.pdf), and is provided below. 
     SpyMacCas9 
       
     
       
         
           
               
            
               
                 (SEQ ID NO: 148) 
               
               
                 MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENP 
               
               
                   
               
               
                 INASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKV 
               
               
                   
               
               
                 MGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV 
               
               
                   
               
               
                 ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDS 
               
               
                   
               
               
                 IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLT 
               
               
                   
               
               
                 KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR 
               
               
                   
               
               
                 EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY 
               
               
                   
               
               
                 PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT 
               
               
                   
               
               
                 LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEIQ 
               
               
                   
               
               
                 TVGQNGGLFDDNPKSPLEVTPSKLVPLKKELNPKKYGGYQKPTTAYPVLL 
               
               
                   
               
               
                 ITDTKQLIPISVMNKKQFEQNPVKFLRDRGYQQVGKNDFIKLPKYTLVDI 
               
               
                   
               
               
                 GDGIKRLWASSKEIHKGNQLVVSKKSQILLYHAHHLDSDLSNDYLQNHNQ 
               
               
                   
               
               
                 QFDVLFNEIISFSKKCKLGKEHIQKIENVYSNKKNSASIEELAESFIKLL 
               
               
                   
               
               
                 GFTQLGATSPFNFLGVKLNQKQYKGKKDYILPCTEGTLIRQSITGLYETR 
               
               
                   
               
               
                 VDLSKIGED. 
               
            
           
         
       
     
     In some embodiments, a variant Cas9 protein harbors, H840A, P475A, W476A, N477A, D1125A, W1126A, and D1218A mutations such that the polypeptide has a reduced ability to cleave a target DNA or RNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g., a single stranded target DNA) but retains the ability to bind a target DNA (e.g., a single stranded target DNA). As another non-limiting example, in some embodiments, the variant Cas9 protein harbors D10A, H840A, P475A, W476A, N477A, D1125A, W1126A, and D1218A mutations such that the polypeptide has a reduced ability to cleave a target DNA. Such a Cas9 protein has a reduced ability to cleave a target DNA (e.g., a single stranded target DNA) but retains the ability to bind a target DNA (e.g., a single stranded target DNA). In some embodiments, when a variant Cas9 protein harbors W476A and W1126A mutations or when the variant Cas9 protein harbors P475A, W476A, N477A, D1125A, W1126A, and D1218A mutations, the variant Cas9 protein does not bind efficiently to a PAM sequence. Thus, in some such cases, when such a variant Cas9 protein is used in a method of binding, the method does not require a PAM sequence. In other words, in some embodiments, when such a variant Cas9 protein is used in a method of binding, the method can include a guide RNA, but the method can be performed in the absence of a PAM sequence (and the specificity of binding is therefore provided by the targeting segment of the guide RNA). Other residues can be mutated to achieve the above effects (i.e., inactivate one or the other nuclease portions). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or A987 can be altered (i.e., substituted). Also, mutations other than alanine substitutions are suitable. 
     In some embodiments, a CRISPR protein-derived domain of a base editor can comprise all or a portion of a Cas9 protein with a canonical PAM sequence (NGG). In other embodiments, a Cas9-derived domain of a base editor can employ a non-canonical PAM sequence. Such sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., et al., “Broadening the targeting range of  Staphylococcus aureus  CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference. 
     Cas9 Domains with Reduced PAM Exclusivity 
     Typically, Cas9 proteins, such as Cas9 from  S. pyogenes  (spCas9), require a canonical NGG PAM sequence to bind a particular nucleic acid region, where the “N” in “NGG” is adenosine (A), thymidine (T), or cytosine (C), and the G is guanosine. This may limit the ability to edit desired bases within a genome. In some embodiments, the base editing fusion proteins provided herein may need to be placed at a precise location, for example a region comprising a target base that is upstream of the PAM. See e.g., Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage”  Nature  533, 420-424 (2016), the entire contents of which are hereby incorporated by reference. Accordingly, in some embodiments, any of the fusion proteins provided herein may contain a Cas9 domain that is capable of binding a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., et al., “Broadening the targeting range of  Staphylococcus aureus  CRISPR-Cas9 by modifying PAM recognition”  Nature Biotechnology  33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference. 
     High Fidelity Cas9 Domains 
     Some aspects of the disclosure provide high fidelity Cas9 domains. In some embodiments, high fidelity Cas9 domains are engineered Cas9 domains comprising one or more mutations that decrease electrostatic interactions between the Cas9 domain and a sugar-phosphate backbone of a DNA, as compared to a corresponding wild-type Cas9 domain. Without wishing to be bound by any particular theory, high fidelity Cas9 domains that have decreased electrostatic interactions with a sugar-phosphate backbone of DNA may have less off-target effects. In some embodiments, a Cas9 domain (e.g., a wild-type Cas9 domain) comprises one or more mutations that decreases the association between the Cas9 domain and a sugar-phosphate backbone of a DNA. In some embodiments, a Cas9 domain comprises one or more mutations that decreases the association between the Cas9 domain and a sugar-phosphate backbone of a DNA by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70%. 
     In some embodiments, any of the Cas9 fusion proteins provided herein comprise one or more of a N497X, a R661X, a Q695X, and/or a Q926X mutation, or a corresponding mutation in any of the amino acid sequences provided herein, wherein X is any amino acid. In some embodiments, any of the Cas9 fusion proteins provided herein comprise one or more of a N497A, a R661A, a Q695A, and/or a Q926A mutation, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the Cas9 domain comprises a D10A mutation, or a corresponding mutation in any of the amino acid sequences provided herein. Cas9 domains with high fidelity are known in the art and would be apparent to the skilled artisan. For example, Cas9 domains with high fidelity have been described in Kleinstiver, B. P., et al. “High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.”  Nature  529, 490-495 (2016); and Slaymaker, I. M., et al. “Rationally engineered Cas9 nucleases with improved specificity.”  Science  351, 84-88 (2015); the entire contents of each are incorporated herein by reference. 
     In some embodiments, the modified Cas9 is a high fidelity Cas9 enzyme. In some embodiments, the high fidelity Cas9 enzyme is SpCas9(K855A), eSpCas9(1.1), SpCas9-HF1, or hyper accurate Cas9 variant (HypaCas9). The modified Cas9 eSpCas9(1.1) contains alanine substitutions that weaken the interactions between the HNH/RuvC groove and the non-target DNA strand, preventing strand separation and cutting at off-target sites. Similarly, SpCas9-HF1 lowers off-target editing through alanine substitutions that disrupt Cas9&#39;s interactions with the DNA phosphate backbone. HypaCas9 contains mutations (SpCas9 N692A/M694A/Q695A/H698A) in the REC3 domain that increase Cas9 proofreading and target discrimination. All three high fidelity enzymes generate less off-target editing than wildtype Cas9. 
     An exemplary high fidelity Cas9 is provided below. 
     High Fidelity Cas9 domain mutations relative to Cas9 are shown in bold and underlined. 
                    (SEQ ID NO: 149)       DKKYSIGL   A   IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL               LEDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL               EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL               RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI               NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN               FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL               LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF               FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK               QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY               VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT   A   FDKN               LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL               LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII               KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL               KRRRYTGWG   A   LSRKLINGIRDKQSGKTILDFLKSDGFANRNFM   M   LIHDDS               LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM               GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV               ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS               IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT               KAERGGLSELDKAGFIKRQLVETR   A   ITKHVAQILDSRMNTKYDENDKLIR               EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY               PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT               LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ               TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK               GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY               SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED               NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP               IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS               ITGLYETRIDLSQLGGD            
Fusion Proteins Comprising a Cas9 Domain and a Cytidine Deaminase and/or Adenosine Deaminase
 
     Some aspects of the disclosure provide fusion proteins comprising a napDNAbp (e.g., a Cas9 domain) and one or more adenosine deaminase, cytidine deaminase domains, and/or DNA glycosylase domains. In some embodiments, the fusion protein comprises a Cas9 domain and an adenosine deaminase domain (e.g., TadA*A). It should be appreciated that the Cas9 domain may be any of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) provided herein. In some embodiments, any of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) provided herein may be fused with any of the cytidine deaminases and/or adenosine deaminases (e.g., TadA*A) provided herein. For example, and without limitation, in some embodiments, the fusion protein comprises the structure: 
     NH 2 -[cytidine deaminase]-[Cas9 domain]-[adenosine deaminase]-COOH; 
     NH 2 -[adenosine deaminase]-[Cas9 domain]-[cytidine deaminase]-COOH; 
     NH 2 -[adenosine deaminase]-[cytidine deaminase]-[Cas9 domain]-COOH; 
     NH 2 -[cytidine deaminase]-[adenosine deaminase]-[Cas9 domain]-COOH; 
     NH 2 -[Cas9 domain]-[adenosine deaminase]-[cytidine deaminase]-COOH; 
     NH 2 -[Cas9 domain]-[cytidine deaminase]-[adenosine deaminase]-COOH; 
     NH 2 -[adenosine deaminase]-[Cas9 domain]-COOH; 
     NH 2 -[Cas9 domain]-[adenosine deaminase]-COOH; 
     NH 2 -[cytidine deaminase]-[Cas9 domain]-COOH; or 
     NH 2 -[Cas9 domain]-[cytidine deaminase]-COOH. 
     In some embodiments, the fusion proteins comprising a cytidine deaminase, abasic editor, and adenosine deaminase and a napDNAbp (e.g., Cas9 domain) do not include a linker sequence. In some embodiments, a linker is present between the cytidine deaminase and/or adenosine deaminase domains and the napDNAbp. In some embodiments, the “-” used in the general architecture above indicates the presence of an optional linker. In some embodiments, the cytidine deaminase and adenosine deaminase and the napDNAbp are fused via any of the linkers provided herein. For example, in some embodiments the cytidine deaminase and/or adenosine deaminase and the napDNAbp are fused via any of the linkers provided herein. 
     Fusion Proteins Comprising a Nuclear Localization Sequence (NLS) 
     In some embodiments, the fusion proteins provided herein further comprise one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, for example a nuclear localization sequence (NLS). In one embodiment, a bipartite NLS is used. In some embodiments, a NLS comprises an amino acid sequence that facilitates the importation of a protein, that comprises an NLS, into the cell nucleus (e.g., by nuclear transport). In some embodiments, any of the fusion proteins provided herein further comprise a nuclear localization sequence (NLS). In some embodiments, the NLS is fused to the N-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the fusion protein. In some embodiments, the NLS is fused to the N-terminus of the Cas9 domain. In some embodiments, the NLS is fused to the C-terminus of an nCas9 domain or a dCas9 domain. In some embodiments, the NLS is fused to the N-terminus of the deaminase. In some embodiments, the NLS is fused to the C-terminus of the deaminase. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker. In some embodiments, the NLS comprises an amino acid sequence of any one of the NLS sequences provided or referenced herein. Additional nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., PCT/EP2000/011690, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, an NLS comprises the amino acid sequence PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 150), KRTADGSEFESPKKKRKV (SEQ ID NO: 80), KRPAATKKAGQAKKKK (SEQ ID NO: 81), KKTELQTTNAENKTKKL (SEQ ID NO: 82), KRGINDRNFWRGENGRKTR (SEQ ID NO: 83), RKSGKIAAIVVKRPRKPKKKRKV (SEQ ID NO: 151), orMDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 86). 
     In some embodiments, the NLS is present in a linker or the NLS is flanked by linkers, for example, the linkers described herein. In some embodiments, the N-terminus or C-terminus NLS is a bipartite NLS. A bipartite NLS comprises two basic amino acid clusters, which are separated by a relatively short spacer sequence (hence bipartite—2 parts, while monopartite NLSs are not). The NLS of nucleoplasmin, KR[PAATKKAGQA]KKKK (SEQ ID NO: 152), is the prototype of the ubiquitous bipartite signal: two clusters of basic amino acids, separated by a spacer of about 10 amino acids. The sequence of an exemplary bipartite NLS follows: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 150) 
               
               
                   
                 PKKKRKVEGADKRTADGSEFESPKKKRKV 
               
            
           
         
       
     
     In some embodiments, the fusion proteins of the invention do not comprise a linker sequence. In some embodiments, linker sequences between one or more of the domains or proteins are present. In some embodiments, the general architecture of exemplary Cas9 fusion proteins with an adenosine deaminase or cytidine deaminase and a Cas9 domain comprises any one of the following structures, where NLS is a nuclear localization sequence (e.g., any NLS provided herein), NH 2  is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein: 
     NH 2 -NLS-[adenosine deaminase]-[Cas9 domain]-COOH; 
     NH 2 -NLS [Cas9 domain]-[adenosine deaminase]-COOH; 
     NH 2 -[adenosine deaminase]-[Cas9 domain]-NLS—COOH; 
     NH 2 -[Cas9 domain]-[adenosine deaminase]-NLS—COOH; 
     NH 2 -NLS-[cytidine deaminase]-[Cas9 domain]-COOH; 
     NH 2 -NLS [Cas9 domain]-[cytidine deaminase]-COOH; 
     NH 2 -[cytidine deaminase]-[Cas9 domain]-NLS—COOH; or 
     NH 2 -[Cas9 domain]-[cytidine deaminase]-NLS—COOH. 
     It should be appreciated that the fusion proteins of the present disclosure may comprise one or more additional features. For example, in some embodiments, the fusion protein may comprise inhibitors, cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art. In some embodiments, the fusion protein comprises one or more His tags. 
     A vector that encodes a CRISPR enzyme comprising one or more nuclear localization sequences (NLSs) can be used. For example, there can be or be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs used. A CRISPR enzyme can comprise the NLSs at or near the ammo-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs at or near the carboxy-terminus, or any combination of these (e.g., one or more NLS at the ammo-terminus and one or more NLS at the carboxy terminus). When more than one NLS is present, each can be selected independently of others, such that a single NLS can be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies. 
     CRISPR enzymes used in the methods can comprise about 6 NLSs. An NLS is considered near the N- or C-terminus when the nearest amino acid to the NLS is within about 50 amino acids along a polypeptide chain from the N- or C-terminus, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, or 50 amino acids. 
     Nucleobase Editing Domain Described herein are base editors comprising a fusion protein that includes a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain (e.g., a deaminase domain). The base editor can be programmed to edit one or more bases in a target polynucleotide sequence by interacting with a guide polynucleotide capable of recognizing the target sequence. Once the target sequence has been recognized, the base editor is anchored on the polynucleotide where editing is to occur and the deaminase domain components of the base editor can then edit a target base. 
     In some embodiments, the nucleobase editing domain includes a deaminase domain. As particularly described herein, the deaminase domain includes a cytosine deaminase or an adenosine deaminase. In some embodiments, the terms “cytosine deaminase” and “cytidine deaminase” can be used interchangeably. In some embodiments, the terms “adenine deaminase” and “adenosine deaminase” can be used interchangeably. Details of nucleobase editing proteins are described in International PCT Application Nos. PCT/2017/045381 (WO2018/027078) and PCT/US2016/058344 (WO2017/070632), each of which is incorporated herein by reference for its entirety. Also see Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), the entire contents of which are hereby incorporated by reference. 
     A to G Editing 
     In some embodiments, a base editor described herein can comprise a deaminase domain which includes an adenosine deaminase. Such an adenosine deaminase domain of a base editor can facilitate the editing of an adenine (A) nucleobase to a guanine (G) nucleobase by deaminating the A to form inosine (I), which exhibits base pairing properties of G. Adenosine deaminase is capable of deaminating (i.e., removing an amine group) adenine of a deoxyadenosine residue in deoxyribonucleic acid (DNA). 
     In some embodiments, the nucleobase editors provided herein can be made by fusing together one or more protein domains, thereby generating a fusion protein. In certain embodiments, the fusion proteins provided herein comprise one or more features that improve the base editing activity (e.g., efficiency, selectivity, and specificity) of the fusion proteins. For example, the fusion proteins provided herein can comprise a Cas9 domain that has reduced nuclease activity. In some embodiments, the fusion proteins provided herein can have a Cas9 domain that does not have nuclease activity (dCas9), or a Cas9 domain that cuts one strand of a duplexed DNA molecule, referred to as a Cas9 nickase (nCas9). Without wishing to be bound by any particular theory, the presence of the catalytic residue (e.g., H840) maintains the activity of the Cas9 to cleave the non-edited (e.g., non-deaminated) strand containing a T opposite the targeted A. Mutation of the catalytic residue (e.g., D10 to A10) of Cas9 prevents cleavage of the edited strand containing the targeted A residue. Such Cas9 variants are able to generate a single-strand DNA break (nick) at a specific location based on the gRNA-defined target sequence, leading to repair of the non-edited strand, ultimately resulting in a T to C change on the non-edited strand. In some embodiments, an A-to-G base editor further comprises an inhibitor of inosine base excision repair, for example, a uracil glycosylase inhibitor (UGI) domain or a catalytically inactive inosine specific nuclease. Without wishing to be bound by any particular theory, the UGI domain or catalytically inactive inosine specific nuclease can inhibit or prevent base excision repair of a deaminated adenosine residue (e.g., inosine), which can improve the activity or efficiency of the base editor. 
     A base editor comprising an adenosine deaminase can act on any polynucleotide, including DNA, RNA and DNA-RNA hybrids. In certain embodiments, a base editor comprising an adenosine deaminase can deaminate a target A of a polynucleotide comprising RNA. For example, the base editor can comprise an adenosine deaminase domain capable of deaminating a target A of an RNA polynucleotide and/or a DNA-RNA hybrid polynucleotide. In an embodiment, an adenosine deaminase incorporated into a base editor comprises all or a portion of adenosine deaminase acting on RNA (ADAR, e.g., ADAR1 or ADAR2). In another embodiment, an adenosine deaminase incorporated into a base editor comprises all or a portion of adenosine deaminase acting on tRNA (ADAT). A base editor comprising an adenosine deaminase domain can also be capable of deaminating an A nucleobase of a DNA polynucleotide. In an embodiment an adenosine deaminase domain of a base editor comprises all or a portion of an ADAT comprising one or more mutations which permit the ADAT to deaminate a target A in DNA. For example, the base editor can comprise all or a portion of an ADAT from  Escherichia coli  (EcTadA) comprising one or more of the following mutations: D108N, A106V, D147Y, E155V, L84F, H123Y, I156F, or a corresponding mutation in another adenosine deaminase. 
     The adenosine deaminase can be derived from any suitable organism (e.g.,  E. coli ). In some embodiments, the adenine deaminase is a naturally-occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA). The corresponding residue in any homologous protein can be identified by e.g., sequence alignment and determination of homologous residues. The mutations in any naturally-occurring adenosine deaminase (e.g., having homology to ecTadA) that corresponds to any of the mutations described herein (e.g., any of the mutations identified in ecTadA) can be generated accordingly. 
     Adenosine Deaminases 
     In some embodiments, a base editor described herein can comprise a deaminase domain which includes an adenosine deaminase. Such an adenosine deaminase domain of a base editor can facilitate the editing of an adenine (A) nucleobase to a guanine (G) nucleobase by deaminating the A to form inosine (I), which exhibits base pairing properties of G. Adenosine deaminase is capable of deaminating (i.e., removing an amine group) adenine of a deoxyadenosine residue in deoxyribonucleic acid (DNA). 
     In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenine. In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenine in a deoxyadenosine residue of DNA. In some embodiments, the adenine deaminase is a naturally-occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA). One of skill in the art will be able to identify the corresponding residue in any homologous protein, e.g., by sequence alignment and determination of homologous residues. Accordingly, one of skill in the art would be able to generate mutations in any naturally-occurring adenosine deaminase (e.g., having homology to ecTadA) that corresponds to any of the mutations described herein, e.g., any of the mutations identified in ecTadA. In some embodiments, the adenosine deaminase is from a prokaryote. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, the adenosine deaminase is from  Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus , or  Bacillus subtilis . In some embodiments, the adenosine deaminase is from  E. coli.    
     The invention provides adenosine deaminase variants that have increased efficiency (&gt;50-60%) and specificity. In particular, the adenosine deaminase variants described herein are more likely to edit a desired base within a polynucleotide, and are less likely to edit bases that are not intended to be altered (i.e., “bystanders”). 
     In particular embodiments, the TadA is any one of the TadA described in PCT/US2017/045381 (WO 2018/027078), which is incorporated herein by reference in its entirety. 
     In some embodiments, the nucleobase editors of the invention are adenosine deaminase variants comprising an alteration in the following sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 20) 
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
               
               
                   
               
               
                 LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG 
               
               
                   
               
               
                 RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFR 
               
               
                   
               
               
                 MPRQVFNAQKKAQSSTD 
               
               
                 (also termed TadA*7.10). 
               
            
           
         
       
     
     In particular embodiments, the fusion proteins comprise a single (e.g., provided as a monomer) TadA*8 variant. In some embodiments, the TadA*8 is linked to a Cas9 nickase. In some embodiments, the fusion proteins of the invention comprise as a heterodimer of a wild-type TadA (TadA(wt)) linked to a TadA*8 variant. In other embodiments, the fusion proteins of the invention comprise as a heterodimer of a TadA*7.10 linked to a TadA*8 variant. In some embodiments, the base editor is ABE8 comprising a TadA*8 variant monomer. In some embodiments, the base editor is ABE8 comprising a heterodimer of a TadA*8 variant and a TadA(wt). In some embodiments, the base editor is ABE8 comprising a heterodimer of a TadA*8 variant and TadA*7.10. In some embodiments, the base editor is ABE8 comprising a heterodimer of a TadA*8 variant. In some embodiments, the TadA*8 variant is selected from Table 7. In some embodiments, the ABE8 is selected from Table 7. The relevant sequences follow: 
     
       
         
           
               
            
               
                 Wild-type TadA (TadA(wt)) or “the TadA reference 
               
               
                 sequence” 
               
               
                 (SEQ ID NO: 2) 
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG 
               
               
                   
               
               
                 RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG 
               
               
                   
               
               
                 RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR 
               
               
                   
               
               
                 MRRQEIKAQKKAQSSTD 
               
               
                   
               
               
                 TadA*7.10: 
               
               
                 (SEQ ID NO: 20) 
               
               
                 MSEVEFSHEYW MRHALTLAKR ARDEREVPVG AVLVLNNRVI 
               
               
                   
               
               
                 GEGWNRAIGL HDPTAHAEIM ALRQGGLVMQ NYRLIDATLY 
               
               
                   
               
               
                 VTFEPCVMCA GAMIHSRIGR VVFGVRNAKT GAAGSLMDVL 
               
               
                   
               
               
                 HYPGMNHRVE ITEGILADEC AALLCYFFRM PRQVFNAQKK 
               
               
                   
               
               
                 AQSSTD 
               
            
           
         
       
     
     In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth in any of the adenosine deaminases provided herein. It should be appreciated that adenosine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein). The disclosure provides any deaminase domains with a certain percent identity plus any of the mutations or combinations thereof described herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to a reference sequence, or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues as compared to any one of the amino acid sequences known in the art or described herein. 
     In some embodiments the TadA deaminase is a full-length  E. coli  TadA deaminase. For example, in certain embodiments, the adenosine deaminase comprises the amino acid sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 22) 
               
               
                 MRRAFITGVFFLSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNR 
               
               
                   
               
               
                 VIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVM 
               
               
                   
               
               
                 CAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILAD 
               
               
                   
               
               
                 ECAALLSDFFRMRRQEIKAQKKAQSSTD. 
               
            
           
         
       
     
     It should be appreciated, however, that additional adenosine deaminases useful in the present application would be apparent to the skilled artisan and are within the scope of this disclosure. For example, the adenosine deaminase may be a homolog of adenosine deaminase acting on tRNA (ADAT). Without limitation, the amino acid sequences of exemplary AD AT homologs include the following: 
     
       
         
           
               
            
               
                   Staphylococcus aureus  TadA: 
               
               
                 (SEQ ID NO: 23) 
               
               
                 MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRET 
               
               
                   
               
               
                 LQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIP 
               
               
                   
               
               
                 RVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFK 
               
               
                   
               
               
                 NLRANKKSTN 
               
               
                   
               
               
                   Bacillus subtilis  TadA: 
               
               
                 (SEQ ID NO: 24) 
               
               
                 MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRS 
               
               
                   
               
               
                 IAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVF 
               
               
                   
               
               
                 GAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRK 
               
               
                   
               
               
                 KKKAARKNLSE 
               
               
                   
               
               
                   Salmonella typhimurium  ( S. typhimurium ) TadA: 
               
               
                 (SEQ ID NO: 25) 
               
               
                 MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHR 
               
               
                   
               
               
                 VIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVM 
               
               
                   
               
               
                 CAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRD 
               
               
                   
               
               
                 ECATLLSDFFRMRRQEIKALKKADRAEGAGPAV 
               
               
                   
               
               
                   Shewanella putrefaciens  ( S. putrefaciens ) TadA: 
               
               
                 (SEQ ID NO: 26) 
               
               
                 MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTA 
               
               
                   
               
               
                 HAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGA 
               
               
                   
               
               
                 RDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEK 
               
               
                   
               
               
                 KALKLAQRAQQGIE 
               
               
                   
               
               
                   Haemophilus influenzae  F3031 ( H. influenzae ) TadA: 
               
               
                 (SEQ ID NO: 27) 
               
               
                 MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWN 
               
               
                   
               
               
                 LSIVQSDPTAHAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILH 
               
               
                   
               
               
                 SRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLS 
               
               
                   
               
               
                 TFFQKRREEKKIEKALLKSLSDK 
               
               
                   
               
               
                   Caulobacter crescentus  ( C. crescentus ) TadA: 
               
               
                 (SEQ ID NO: 28) 
               
               
                 MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGN 
               
               
                   
               
               
                 GPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISH 
               
               
                   
               
               
                 ARIGRVVFGADDPKGGAWHGPKFFAQPTCHWRPEVTGGVLADESADLLRG 
               
               
                   
               
               
                 FFRARRKAKI 
               
               
                   
               
               
                   Geobacter sulfurreducens  ( G. sulfurreducens ) TadA: 
               
               
                 (SEQ ID NO: 29) 
               
               
                 MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHN 
               
               
                   
               
               
                 LREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIIL 
               
               
                   
               
               
                 ARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLS 
               
               
                   
               
               
                 DFFRDLRRRKKAKATPALFIDERKVPPEP 
               
               
                   
               
               
                 An embodiment of  E. Coli  TadA (ecTadA) includes 
               
               
                 the following: 
               
               
                 (SEQ ID NO: 153) 
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
               
               
                   
               
               
                 LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG 
               
               
                   
               
               
                 RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFR 
               
               
                   
               
               
                 MPRQVFNAQKKAQSSTD 
               
            
           
         
       
     
     In some embodiments, the adenosine deaminase is from a prokaryote. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, the adenosine deaminase is from  Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus , or  Bacillus subtilis . In some embodiments, the adenosine deaminase is from  E. coli.    
     In one embodiment, a fusion protein of the invention comprises a wild-type TadA linked to TadA7.10, which is linked to Cas9 nickase. In particular embodiments, the fusion proteins comprise a single TadA7.10 domain (e.g., provided as a monomer). In other embodiments, the ABE7.10 editor comprises TadA7.10 and TadA(wt), which are capable of forming heterodimers. 
     In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth in any of the adenosine deaminases provided herein. It should be appreciated that adenosine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein). The disclosure provides any deaminase domains with a certain percent identity plus any of the mutations or combinations thereof described herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to a reference sequence, or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues as compared to any one of the amino acid sequences known in the art or described herein. 
     It should be appreciated that any of the mutations provided herein (e.g., based on the TadA reference sequence) can be introduced into other adenosine deaminases, such as  E. coli  TadA (ecTadA),  S. aureus  TadA (saTadA), or other adenosine deaminases (e.g., bacterial adenosine deaminases). It would be apparent to the skilled artisan that additional deaminases may similarly be aligned to identify homologous amino acid residues that can be mutated as provided herein. Thus, any of the mutations identified in the TadA reference sequence can be made in other adenosine deaminases (e.g., ecTadA) that have homologous amino acid residues. It should also be appreciated that any of the mutations provided herein can be made individually or in any combination in the TadA reference sequence or another adenosine deaminase. It should be appreciated that the amino acid substitutions in a TadA variant are as numbered in the TadA reference sequence (SEQ ID NO: 2), and can be a corresponding amino acid substitution or position in any other TadA variant that have homologous amino acid residues. It should be appreciated that the numbering of the specific positions or residues in the respective sequences depends on the particular protein and numbering scheme used; numbering might be different in a TadA varaiant sharing homology with the TadA reference sequence, and differences in sequences from species to species may affect numbering. One of skill in the art will be able to identify the respective residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues. 
     In some embodiments, the adenosine deaminase comprises a D108X mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D108G, D108N, D108V, D108A, or D108Y mutation, or a corresponding mutation in another adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises an A106X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A106V mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., wild-type TadA or ecTadA). 
     In some embodiments, the adenosine deaminase comprises a E155X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a E155D, E155G, or E155V mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises a D147X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D147Y, mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises an A106X, E155X, or D147X, mutation in the TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an E155D, E155G, or E155V mutation. In some embodiments, the adenosine deaminase comprises a D147Y. 
     For example, an adenosine deaminase can contain a D108N, a A106V, a E155V, and/or a D147Y mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). In some embodiments, an adenosine deaminase comprises the following group of mutations (groups of mutations are separated by a “;”) in TadA reference sequence, or corresponding mutations in another adenosine deaminase (e.g., ecTadA): D108N and A106V; D108N and E155V; D108N and D147Y; A106V and E155V; A106V and D147Y; E155V and D147Y; D108N, A106V, and E155V; D108N, A106V, and D147Y; D108N, E155V, and D147Y; A106V, E155V, and D 147Y; and D108N, A106V, E155V, and D147Y. It should be appreciated, however, that any combination of corresponding mutations provided herein can be made in an adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises one or more of a H8X, T17X, L18X, W23X, L34X, W45X, R51X, A56X, E59X, E85X, M94X, I95X, V102X, F104X, A106X, R107X, D108X, K110X, M118X, N127X, A138X, F149X, M151X, R153X, Q154X, I156X, and/or K157X mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase (e.g., ecTadA), where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of H8Y, T17S, L18E, W23L, L34S, W45L, R51H, A56E, or A56S, E59G, E85K, or E85G, M94L, I95L, V102A, F104L, A106V, R107C, or R107H, or R107P, D108G, or D108N, or D108V, or D108A, or D108Y, K110I, M118K, N127S, A138V, F149Y, M151V, R153C, Q154L, I156D, and/or K157R mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises one or more of a H8X, D108X, and/or N127X mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase (e.g., ecTadA), where X indicates the presence of any amino acid. In some embodiments, the adenosine deaminase comprises one or more of a H8Y, D108N, and/or N127S mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises one or more of H8X, R26X, M61X, L68X, M70X, A106X, D108X, A109X, N127X, D147X, R152X, Q154X, E155X, K161X, Q163X, and/or T166X mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase (e.g., ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of H8Y, R26W, M61I, L68Q, M70V, A106T, D108N, A109T, N127S, D147Y, R152C, Q154H or Q154R, E155G or E155V or E155D, K161Q, Q163H, and/or T166P mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8X, D108X, N127X, D147X, R152X, and Q154X in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8X, M61X, M70X, D108X, N127X, Q154X, E155X, and Q163X in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five, mutations selected from the group consisting of H8X, D108X, N127X, E155X, and T166X in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8X, A106X, D108X, mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8X, R26X, L68X, D108X, N127X, D147X, and E155X, or a corresponding mutation or mutations in another adenosine deaminase, where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five, mutations selected from the group consisting of H8X, D108X, A109X, N127X, and E155X in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8Y, D108N, N127S, D147Y, R152C, and Q154H in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8Y, M61I, M70V, D108N, N127S, Q154R, E155G and Q163H in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, or five, mutations selected from the group consisting of H8Y, D108N, N127S, E155V, and T166P in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of H8Y, A106T, D108N, N127S, E155D, and K161Q in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, seven, or eight mutations selected from the group consisting of H8Y, R26W, L68Q, D108N, N127S, D147Y, and E155V in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, or five, mutations selected from the group consisting of H8Y, D108N, A109T, N127S, and E155G in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA). 
     Any of the mutations provided herein and any additional mutations (e.g., based on the ecTadA amino acid sequence) can be introduced into any other adenosine deaminases. Any of the mutations provided herein can be made individually or in any combination in TadA reference sequence or another adenosine deaminase (e.g., ecTadA). 
     Details of A to G nucleobase editing proteins are described in International PCT Application No. PCT/2017/045381 (WO2018/027078) and Gaudelli, N. M., et al., “Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage” Nature, 551, 464-471 (2017), the entire contents of which are hereby incorporated by reference. 
     In some embodiments, the adenosine deaminase comprises one or more corresponding mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises a D108N, D108G, or D108V mutation in TadA reference sequence, or corresponding mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises a A106V and D108N mutation in TadA reference sequence, or corresponding mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises R107C and D108N mutations in TadA reference sequence, or corresponding mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises a H8Y, D108N, N127S, D147Y, and Q154H mutation in TadA reference sequence, or corresponding mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises a H8Y, D108N, N127S, D147Y, and E155V mutation in TadA reference sequence, or corresponding mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises a D108N, D147Y, and E155V mutation in TadA reference sequence, or corresponding mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises a H8Y, D108N, and N127S mutation in TadA reference sequence, or corresponding mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises a A106V, D108N, D147Y and E155V mutation in TadA reference sequence, or corresponding mutations in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises one or more of a S2X, H8X, I49X, L84X, H123X, N127X, I156X and/or K160X mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of S2A, H8Y, I49F, L84F, H123Y, N127S, I156F and/or K160S mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises an L84X mutation adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an L84F mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises an H123X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an H123Y mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises an I156X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an I156F mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, or seven mutations selected from the group consisting of L84X, A106X, D108X, H123X, D147X, E155X, and I156X in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of S2X, I49X, A106X, D108X, D147X, and E155X in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one, two, three, four, or five, mutations selected from the group consisting of H8X, A106X, D108X, N127X, and K160X in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA), where X indicates the presence of any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. 
     In some embodiments, the adenosine deaminase comprises one, two, three, four, five, six, or seven mutations selected from the group consisting of L84F, A106V, D108N, H123Y, D147Y, E155V, and I156F in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises one, two, three, four, five, or six mutations selected from the group consisting of S2A, I49F, A106V, D108N, D147Y, and E155V in TadA reference sequence. 
     In some embodiments, the adenosine deaminase comprises one, two, three, four, or five, mutations selected from the group consisting of H8Y, A106T, D108N, N127S, and K160S in TadA reference sequence, or a corresponding mutation or mutations in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises one or more of a E25X, R26X, R107X, A142X, and/or A143X mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase (e.g., ecTadA), where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of E25M, E25D, E25A, E25R, E25V, E25S, E25Y, R26G, R26N, R26Q, R26C, R26L, R26K, R107P, R107K, R107A, R107N, R107W, R107H, R107S, A142N, A142D, A142G, A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q and/or A143R mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase (e.g., ecTadA). In some embodiments, the adenosine deaminase comprises one or more of the mutations described herein corresponding to TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises an E25X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an E25M, E25D, E25A, E25R, E25V, E25S, or E25Y mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises an R26X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises R26G, R26N, R26Q, R26C, R26L, or R26K mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises an R107X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R107P, R107K, R107A, R107N, R107W, R107H, or R107S mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises an A142X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A142N, A142D, A142G, mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises an A143X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q and/or A143R mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises one or more of a H36X, N37X, P48X, I49X, R51X, M70X, N72X, D77X, E134X, S 146X, Q154X, K157X, and/or K161X mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase (e.g., ecTadA), where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises one or more of H36L, N37T, N37S, P48T, P48L, I49V, R51H, R51L, M70L, N72S, D77G, E134G, S146R, S146C, Q154H, K157N, and/or K161T mutation in TadA reference sequence, or one or more corresponding mutations in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises an H36X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an H36L mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises an N37X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an N37T, or N37S mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises an P48X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an P48T, or P48L mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises an R51X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an R51H, or R51L mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises an S146X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an S146R, or S146C mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises an K157X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a K157N mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises an P48X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a P48S, P48T, or P48A mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises an A142X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a A142N mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises an W23X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a W23R, or W23L mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In some embodiments, the adenosine deaminase comprises an R152X mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA), where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a R152P, or R52H mutation in TadA reference sequence, or a corresponding mutation in another adenosine deaminase (e.g., ecTadA). 
     In one embodiment, the adenosine deaminase may comprise the mutations H36L, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, E155V, I156F, and K157N. In some embodiments, the adenosine deaminase comprises the following combination of mutations relative to TadA reference sequence, where each mutation of a combination is separated by a “_” and each combination of mutations is between parentheses: 
     (A106V_D108N), 
     (R107C_D108N), 
     (H8Y_D108N_N127S_D147Y_Q154H), 
     (H8Y_D108N_N127S_D147Y_E155V), 
     (D108N_D147Y_E155V), 
     (H8Y_D108N_N127S), 
     (H8Y_D108N_N127S_D147Y_Q154H), 
     (A106V_D108N_D147Y_E155V), 
     (D108Q_D147Y_E155V), 
     (D108M_D147Y_E155V), 
     (D108L_D147Y_E155V), 
     (D108K_D147Y_E155V), 
     (D108I_D147Y_E155V), 
     (D108F_D147Y_E155V), 
     (A106V_D108N_D147Y), 
     (A106V_D108M_D147Y_E155V), 
     (E59A_A106V_D108N_D147Y_E155V), 
     (E59A cat dead_A106V_D108N_D147Y_E155V), 
     (L84F_A106V_D108N_H123Y_D147Y_E155V_I156Y), 
     (L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), 
     (R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F), 
     (E25G_R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F), 
     (E25D_R26G_L84F_A106V_R107K_D108N_H123Y_A142N_A143G_D147Y_E155V_I156F), 
     (R26Q_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F), 
     (E25M_R26G_L84F_A106V_R107P_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F), 
     (R26C_L84F_A106V_R107H_D108N_H123Y_A142N_D147Y_E155V_I156F), 
     (L84F_A106V_D108N_H123Y_A142N_A143L_D147Y_E155V_I156F), 
     (R26G_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F), 
     (E25A_R26G_L84F_A106V_R107N_D108N_H123Y_A142N_A143E_D147Y_E155V_I156F), 
     (R26G_L84F_A106V_R107H_D108N_H123Y_A142N_A143D_D147Y_E155V_I156F), 
     (A106V_D108N_A142N_D147Y_E155V), 
     (R26G_A106V_D108N_A142N_D147Y_E155V), 
     (E25D_R26G_A106V_R107K_D108N_A142N_A143G_D147Y_E155V), 
     (R26G_A106V_D108N_R107H_A142N_A143D_D147Y_E155V), 
     (E25D_R26G_A106V_D108N_A142N_D147Y_E155V), 
     (A106V_R107K_D108N_A142N_D147Y_E155V), 
     (A106V_D108N_A142N_A143G_D147Y_E155V), 
     (A106V_D108N_A142N_A143L_D147Y_E155V), 
     (H36L_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N), 
     (N37T_P48T_M70L_L84F_A106V_D108N_H123Y_D147Y_I49V_E155V_I156F), 
     (N37S_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K161T), 
     (H36L_L84F_A106V_D108N_H123Y_D147Y_Q154H_E155V_I156F), 
     (N72S_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F), 
     (H36L_P48L_L84F_A106V_D108N_H123Y_E134G_D147Y_E155V_I156F), 
     (H36L_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K157N), 
     (H36L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F), 
     (L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T), 
     (N37S_R51H_D77G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), 
     (R51L_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_K157N), 
     (D24G_Q71R_L84F_H96L_A106V_D108N_H123Y_D147Y_E155V_I156F_K160E), 
     (H36L_G67V_L84F_A106V_D108N_H123Y_S146T_D147Y_E155V_I156F), 
     (Q71L_L84F_A106V_D108N_H123Y_L137M_A143E_D147Y_E155V_I156F), 
     (E25G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_Q159L), 
     (L84F_A91T_F104I_A106V_D108N_H123Y_D147Y_E155V_I156F), 
     (N72D_L84F_A106V_D108N_H123Y_G125A_D147Y_E155V_I156F), 
     (P48S_L84F_S97C_A106V_D108N_H123Y_D147Y_E155V_I156F), 
     (W23G_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), 
     (D24G_P48L_Q71R_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F_Q159L), 
     (L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F), 
     (H36L_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N), (N37S_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F_K161T), 
     (L84F_A106V_D108N_D147Y_E155V_I156F), 
     (R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K161T), 
     (L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K161T), 
     (L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K160E_K161T), 
     (L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N_K160E), 
     (R74Q_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), 
     (R74A_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), 
     (L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), 
     (R74Q_L84F_A106V_D108N_H123Y_D147Y_E155V_I156F), 
     (L84F_R98Q_A106V_D108N_H123Y_D147Y_E155V_I156F), 
     (L84F_A106V_D108N_H123Y_R129Q_D147Y_E155V_I156F), 
     (P48S_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F), 
     (P48S_A142N), 
     (P48T_I49V_L84F_A106V_D108N_H123Y_A142N_D147Y_E155V_I156F_L157N), 
     (P48T_I49V_A142N), 
     (H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N), 
     (H36L_P48S_R51L_L84F_A106V_D108N_H123Y_S146C_A142N_D147Y_E155V_I156F 
     (H36L_P48T_I49V_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N), 
     (H36L_P48T_I49V_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N), 
     (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N), 
     (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_E155V_I156F_K157N), 
     (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_A142N_D147Y_E155V_I156F_K157N), 
     (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N), 
     (W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_E155V_I156F_K157N), 
     (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T), 
     (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152H_E155V_I156F_K157N), 
     (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N), 
     (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N), 
     (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_E155V_I156F_K157N), 
     (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142A_S146C_D147Y_R152P_E155V_I156F_K157N), 
     (W23L_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146R_D147Y_E155V_I156F_K161T), 
     (W23R_H36L_P48A_R51L_L84F_A106V_D108N_H123Y_S146C_D147Y_R152P_E155V_I156F_K157N), 
     (H36L_P48A_R51L_L84F_A106V_D108N_H123Y_A142N_S146C_D147Y_R152P_E155V_I156F_K157N). 
     In certain embodiments, the fusion proteins provided herein comprise one or more features that improve the base editing activity of the fusion proteins. For example, any of the fusion proteins provided herein may comprise a Cas9 domain that has reduced nuclease activity. In some embodiments, any of the fusion proteins provided herein may have a Cas9 domain that does not have nuclease activity (dCas9), or a Cas9 domain that cuts one strand of a duplexed DNA molecule, referred to as a Cas9 nickase (nCas9). 
     In some embodiments, the adenosine deaminase is TadA*7.10. In some embodiments, TadA*7.10 comprises at least one alteration. In particular embodiments, TadA*7.10 comprises one or more of the following alterations or additional alterations to TadA*7.10: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and Q154R. The alteration Y123H is also referred to herein as H123H (the alteration H123Y in TadA*7.10 reverted back to Y123H (wt)). In other embodiments, the TadA*7.10 comprises a combination of alterations selected from the group of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. In particular embodiments, an adenosine deaminase variant comprises a deletion of the C terminus beginning at residue 149, 150, 151, 152, 153, 154, 155, 156, and 157. 
     In some embodiments, a TadA variant comprises at least one alteration relative to TadA7.10. In some embodiments, a TadA variant comprises at least one alteration relative to a wild type TadA. Amino acid alterations in a TadA variant may be any one of the amino acid substitutions as described herein relative to TadA7.10 or wild type TadA. In some embodiments, a TadA variant, e.g. a TadA8, comprises an amino acid alteration at amino acid position 23, 26, 36, 37, 48, 49, 51, 72, 84, 87, 105, 108, 123, 125, 142, 145, 147, 152, 155, 16, 157, 161, or any combination thereof. In some embodiments, the TadA variant comprises amino acid alteration V82X relative to TadA7.10, wherein X is any amino acid other than V. In some embodiments, the TadA variant comprises a V82S alteration relative to TadA7.10. In some embodiments, amino acid X is an acidic amino acid, a basic amino acid, or a neutral amino acid. In some embodiments, a TadA variant comprises amino acid alteration T166X relative to TadA7.10, wherein X is any amino acid other than T. In some embodiments, amino acid X is an acidic amino acid, a basic amino acid, or a neutral amino acid. In some embodiments, a TadA variant comprises amino acid alteration V82X, Y147X, Q154X, I76X, Y123X, R23X, L36X, A48X, L51X, F84X, V106X, N108X, Y123X, C146X, Y147X, P152X, Q154X, V155X, F156X, N157X, T166X relative to TadA7.10, or any combination thereof, wherein X is any amino acid other than the amino acid in TadA7.10. In some embodiments, X is an acidic amino acid, a basic amino acid, or a neutral amino acid. In some embodiments, X reverts the amino acid to a wild type amino acid in the TadA reference sequence. 
     In other embodiments, a base editor of the invention is a monomer comprising an adenosine deaminase variant (e.g., TadA*8) comprising one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and/or Q154R compared to TadA*7.10 or the reference TadA sequence. In other embodiments, the adenosine deaminase variant (TadA*8) is a monomer comprising a combination of alterations selected from the group of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. In other embodiments, a base editor is a heterodimer comprising a wild-type adenosine deaminase and an adenosine deaminase variant (e.g., TadA*8) comprising one or more of the following alterations Y147T, Y147R, Q154S, Y123H, V82S, T166R, and/or Q154R. In other embodiments, the base editor is a heterodimer comprising a TadA*7.10 domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising a combination of alterations selected from the group of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. 
     In one embodiment, an adenosine deaminase is a TadA*8 that comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 21) 
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
               
               
                   
               
               
                 LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG 
               
               
                   
               
               
                 RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFR 
               
               
                   
               
               
                 MPRQVFNAQKKAQSSTD 
               
            
           
         
       
     
     In some embodiments, the TadA*8 is a truncated. In some embodiments, the truncated TadA*8 is missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to the full length TadA*8. In some embodiments, the truncated TadA*8 is missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to the full length TadA*8. In some embodiments the adenosine deaminase variant is a full-length TadA*8. 
     In some embodiments the TadA*8 is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, TadA*8.24. 
     In one embodiment, a fusion protein of the invention comprises a wild-type TadA is linked to an adenosine deaminase variant described herein (e.g., TadA*8), which is linked to Cas9 nickase. In particular embodiments, the fusion proteins comprise a single TadA*8 domain (e.g., provided as a monomer). In other embodiments, the base editor comprises TadA*8 and TadA(wt), which are capable of forming heterodimers. Exemplary sequences follow: 
     
       
         
           
               
            
               
                 TadA(wt): 
               
               
                 (SEQ ID NO: 2) 
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG 
               
               
                   
               
               
                 RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG 
               
               
                   
               
               
                 RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR 
               
               
                   
               
               
                 MRRQEIKAQKKAQSSTD 
               
               
                   
               
               
                 TadA*7.10: 
               
               
                 (SEQ ID NO: 20) 
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
               
               
                   
               
               
                 LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG 
               
               
                   
               
               
                 RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFR 
               
               
                   
               
               
                 MPRQVFNAQKKAQSSTD 
               
               
                   
               
               
                 Tad A*8: 
               
               
                 (SEQ ID NO: 21) 
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
               
               
                   
               
               
                 LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG 
               
               
                   
               
               
                 RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFR 
               
               
                   
               
               
                 MPRQVFNAQKKAQSSTD. 
               
            
           
         
       
     
     In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences set forth in any of the adenosine deaminases provided herein. It should be appreciated that adenosine deaminases provided herein may include one or more mutations (e.g., any of the mutations provided herein). The disclosure provides any deaminase domains with a certain percent identity plus any of the mutations or combinations thereof described herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to a reference sequence, or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues as compared to any one of the amino acid sequences known in the art or described herein. 
     In particular embodiments, a TadA*8 comprises one or more mutations at any of the following positions shown in bold. In other embodiments, a TadA*8 comprises one or more mutations at any of the positions shown with underlining: 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 20) 
                   
               
            
           
           
               
               
               
            
               
                 MSEVEFSHEY WMRHALTLAK RARDEREVPV GAVLVLNNRV IGEGWNRAIG 
                 50 
                   
               
               
                   
               
               
                 LHDPTAHAEI MALRQGGLVM QN Y RLIDATL Y   V   TFEPCVMC AGAMIHSRIG 
                 100 
               
               
                   
               
               
                 RVVFGVRNAK TGAAGSLMDV LH   Y   PGMNHRV EITEGILADE CAALLC   Y   FFR 
                 150 
               
               
                   
               
               
                 MPR   Q   VFNAQK KAQSS   T   D 
                   
               
            
           
         
       
     
     For example, the TadA*8 comprises alterations at amino acid position 82 and/or 166 (e.g., V82S, T166R) alone or in combination with any one or more of the following Y147T, Y147R, Q154S, Y123H, and/or Q154R. In particular embodiments, a combination of alterations is selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R. 
     In some embodiments, the adenosine deaminase is TadA*8, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 21) 
               
               
                   
                 MSEVEESHEY WMRHALTLAK RARDEREVPV GAVLVLNNRV 
               
               
                   
               
               
                   
                 IGEGWNRAIG LHDPTAHAEI MALRQGGLVM QNYRLIDATL 
               
               
                   
               
               
                   
                 YVTFEPCVMC AGAMIHSRIG RVVFGVRNAK TGAAGSLMDV 
               
               
                   
               
               
                   
                 LHYPGMNHRV EITEGILADE CAALLCTFFR MPRQVFNAQK 
               
               
                   
               
               
                   
                 KAQSSTD 
               
            
           
         
       
     
     In some embodiments, the TadA*8 is truncated. In some embodiments, the truncated TadA*8 is missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to the full length TadA*8. In some embodiments, the truncated TadA*8 is missing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to the full length TadA*8. In some embodiments the adenosine deaminase variant is a full-length TadA*8. 
     In one embodiment, a fusion protein of the invention comprises a wild-type TadA is linked to an adenosine deaminase variant described herein (e.g., TadA*8), which is linked to Cas9 nickase. In particular embodiments, the fusion proteins comprise a single TadA*8 domain (e.g., provided as a monomer). In other embodiments, the base editor comprises TadA*8 and TadA(wt), which are capable of forming heterodimers. 
     In some embodiments, a synthetic library of adenosine deaminases alleles, e.g., TadA alleles can be utilized to generate an adenosine base editor with modified base editing efficiency and/or specificity. In some embodiments, an adenosine base editor generated from a synthetic library comprises higher base editing efficiency and/or specificity. In some embodiments, an adenosine base editor generated from a synthetic library exhibits increased base editing efficiency, increased base editing specificity, reduced off-target editing, reduced bystander editing, reduced indel formation, and/or reduced spurious editing compared to an adenosine base editor with a wild type TadA. In some embodiments, an adenosine base editor generated from a synthetic library exhibits increased base editing efficiency, increased base editing specificity, reduced off-target editing, reduced bystander editing, reduced indel formation, and/or reduced spurious editing compared to an adenosine base editor with a TadA*7.10. In some embodiments, the synthetic library comprises randomized TadA portion of ABE. In some embodiments, the synthetic library comprises all 20 canonical amino acid substitutions at each position of TadA. In some embodiments, the synthetic library comprises an average frequency of 1-2 nucleotide substitution mutations per library member. In some embodiments, the synthetic library comprises background mutations found in TadA*7.10. 
     In some embodiments, the base editing system described herein comprises an ABE with TadA inserted into a Cas9. Sequences of relevant ABEs with TadA inserted into a Cas9 are provided. 
     
       
         
           
               
            
               
                 101 Cas9 TadAins 1015 
               
               
                 (SEQ ID NO: 154) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVGSSGSETPGTSESATPESSGSEVEFSHEYWMRHAL 
               
               
                   
               
               
                 TLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQG 
               
               
                   
               
               
                 GLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGS 
               
               
                   
               
               
                 LMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSST 
               
               
                   
               
               
                 DYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE 
               
               
                   
               
               
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR 
               
               
                   
               
               
                 NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL 
               
               
                   
               
               
                 GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML 
               
               
                   
               
               
                 ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 102 Cas9 TadAins 1022 
               
               
                 (SEQ ID NO: 155) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIGSSGSETPGTSESATPESSGSEVEFSHE 
               
               
                   
               
               
                 YWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAE 
               
               
                   
               
               
                 IMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNA 
               
               
                   
               
               
                 KTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQ 
               
               
                   
               
               
                 KKAQSSTDAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE 
               
               
                   
               
               
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR 
               
               
                   
               
               
                 NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL 
               
               
                   
               
               
                 GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML 
               
               
                   
               
               
                 ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 103 Cas9 TadAins 1029 
               
               
                 (SEQ ID NO: 156) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGSSGSETPGTSESATPESSGS 
               
               
                   
               
               
                 EVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLH 
               
               
                   
               
               
                 DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV 
               
               
                   
               
               
                 VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMP 
               
               
                   
               
               
                 RQVFNAQKKAQSSTDGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE 
               
               
                   
               
               
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR 
               
               
                   
               
               
                 NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL 
               
               
                   
               
               
                 GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML 
               
               
                   
               
               
                 ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 103 Cas9 TadAins 1040 
               
               
                 (SEQ ID NO: 157) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSGSSGSETPGT 
               
               
                   
               
               
                 SESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVI 
               
               
                   
               
               
                 GEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCA 
               
               
                   
               
               
                 GAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADEC 
               
               
                   
               
               
                 AALLCYFFRMPRQVFNAQKKAQSSTDNIMNFFKTEITLANGEIRKRPLIE 
               
               
                   
               
               
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR 
               
               
                   
               
               
                 NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL 
               
               
                   
               
               
                 GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML 
               
               
                   
               
               
                 ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 105 Cas9 TadAins 1068 
               
               
                 (SEQ ID NO: 158) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGEGSSGSETPGTSESATPESSGSEVEFSHEYWMR 
               
               
                   
               
               
                 HALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMAL 
               
               
                   
               
               
                 RQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGA 
               
               
                   
               
               
                 AGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQ 
               
               
                   
               
               
                 SSTDTGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR 
               
               
                   
               
               
                 NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL 
               
               
                   
               
               
                 GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML 
               
               
                   
               
               
                 ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 106 Cas9 TadAins 1247 
               
               
                 (SEQ ID NO: 159) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGGSS 
               
               
                   
               
               
                 GSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVL 
               
               
                   
               
               
                 VLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTF 
               
               
                   
               
               
                 EPCVMCAGAMIHSRIGRVVFGVRNARTGAAGSLMDVLHYPGMNHRVEITE 
               
               
                   
               
               
                 GILADECAALLCYFFRMPRQVFNAQKKAQSSTDSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 107 Cas9 TadAins 1054 
               
               
                 (SEQ ID NO: 160) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDERE 
               
               
                   
               
               
                 VPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLID 
               
               
                   
               
               
                 ATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMN 
               
               
                   
               
               
                 HRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDGEIRKRPLIE 
               
               
                   
               
               
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR 
               
               
                   
               
               
                 NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL 
               
               
                   
               
               
                 GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML 
               
               
                   
               
               
                 ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 108 Cas9 TadAins 1026 
               
               
                 (SEQ ID NO: 161) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEGSSGSETPGTSESATPESSGSEVE 
               
               
                   
               
               
                 FSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPT 
               
               
                   
               
               
                 AHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFG 
               
               
                   
               
               
                 VRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQV 
               
               
                   
               
               
                 FNAQKKAQSSTDQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE 
               
               
                   
               
               
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR 
               
               
                   
               
               
                 NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL 
               
               
                   
               
               
                 GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML 
               
               
                   
               
               
                 ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 109 Cas9 TadAins 768 
               
               
                 (SEQ ID NO: 162) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQGSSGSETPGTSESATPESSGSEVEFSHEYWMR 
               
               
                   
               
               
                 HALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMAL 
               
               
                   
               
               
                 RQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGA 
               
               
                   
               
               
                 AGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRTTQKGQKNSR 
               
               
                   
               
               
                 ERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQEL 
               
               
                   
               
               
                 DINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKK 
               
               
                   
               
               
                 MKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQIT 
               
               
                   
               
               
                 KHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREI 
               
               
                   
               
               
                 NNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQE 
               
               
                   
               
               
                 IGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGR 
               
               
                   
               
               
                 DFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP 
               
               
                   
               
               
                 KKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNP 
               
               
                   
               
               
                 IDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELAL 
               
               
                   
               
               
                 PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSK 
               
               
                   
               
               
                 RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFD 
               
               
                   
               
               
                 TTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 110.1 Cas9 TadAins 1250 
               
               
                 (SEQ ID NO: 163) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPG 
               
               
                   
               
               
                 SSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGA 
               
               
                   
               
               
                 VLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYV 
               
               
                   
               
               
                 TFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEI 
               
               
                   
               
               
                 TEGILADECAALLCYFFRMPREDNEQKQLFVEQHKHYLDEIIEQISEFSK 
               
               
                   
               
               
                 RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFD 
               
               
                   
               
               
                 TTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 110.2 Cas9 TadAins 1250 
               
               
                 (SEQ ID NO: 164) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPG 
               
               
                   
               
               
                 SSGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVP 
               
               
                   
               
               
                 VGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDAT 
               
               
                   
               
               
                 LYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHR 
               
               
                   
               
               
                 VEITEGILADECAALLCYFFRMPREDNEQKQLFVEQHKHYLDEIIEQISE 
               
               
                   
               
               
                 FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFK 
               
               
                   
               
               
                 YFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 110.3 Cas9 TadAins 1250 
               
               
                 (SEQ ID NO: 165) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQEYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPG 
               
               
                   
               
               
                 SSGSSGSETPGTSESATPESGSSSGSEVEFSHEYWMRHALTLAKRARDER 
               
               
                   
               
               
                 EVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLI 
               
               
                   
               
               
                 DATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNARTGAAGSLMDVLHYPGM 
               
               
                   
               
               
                 NHRVEITEGILADECAALLCYFFRMPREDNEQKQLFVEQHKHYLDEIIEQ 
               
               
                   
               
               
                 ISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
               
               
                   
               
               
                 AFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 110.4 Cas9 TadAins 1250 
               
               
                 (SEQ ID NO: 166) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPG 
               
               
                   
               
               
                 SSGSSGSETPGTSESATPESGSSSGSEVEFSHEYWMRHALTLAKRARDER 
               
               
                   
               
               
                 EVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLI 
               
               
                   
               
               
                 DATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNARTGAAGSLMDVLHYPGM 
               
               
                   
               
               
                 NHRVEITEGILADECAALLCYFFRMRREDNEQKQLFVEQHKHYLDEIIEQ 
               
               
                   
               
               
                 ISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
               
               
                   
               
               
                 AFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 110.5 Cas9 TadAins 1249 
               
               
                 (SEQ ID NO: 167) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSGS 
               
               
                   
               
               
                 SGSSGSETPGTSESATPESGSSSGSEVEFSHEYWMRHALTLAKRARDERE 
               
               
                   
               
               
                 VPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLID 
               
               
                   
               
               
                 ATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMN 
               
               
                   
               
               
                 HRVEITEGILADECAALLCYFFRMRRPEDNEQKQLFVEQHKHYLDEIIEQ 
               
               
                   
               
               
                 ISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
               
               
                   
               
               
                 AFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 110.5 Cas9 TadAins delta 59-66 1250 
               
               
                 (SEQ ID NO: 168) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPG 
               
               
                   
               
               
                 SSGSSGSETPGTSESATPESGSSGSEVEFSHEYWMRHALTLAKRARDERE 
               
               
                   
               
               
                 VPVGAVLVLNNRVIGEGWNRAHAEIMALRQGGLVMQNYRLIDATLYVTFE 
               
               
                   
               
               
                 PCVMCAGAMIHSRIGRVVFGVRNARTGAAGSLMDVLHYPGMNHRVEITEG 
               
               
                   
               
               
                 ILADECAALLCYFFRMPRQVFNAQKKAQSSTDEDNEQKQLFVEQHKHYLD 
               
               
                   
               
               
                 EIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTN 
               
               
                   
               
               
                 LGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGG 
               
               
                   
               
               
                 D 
               
               
                   
               
               
                 110.6 Cas9 TadAins 1251 
               
               
                 (SEQ ID NO: 169) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE 
               
               
                   
               
               
                 GSSGSSGSETPGTSESATPESGSSSGSEVEFSHEYWMRHALTLAKRARDE 
               
               
                   
               
               
                 REVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRL 
               
               
                   
               
               
                 IDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPG 
               
               
                   
               
               
                 MNHRVEITEGILADECAALLCYFFRMRRDNEQKQLFVEQHKHYLDEIIEQ 
               
               
                   
               
               
                 ISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
               
               
                   
               
               
                 AFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 110.7 Cas9 TadAins 1252 
               
               
                 (SEQ ID NO: 170) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE 
               
               
                   
               
               
                 DGSSGSSGSETPGTSESATPESGSSSGSEVEFSHEYWMRHALTLAKRARD 
               
               
                   
               
               
                 EREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYR 
               
               
                   
               
               
                 LIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP 
               
               
                   
               
               
                 GMNHRVEITEGILADECAALLCYFFRMRRNEQKQLFVEQHKHYLDEIIEQ 
               
               
                   
               
               
                 ISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
               
               
                   
               
               
                 AFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 110.8 Cas9 TadAins delta 59-66 C-truncate 1250 
               
               
                 (SEQ ID NO: 171) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPG 
               
               
                   
               
               
                 SSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGA 
               
               
                   
               
               
                 VLVLNNRVIGEGWNRAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMC 
               
               
                   
               
               
                 AGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADE 
               
               
                   
               
               
                 CAALLCYFFRMPRQEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA 
               
               
                   
               
               
                 NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKR 
               
               
                   
               
               
                 YTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 111.1 Cas9 TadAins 997 
               
               
                 (SEQ ID NO: 172) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALSHE 
               
               
                   
               
               
                 YWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAE 
               
               
                   
               
               
                 IMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNA 
               
               
                   
               
               
                 KTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQ 
               
               
                   
               
               
                 KKAQSSTDGSSGSETPGTSESATPESSGIKKYPKLESEFVYGDYKVYDVR 
               
               
                   
               
               
                 KMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET 
               
               
                   
               
               
                 GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKL 
               
               
                   
               
               
                 IARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIM 
               
               
                   
               
               
                 ERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGE 
               
               
                   
               
               
                 LQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI 
               
               
                   
               
               
                 IEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLG 
               
               
                   
               
               
                 APAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 111.2 Cas9 TadAins 997 
               
               
                 (SEQ ID NO: 173) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALSHE 
               
               
                   
               
               
                 YWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAE 
               
               
                   
               
               
                 IMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNA 
               
               
                   
               
               
                 KTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQ 
               
               
                   
               
               
                 KKAQSSTDGSSGSSGSETPGTSESATPESSGGSSIKKYPKLESEFVYGDY 
               
               
                   
               
               
                 KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLI 
               
               
                   
               
               
                 ETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPK 
               
               
                   
               
               
                 RNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKEL 
               
               
                   
               
               
                 LGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRM 
               
               
                   
               
               
                 LASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHK 
               
               
                   
               
               
                 HYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLF 
               
               
                   
               
               
                 TLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLS 
               
               
                   
               
               
                 QLGGD 
               
               
                   
               
               
                 112 delta HNH TadA 
               
               
                 (SEQ ID NO: 174) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSEVEFSHE 
               
               
                   
               
               
                 YWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAE 
               
               
                   
               
               
                 IMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNA 
               
               
                   
               
               
                 KTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQ 
               
               
                   
               
               
                 KKAQSSTDGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEND 
               
               
                   
               
               
                 KLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTAL 
               
               
                   
               
               
                 IKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFK 
               
               
                   
               
               
                 TEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKK 
               
               
                   
               
               
                 TEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVA 
               
               
                   
               
               
                 KVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIK 
               
               
                   
               
               
                 LPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKG 
               
               
                   
               
               
                 SPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH 
               
               
                   
               
               
                 RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL 
               
               
                   
               
               
                 IHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 113 N-term single TadA helix trunc 165-end 
               
               
                 (SEQ ID NO: 175) 
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
               
               
                   
               
               
                 LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG 
               
               
                   
               
               
                 RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFR 
               
               
                   
               
               
                 MPRSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSV 
               
               
                   
               
               
                 GWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR 
               
               
                   
               
               
                 TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH 
               
               
                   
               
               
                 PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRG 
               
               
                   
               
               
                 HFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARL 
               
               
                   
               
               
                 SKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQL 
               
               
                   
               
               
                 SKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAP 
               
               
                   
               
               
                 LSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG 
               
               
                   
               
               
                 ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHL 
               
               
                   
               
               
                 GELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMT 
               
               
                   
               
               
                 RKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYE 
               
               
                   
               
               
                 YFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKE 
               
               
                   
               
               
                 DYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDIL 
               
               
                   
               
               
                 EDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKL 
               
               
                   
               
               
                 INGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQ 
               
               
                   
               
               
                 GDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE 
               
               
                   
               
               
                 NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL 
               
               
                   
               
               
                 QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGK 
               
               
                   
               
               
                 SDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGF 
               
               
                   
               
               
                 IKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF 
               
               
                   
               
               
                 RKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY 
               
               
                   
               
               
                 DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN 
               
               
                   
               
               
                 GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNS 
               
               
                   
               
               
                 DKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI 
               
               
                   
               
               
                 TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS 
               
               
                   
               
               
                 AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYL 
               
               
                   
               
               
                 DEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT 
               
               
                   
               
               
                 NLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG 
               
               
                   
               
               
                 GD 
               
               
                   
               
               
                 114 N-term single TadA helix trunc 165-end delta 
               
               
                 59-65 
               
               
                 (SEQ ID NO: 176) 
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRTAH 
               
               
                   
               
               
                 AEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVR 
               
               
                   
               
               
                 NAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRSGGS 
               
               
                   
               
               
                 SGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITD 
               
               
                   
               
               
                 EYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYT 
               
               
                   
               
               
                 RRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIV 
               
               
                   
               
               
                 DEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGD 
               
               
                   
               
               
                 LNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE 
               
               
                   
               
               
                 NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDD 
               
               
                   
               
               
                 DLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIK 
               
               
                   
               
               
                 RYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFY 
               
               
                   
               
               
                 KFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAIL 
               
               
                   
               
               
                 RRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI 
               
               
                   
               
               
                 TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNE 
               
               
                   
               
               
                 LTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE 
               
               
                   
               
               
                 CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTL 
               
               
                   
               
               
                 TLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK 
               
               
                   
               
               
                 QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEH 
               
               
                   
               
               
                 IANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKG 
               
               
                   
               
               
                 QKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMY 
               
               
                   
               
               
                 VDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSE 
               
               
                   
               
               
                 EVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVE 
               
               
                   
               
               
                 TRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFY 
               
               
                   
               
               
                 KVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIA 
               
               
                   
               
               
                 KSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIV 
               
               
                   
               
               
                 WDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARK 
               
               
                   
               
               
                 KDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS 
               
               
                   
               
               
                 FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKG 
               
               
                   
               
               
                 NELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQI 
               
               
                   
               
               
                 SEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAA 
               
               
                   
               
               
                 FKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 115.1 Cas9 TadAins1004 
               
               
                 (SEQ ID NO: 177) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREV 
               
               
                   
               
               
                 PVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDA 
               
               
                   
               
               
                 TLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPRQLESEFVYGDYKVYDVRKMIAKSEQ 
               
               
                   
               
               
                 EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG 
               
               
                   
               
               
                 RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD 
               
               
                   
               
               
                 PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN 
               
               
                   
               
               
                 PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA 
               
               
                   
               
               
                 LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS 
               
               
                   
               
               
                 KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF 
               
               
                   
               
               
                 DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 115.2 Cas9 TadAins1005 
               
               
                 (SEQ ID NO: 178) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDERE 
               
               
                   
               
               
                 VPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLID 
               
               
                   
               
               
                 ATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMN 
               
               
                   
               
               
                 HRVEITEGILADECAALLCYFFRMPRQESEFVYGDYKVYDVRKMIAKSEQ 
               
               
                   
               
               
                 EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG 
               
               
                   
               
               
                 RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD 
               
               
                   
               
               
                 PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN 
               
               
                   
               
               
                 PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA 
               
               
                   
               
               
                 LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS 
               
               
                   
               
               
                 KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF 
               
               
                   
               
               
                 DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 115.3 Cas9 TadAins1006 
               
               
                 (SEQ ID NO: 179) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLEGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDER 
               
               
                   
               
               
                 EVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLI 
               
               
                   
               
               
                 DATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGM 
               
               
                   
               
               
                 NHRVEITEGILADECAALLCYFFRMPRQSEFVYGDYKVYDVRKMIAKSEQ 
               
               
                   
               
               
                 EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG 
               
               
                   
               
               
                 RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD 
               
               
                   
               
               
                 PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN 
               
               
                   
               
               
                 PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA 
               
               
                   
               
               
                 LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS 
               
               
                   
               
               
                 KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF 
               
               
                   
               
               
                 DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 115.4 Cas9 TadAins1007 
               
               
                 (SEQ ID NO: 180) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDE 
               
               
                   
               
               
                 REVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRL 
               
               
                   
               
               
                 IDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPG 
               
               
                   
               
               
                 MNHRVEITEGILADECAALLCYFFRMPRQEFVYGDYKVYDVRKMIAKSEQ 
               
               
                   
               
               
                 EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG 
               
               
                   
               
               
                 RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD 
               
               
                   
               
               
                 PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN 
               
               
                   
               
               
                 PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA 
               
               
                   
               
               
                 LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS 
               
               
                   
               
               
                 KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF 
               
               
                   
               
               
                 DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 116.1 Cas9 TadAins C-term truncate2 792 
               
               
                 (SEQ ID NO: 181) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGGSSGSETP 
               
               
                   
               
               
                 GTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNR 
               
               
                   
               
               
                 VIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVM 
               
               
                   
               
               
                 CAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILAD 
               
               
                   
               
               
                 ECAALLCYFFRMPRQSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE 
               
               
                   
               
               
                 LDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVK 
               
               
                   
               
               
                 KMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI 
               
               
                   
               
               
                 TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVRE 
               
               
                   
               
               
                 INNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ 
               
               
                   
               
               
                 EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG 
               
               
                   
               
               
                 RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD 
               
               
                   
               
               
                 PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN 
               
               
                   
               
               
                 PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA 
               
               
                   
               
               
                 LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS 
               
               
                   
               
               
                 KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF 
               
               
                   
               
               
                 DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 116.2 Cas9 TadAins C-term truncate2 791 
               
               
                 (SEQ ID NO: 182) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSSGSETPG 
               
               
                   
               
               
                 TSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV 
               
               
                   
               
               
                 IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMC 
               
               
                   
               
               
                 AGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADE 
               
               
                   
               
               
                 CAALLCYFFRMPRQGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE 
               
               
                   
               
               
                 LDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVK 
               
               
                   
               
               
                 KMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI 
               
               
                   
               
               
                 TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVRE 
               
               
                   
               
               
                 INNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ 
               
               
                   
               
               
                 EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG 
               
               
                   
               
               
                 RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD 
               
               
                   
               
               
                 PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN 
               
               
                   
               
               
                 PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA 
               
               
                   
               
               
                 LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS 
               
               
                   
               
               
                 KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF 
               
               
                   
               
               
                 DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 116.3 Cas9 TadAins C-term truncate2 790 
               
               
                 (SEQ ID NO: 183) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKEGSSGSETPGT 
               
               
                   
               
               
                 SESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVI 
               
               
                   
               
               
                 GEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCA 
               
               
                   
               
               
                 GAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADEC 
               
               
                   
               
               
                 AALLCYFFRMPRQLGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE 
               
               
                   
               
               
                 LDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVK 
               
               
                   
               
               
                 KMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI 
               
               
                   
               
               
                 TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVRE 
               
               
                   
               
               
                 INNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ 
               
               
                   
               
               
                 EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG 
               
               
                   
               
               
                 RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD 
               
               
                   
               
               
                 PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN 
               
               
                   
               
               
                 PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELA 
               
               
                   
               
               
                 LPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS 
               
               
                   
               
               
                 KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF 
               
               
                   
               
               
                 DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 117 Cas9 delta 1017-1069 
               
               
                 (SEQ ID NO: 184) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYSSGSEVEFSHEYWMRHALTLAKRARDEREVPVGA 
               
               
                   
               
               
                 VLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYV 
               
               
                   
               
               
                 TFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEI 
               
               
                   
               
               
                 TEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDGEIVWDKGRDFATVR 
               
               
                   
               
               
                 KVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGF 
               
               
                   
               
               
                 DSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEA 
               
               
                   
               
               
                 KGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVN 
               
               
                   
               
               
                 FLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD 
               
               
                   
               
               
                 ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK 
               
               
                   
               
               
                 RYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 118 Cas9 TadA-CP116ins 1067 
               
               
                 (SEQ ID NO: 185) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNMNHRVEITEGILADECAALLCYFFRMPRQVFNAQ 
               
               
                   
               
               
                 KKAQSSTDGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRAR 
               
               
                   
               
               
                 DEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNY 
               
               
                   
               
               
                 RLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHY 
               
               
                   
               
               
                 PGGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR 
               
               
                   
               
               
                 NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL 
               
               
                   
               
               
                 GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML 
               
               
                   
               
               
                 ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 119 Cas9 TadAins 701 
               
               
                 (SEQ ID NO: 186) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPV 
               
               
                   
               
               
                 GAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATL 
               
               
                   
               
               
                 YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRV 
               
               
                   
               
               
                 EITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDLTFKEDIQKAQVS 
               
               
                   
               
               
                 GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA 
               
               
                   
               
               
                 RENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLY 
               
               
                   
               
               
                 YLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNR 
               
               
                   
               
               
                 GKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA 
               
               
                   
               
               
                 GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVS 
               
               
                   
               
               
                 DFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYK 
               
               
                   
               
               
                 VYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE 
               
               
                   
               
               
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR 
               
               
                   
               
               
                 NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL 
               
               
                   
               
               
                 GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML 
               
               
                   
               
               
                 ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 120 Cas9 TadACP136ins 1248 
               
               
                 (SEQ ID NO: 187) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSMN 
               
               
                   
               
               
                 HRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDGSSGSETPGT 
               
               
                   
               
               
                 SESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVI 
               
               
                   
               
               
                 GEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCA 
               
               
                   
               
               
                 GAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 121 Cas9 TadACP136ins 1052 
               
               
                 (SEQ ID NO: 188) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLAMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDGSSGS 
               
               
                   
               
               
                 ETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVL 
               
               
                   
               
               
                 NNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEP 
               
               
                   
               
               
                 CVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGNGEIRKRPLIE 
               
               
                   
               
               
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR 
               
               
                   
               
               
                 NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL 
               
               
                   
               
               
                 GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML 
               
               
                   
               
               
                 ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 122 Cas9 TadACP136ins 1041 
               
               
                 (SEQ ID NO: 189) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSMNHRVEITEG 
               
               
                   
               
               
                 ILADECAALLCYFFRMPRQVFNAQKKAQSSTDGSSGSETPGTSESATPES 
               
               
                   
               
               
                 SGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAI 
               
               
                   
               
               
                 GLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRI 
               
               
                   
               
               
                 GRVVFGVRNAKTGAAGSLMDVLHYPGNIMNFFKTEITLANGEIRKRPLIE 
               
               
                   
               
               
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR 
               
               
                   
               
               
                 NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL 
               
               
                   
               
               
                 GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML 
               
               
                   
               
               
                 ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 123 Cas9 TadACP139ins 1299 
               
               
                 (SEQ ID NO: 190) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE 
               
               
                   
               
               
                 DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRMN 
               
               
                   
               
               
                 HRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDGSSGSETPGT 
               
               
                   
               
               
                 SESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVI 
               
               
                   
               
               
                 GEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCA 
               
               
                   
               
               
                 GAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 124 Cas9 delta 792-872 TadAins 
               
               
                 (SEQ ID NO: 191) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSEVEFSHE 
               
               
                   
               
               
                 YWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAE 
               
               
                   
               
               
                 IMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNA 
               
               
                   
               
               
                 KTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQ 
               
               
                   
               
               
                 KKAQSSTDEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA 
               
               
                   
               
               
                 GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVS 
               
               
                   
               
               
                 DFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYK 
               
               
                   
               
               
                 VYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE 
               
               
                   
               
               
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR 
               
               
                   
               
               
                 NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL 
               
               
                   
               
               
                 GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML 
               
               
                   
               
               
                 ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 125 Cas9 delta 792-906 TadAins 
               
               
                 (SEQ ID NO: 192) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSEVEFSHE 
               
               
                   
               
               
                 YWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAE 
               
               
                   
               
               
                 IMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNA 
               
               
                   
               
               
                 KTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQ 
               
               
                   
               
               
                 KKAQSSTDGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK 
               
               
                   
               
               
                 LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALI 
               
               
                   
               
               
                 KKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT 
               
               
                   
               
               
                 EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT 
               
               
                   
               
               
                 EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAK 
               
               
                   
               
               
                 VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKL 
               
               
                   
               
               
                 PKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGS 
               
               
                   
               
               
                 PEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHR 
               
               
                   
               
               
                 DKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI 
               
               
                   
               
               
                 HQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 126 TadA CP65ins 1003 
               
               
                 (SEQ ID NO: 193) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGR 
               
               
                   
               
               
                 VVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRM 
               
               
                   
               
               
                 PRQVFNAQKKAQSSTDGSSGSETPGTSESATPESSGSEVEFSHEYWMRHA 
               
               
                   
               
               
                 LTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPLESEFVYGDYK 
               
               
                   
               
               
                 VYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE 
               
               
                   
               
               
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR 
               
               
                   
               
               
                 NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL 
               
               
                   
               
               
                 GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML 
               
               
                   
               
               
                 ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 127 TadA CP65ins 1016 
               
               
                 (SEQ ID NO: 194) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVM 
               
               
                   
               
               
                 CAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILAD 
               
               
                   
               
               
                 ECAALLCYFFRMPRQVFNAQKKAQSSTDGSSGSETPGTSESATPESSGSE 
               
               
                   
               
               
                 VEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHD 
               
               
                   
               
               
                 PYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE 
               
               
                   
               
               
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR 
               
               
                   
               
               
                 NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL 
               
               
                   
               
               
                 GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML 
               
               
                   
               
               
                 ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 128 TadA CP65ins 1022 
               
               
                 (SEQ ID NO: 195) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMITAHAEIMALRQGGLVMQNYRLIDATLYV 
               
               
                   
               
               
                 TFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEI 
               
               
                   
               
               
                 TEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDGSSGSETPGTSESAT 
               
               
                   
               
               
                 PESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWN 
               
               
                   
               
               
                 RAIGLHDPAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE 
               
               
                   
               
               
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR 
               
               
                   
               
               
                 NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL 
               
               
                   
               
               
                 GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML 
               
               
                   
               
               
                 ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 129 TadA CP65ins 1029 
               
               
                 (SEQ ID NO: 196) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEITAHAEIMALRQGGLVMQNYRL 
               
               
                   
               
               
                 IDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPG 
               
               
                   
               
               
                 MNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDGSSGSETP 
               
               
                   
               
               
                 GTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNR 
               
               
                   
               
               
                 VIGEGWNRAIGLHDPGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE 
               
               
                   
               
               
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR 
               
               
                   
               
               
                 NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL 
               
               
                   
               
               
                 GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML 
               
               
                   
               
               
                 ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 130 TadA CP65ins 1041 
               
               
                 (SEQ ID NO: 197) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSTAHAEIMALR 
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAA 
               
               
                   
               
               
                 GSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQS 
               
               
                   
               
               
                 STDGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKRARDEREV 
               
               
                   
               
               
                 PVGAVLVLNNRVIGEGWNRAIGLHDPNIMNFFKTEITLANGEIRKRPLIE 
               
               
                   
               
               
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR 
               
               
                   
               
               
                 NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL 
               
               
                   
               
               
                 GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML 
               
               
                   
               
               
                 ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 131 TadA CP65ins 1054 
               
               
                 (SEQ ID NO: 198) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG 
               
               
                   
               
               
                 RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFR 
               
               
                   
               
               
                 MPRQVFNAQKKAQSSTDGSSGSETPGTSESATPESSGSEVEFSHEYWMRH 
               
               
                   
               
               
                 ALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPGEIRKRPLIE 
               
               
                   
               
               
                 TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKR 
               
               
                   
               
               
                 NSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL 
               
               
                   
               
               
                 GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML 
               
               
                   
               
               
                 ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
               
                   
               
               
                 132 TadA CP65ins 1246 
               
               
                 (SEQ ID NO: 199) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP 
               
               
                   
               
               
                 INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV 
               
               
                   
               
               
                 MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP 
               
               
                   
               
               
                 VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD 
               
               
                   
               
               
                 SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI 
               
               
                   
               
               
                 REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK 
               
               
                   
               
               
                 YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV 
               
               
                   
               
               
                 QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE 
               
               
                   
               
               
                 KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
               
               
                   
               
               
                 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGTAH 
               
               
                   
               
               
                 AEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVR 
               
               
                   
               
               
                 NAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFN 
               
               
                   
               
               
                 AQKKAQSSTDGSSGSETPGTSESATPESSGSEVEFSHEYWMRHALTLAKR 
               
               
                   
               
               
                 ARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPSPEDNEQKQLFVEQHKH 
               
               
                   
               
               
                 YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFT 
               
               
                   
               
               
                 LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQ 
               
               
                   
               
               
                 LGGD 
               
            
           
         
       
     
     In some embodiments, Accordingly, adenosine deaminase base editors were generated to insert TadA or variants thereof into the Cas9 polypeptide at the identified positions. 
     In some embodiments, a synthetic library of adenosine deaminases alleles, e.g., TadA alleles can be utilized to generate an adenosine base editor with modified base editing efficiency and/or specificity. In some embodiments, an adenosine base editor generated from a synthetic library comprises higher base editing efficiency and/or specificity. In some embodiments, an adenosine base editor generated from a synthetic library exhibits increased base editing efficiency, increased base editing specificity, reduced off-target editing, reduced bystander editing, reduced indel formation, and/or reduced spurious editing compared to an adenosine base editor with a wild type TadA. In some embodiments, an adenosine base editor generated from a synthetic library exhibits increased base editing efficiency, increased base editing specificity, reduced off-target editing, reduced bystander editing, reduced indel formation, and/or reduced spurious editing compared to an adenosine base editor with a TadA*7.10. In some embodiments, the synthetic library comprises randomized TadA portion of ABE. In some embodiments, the synthetic library comprises all 20 canonical amino acid substitutions at each position of TadA. In some embodiments, the synthetic library comprises an average frequency of 1-2 nucleotide substitution mutations per library member. In some embodiments, the synthetic library comprises background mutations found in TadA*7.10. 
     C to T Editing 
     In some embodiments, a base editor disclosed herein comprises a fusion protein comprising a cytidine deaminase capable of deaminating a target cytidine (C) base of a polynucleotide to produce uridine (U), which has the base pairing properties of thymine. In some embodiments, for example, when the polynucleotide is double-stranded (e.g., DNA), the uridine base is substituted with a thymidine base (e.g., by the cellular repair machinery) to give rise to a C:G to a T:A transition. In other embodiments, deamination of a C to U in a nucleic acid by a base editor cannot be accompanied by substitution of the U to a T. 
     The deamination of a target C in a polynucleotide to give rise to a U is a non-limiting example of a type of base editing that can be executed by a base editor described herein. In another example, a base editor comprising a cytidine deaminase domain can mediate conversion of a cytidine (C) base to a guanine (G) base. For example, a U of a polynucleotide produced by deamination of a cytidine by a cytidine deaminase domain of a base editor can be excised from the polynucleotide by a base excision repair mechanism (e.g., by a uracil DNA glycosylase (UDG) domain), producing an abasic site. The nucleobase opposite the abasic site can then be substituted (e.g., by base repair machinery) with another base, such as a C, by, for example, a translesion polymerase. Although it is typical for a nucleobase opposite an abasic site to be replaced with a C, other substitutions (e.g., A, G, or T) can also occur. 
     Accordingly, in some embodiments a base editor described herein comprises a deamination domain (e.g., cytidine deaminase domain) capable of deaminating a target C to a U in a polynucleotide. Further, as described below, the base editor can comprise additional domains which facilitate conversion of the U resulting from deamination to, in some embodiments, a T or a G. For example, a base editor comprising a cytidine deaminase domain can further comprise a uracil glycosylase inhibitor (UGI) domain to mediate substitution of a U by a T, completing a C-to-T base editing event. In another example, a base editor can incorporate a translesion polymerase to improve the efficiency of C-to-G base editing, since a translesion polymerase can facilitate incorporation of a C opposite an abasic site (i.e., resulting in incorporation of a G at the abasic site and completing the C-to-G base editing event). In some embodiments, the additional domains are internally fused along with the deaminase domain. 
     A base editor comprising a cytidine deaminase as a domain can deaminate a target C in any polynucleotide, including DNA, RNA and DNA-RNA hybrids. Typically, a cytidine deaminase catalyzes a C nucleobase that is positioned in the context of a single-stranded portion of a polynucleotide. In some embodiments, the entire polynucleotide comprising a target C can be single-stranded. For example, a cytidine deaminase incorporated into the base editor can deaminate a target C in a single-stranded RNA polynucleotide. In other embodiments, a base editor comprising a cytidine deaminase domain can act on a double-stranded polynucleotide, but the target C can be positioned in a portion of the polynucleotide which at the time of the deamination reaction is in a single-stranded state. For example, in embodiments where the napDNAbp domain comprises a Cas12 domain, several nucleotides can be left unpaired during formation of the Cas12-gRNA-target DNA complex, resulting in formation of a Cas12 “R-loop complex.” These unpaired nucleotides can form a bubble of single-stranded DNA that can serve as a substrate for a single-strand specific nucleotide deaminase enzyme (e.g., a cytidine deaminase). 
     In some embodiments, a cytidine deaminase of a base editor can comprise all or a portion of an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. The APOBEC family comprises evolutionarily conserved cytidine deaminases. Members of this family are C-to-U editing enzymes. The N-terminal domain of APOBEC-like proteins is the catalytic domain, while the C-terminal domain is a pseudocatalytic domain. More specifically, the catalytic domain is a zinc dependent cytidine deaminase domain and is important for cytidine deamination. APOBEC family members include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D (now referred to as “APOBEC3E”), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and Activation-induced (cytidine) deaminase (AID). A number of base editors comprising modified cytidine deaminases are commercially available, including but not limited to SaBE3, SaKKH-BE3, VQR-BE3, EQR-BE3, VRER-BE3, YE1-BE3, EE-BE3, YE2-BE3, and YEE-BE3, which are available from Addgene (plasmids 85169, 85170, 85171, 85172, 85173, 85174, 85175, 85176, 85177). In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of an APOBEC1 deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC2 deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of is an APOBEC3 deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of an APOBEC3A deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC3B deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC3C deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC3D deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC3E deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC3F deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC3G deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC3H deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of APOBEC4 deaminase. In some embodiments, a deaminase incorporated into a base editor comprises all or a portion of activation-induced deaminase (AID). 
     In some embodiments a deaminase incorporated into a base editor comprises all or a portion of cytidine deaminase 1 (CDA1). It will be appreciated that a base editor can comprise a deaminase from any suitable organism (e.g., a human or a rat). In some embodiments, a deaminase domain of a base editor is from a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase domain of the base editor is derived from rat (e.g., rat APOBEC1). In some embodiments, the deaminase domain of the base editor is human APOBEC1. In some embodiments, the deaminase domain of the base editor is pmCDA1. 
     The base sequence and amino acid sequence of PmCDA1 and the base sequence and amino acid sequence of CDS of human AID are shown herein below. 
     
       
         
           
               
               
            
               
                   
                 &gt;tr|A5H718|A5H718_PETMA Cytosine deaminase 
               
               
                   
                 OS =  Petromyzon marinus  OX = 7757 
               
               
                   
                 PE = 2 SV = 1 
               
               
                   
                 (SEQ ID NO: 200) 
               
               
                   
                 MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYV 
               
               
                   
                   
               
               
                   
                 LFELKRRGERRACFWGYAVNKPQSGTERGIHAEIF 
               
               
                   
                   
               
               
                   
                 SIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKIL 
               
               
                   
                   
               
               
                   
                 EWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWN 
               
               
                   
                   
               
               
                   
                 LRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENR 
               
               
                   
                   
               
               
                   
                 WLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV 
               
               
                   
                   
               
               
                   
                 &gt;EF094822.1  Petromyzon marinus  isolate 
               
               
                   
                 PmCDA.21 cytosine deaminase mRNA, complete 
               
               
                   
                 cds 
               
               
                   
                 (SEQ ID NO: 201) 
               
               
                   
                 TGACACGACACAGCCGTGTATATGAGGAAGGGTAG 
               
               
                   
                   
               
               
                   
                 CTGGATGGGGGGGGGGGGAATACGTTCAGAGAGGA 
               
               
                   
                   
               
               
                   
                 CATTAGCGAGCGTCTTGTTGGTGGCCTTGAGTCTA 
               
               
                   
                   
               
               
                   
                 GACACCTGCAGACATGACCGACGCTGAGTACGTGA 
               
               
                   
                   
               
               
                   
                 GAATCCATGAGAAGTTGGACATCTACACGTTTAAG 
               
               
                   
                   
               
               
                   
                 AAACAGTTTTTCAACAACAAAAAATCCGTGTCGCA 
               
               
                   
                   
               
               
                   
                 TAGATGCTACGTTCTCTTTGAATTAAAACGACGGG 
               
               
                   
                   
               
               
                   
                 GTGAACGTAGAGCGTGTTTTTGGGGCTATGCTGTG 
               
               
                   
                   
               
               
                   
                 AATAAACCACAGAGCGGGACAGAACGTGGAATTCA 
               
               
                   
                   
               
               
                   
                 CGCCGAAATCTTTAGCATTAGAAAAGTCGAAGAAT 
               
               
                   
                   
               
               
                   
                 ACCTGCGCGACAACCCCGGACAATTCACGATAAAT 
               
               
                   
                   
               
               
                   
                 TGGTACTCATCCTGGAGTCCTTGTGCAGATTGCGC 
               
               
                   
                   
               
               
                   
                 TGAAAAGATCTTAGAATGGTATAACCAGGAGCTGC 
               
               
                   
                   
               
               
                   
                 GGGGGAACGGCCACACTTTGAAAATCTGGGCTTGC 
               
               
                   
                   
               
               
                   
                 AAACTCTATTACGAGAAAAATGCGAGGAATCAAAT 
               
               
                   
                   
               
               
                   
                 TGGGCTGTGGAACCTCAGAGATAACGGGGTTGGGT 
               
               
                   
                   
               
               
                   
                 TGAATGTAATGGTAAGTGAACACTACCAATGTTGC 
               
               
                   
                   
               
               
                   
                 AGGAAAATATTCATCCAATCGTCGCACAATCAATT 
               
               
                   
                   
               
               
                   
                 GAATGAGAATAGATGGCTTGAGAAGACTTTGAAGC 
               
               
                   
                   
               
               
                   
                 GAGCTGAAAAACGACGGAGCGAGTTGTCCATTATG 
               
               
                   
                   
               
               
                   
                 ATTCAGGTAAAAATACTCCACACCACTAAGAGTCC 
               
               
                   
                   
               
               
                   
                 TGCTGTTTAAGAGGCTATGCGGATGGTTTTC 
               
               
                   
                   
               
               
                   
                 &gt;tr|Q6QJ80|Q6QJ80_HUMAN Activation- 
               
               
                   
                 induced cytidine deaminase 
               
               
                   
                 OS =  Homo sapiens  OX = 9606 
               
               
                   
                 GN = AICDA PE = 2 SV = 1 
               
               
                   
                 (SEQ ID NO: 202) 
               
               
                   
                 MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYWKRR 
               
               
                   
                   
               
               
                   
                 DSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLD 
               
               
                   
                   
               
               
                   
                 PGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSL 
               
               
                   
                   
               
               
                   
                 RIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTF 
               
               
                   
                   
               
               
                   
                 KAPV 
               
               
                   
                   
               
               
                   
                 &gt;NG_011588.1:5001-15681  Homo sapiens   
               
               
                   
                 activation induced cytidine deaminase 
               
               
                   
                 (AICDA), 
               
               
                   
                 RefSeqGene (LRG17) on chromosome 12 
               
               
                   
                 (SEQ ID NO: 203) 
               
               
                   
                 AGAGAACCATCATTAATTGAAGTGAGATTTTTCTG 
               
               
                   
                   
               
               
                   
                 GCCTGAGACTTGCAGGGAGGCAAGAAGACACTCTG 
               
               
                   
                   
               
               
                   
                 GACACCACTATGGACAGGTAAAGAGGCAGTCTTCT 
               
               
                   
                   
               
               
                   
                 CGTGGGTGATTGCACTGGCCTTCCTCTCAGAGCAA 
               
               
                   
                   
               
               
                   
                 ATCTGAGTAATGAGACTGGTAGCTATCCCTTTCTC 
               
               
                   
                   
               
               
                   
                 TCATGTAACTGTCTGACTGATAAGATCAGCTTGAT 
               
               
                   
                   
               
               
                   
                 CAATATGCATATATATTTTTTGATCTGTCTCCTTT 
               
               
                   
                   
               
               
                   
                 TCTTCTATTCAGATCTTATACGCTGTCAGCCCAAT 
               
               
                   
                   
               
               
                   
                 TCTTTCTGTTTCAGACTTCTCTTGATTTCCCTCTT 
               
               
                   
                   
               
               
                   
                 TTTCATGTGGCAAAAGAAGTAGTGCGTACAATGTA 
               
               
                   
                   
               
               
                   
                 CTGATTCGTCCTGAGATTTGTACCATGGTTGAAAC 
               
               
                   
                   
               
               
                   
                 TAATTTATGGTAATAATATTAACATAGCAAATCTT 
               
               
                   
                   
               
               
                   
                 TAGAGACTCAAATCATGAAAAGGTAATAGCAGTAC 
               
               
                   
                   
               
               
                   
                 TGTACTAAAAACGGTAGTGCTAATTTTCGTAATAA 
               
               
                   
                   
               
               
                   
                 TTTTGTAAATATTCAACAGTAAAACAACTTGAAGA 
               
               
                   
                   
               
               
                   
                 CACACTTTCCTAGGGAGGCGTTACTGAAATAATTT 
               
               
                   
                   
               
               
                   
                 AGCTATAGTAAGAAAATTTGTAATTTTAGAAATGC 
               
               
                   
                   
               
               
                   
                 CAAGCATTCTAAATTAATTGCTTGAAAGTCACTAT 
               
               
                   
                   
               
               
                   
                 GATTGTGTCCATTATAAGGAGACAAATTCATTCAA 
               
               
                   
                   
               
               
                   
                 GCAAGTTATTTAATGTTAAAGGCCCAATTGTTAGG 
               
               
                   
                   
               
               
                   
                 CAGTTAATGGCACTTTTACTATTAACTAATCTTTC 
               
               
                   
                   
               
               
                   
                 CATTTGTTCAGACGTAGCTTAACTTACCTCTTAGG 
               
               
                   
                   
               
               
                   
                 TGTGAATTTGGTTAAGGTCCTCATAATGTCTTTAT 
               
               
                   
                   
               
               
                   
                 GTGCAGTTTTTGATAGGTTATTGTCATAGAACTTA 
               
               
                   
                   
               
               
                   
                 TTCTATTCCTACATTTATGATTACTATGGATGTAT 
               
               
                   
                   
               
               
                   
                 GAGAATAACACCTAATCCTTATACTTTACCTCAAT 
               
               
                   
                   
               
               
                   
                 TTAACTCCTTTATAAAGAACTTACATTACAGAATA 
               
               
                   
                   
               
               
                   
                 AAGATTTTTTAAAAATATATTTTTTTGTAGAGACA 
               
               
                   
                   
               
               
                   
                 GGGTCTTAGCCCAGCCGAGGCTGGTCTCTAAGTCC 
               
               
                   
                   
               
               
                   
                 TGGCCCAAGCGATCCTCCTGCCTGGGCCTCCTAAA 
               
               
                   
                   
               
               
                   
                 GTGCTGGAATTATAGACATGAGCCATCACATCCAA 
               
               
                   
                   
               
               
                   
                 TATACAGAATAAAGATTTTTAATGGAGGATTTAAT 
               
               
                   
                   
               
               
                   
                 GTTCTTCAGAAAATTTTCTTGAGGTCAGACAATGT 
               
               
                   
                   
               
               
                   
                 CAAATGTCTCCTCAGTTTACACTGAGATTTTGAAA 
               
               
                   
                   
               
               
                   
                 ACAAGTCTGAGCTATAGGTCCTTGTGAAGGGTCCA 
               
               
                   
                   
               
               
                   
                 TTGGAAATACTTGTTCAAAGTAAAATGGAAAGCAA 
               
               
                   
                   
               
               
                   
                 AGGTAAAATCAGCAGTTGAAATTCAGAGAAAGACA 
               
               
                   
                   
               
               
                   
                 GAAAAGGAGAAAAGATGAAATTCAACAGGACAGAA 
               
               
                   
                   
               
               
                   
                 GGGAAATATATTATCATTAAGGAGGACAGTATCTG 
               
               
                   
                   
               
               
                   
                 TAGAGCTCATTAGTGATGGCAAAATGACTTGGTCA 
               
               
                   
                   
               
               
                   
                 GGATTATTTTTAACCCGCTTGTTTCTGGTTTGCAC 
               
               
                   
                   
               
               
                   
                 GGCTGGGGATGCAGCTAGGGTTCTGCCTCAGGGAG 
               
               
                   
                   
               
               
                   
                 CACAGCTGTCCAGAGCAGCTGTCAGCCTGCAAGCC 
               
               
                   
                   
               
               
                   
                 TGAAACACTCCCTCGGTAAAGTCCTTCCTACTCAG 
               
               
                   
                   
               
               
                   
                 GACAGAAATGACGAGAACAGGGAGCTGGAAACAGG 
               
               
                   
                   
               
               
                   
                 CCCCTAACCAGAGAAGGGAAGTAATGGATCAACAA 
               
               
                   
                   
               
               
                   
                 AGTTAACTAGCAGGTCAGGATCACGCAATTCATTT 
               
               
                   
                   
               
               
                   
                 CACTCTGACTGGTAACATGTGACAGAAACAGTGTA 
               
               
                   
                   
               
               
                   
                 GGCTTATTGTATTTTCATGTAGAGTAGGACCCAAA 
               
               
                   
                   
               
               
                   
                 AATCCACCCAAAGTCCTTTATCTATGCCACATCCT 
               
               
                   
                   
               
               
                   
                 TCTTATCTATACTTCCAGGACACTTTTTCTTCCTT 
               
               
                   
                   
               
               
                   
                 ATGATAAGGCTCTCTCTCTCTCCACACACACACAC 
               
               
                   
                   
               
               
                   
                 ACACACACACACACACACACACACACACACACAAA 
               
               
                   
                   
               
               
                   
                 CACACACCCCGCCAACCAAGGTGCATGTAAAAAGA 
               
               
                   
                   
               
               
                   
                 TGTAGATTCCTCTGCCTTTCTCATCTACACAGCCC 
               
               
                   
                   
               
               
                   
                 AGGAGGGTAAGTTAATATAAGAGGGATTTATTGGT 
               
               
                   
                   
               
               
                   
                 AAGAGATGATGCTTAATCTGTTTAACACTGGGCCT 
               
               
                   
                   
               
               
                   
                 CAAAGAGAGAATTTCTTTTCTTCTGTACTTATTAA 
               
               
                   
                   
               
               
                   
                 GCACCTATTATGTGTTGAGCTTATATATACAAAGG 
               
               
                   
                   
               
               
                   
                 GTTATTATATGCTAATATAGTAATAGTAATGGTGG 
               
               
                   
                   
               
               
                   
                 TTGGTACTATGGTAATTACCATAAAAATTATTATC 
               
               
                   
                   
               
               
                   
                 CTTTTAAAATAAAGCTAATTATTATTGGATCTTTT 
               
               
                   
                   
               
               
                   
                 TTAGTATTCATTTTATGTTTTTTATGTTTTTGATT 
               
               
                   
                   
               
               
                   
                 TTTTAAAAGACAATCTCACCCTGTTACCCAGGCTG 
               
               
                   
                   
               
               
                   
                 GAGTGCAGTGGTGCAATCATAGCTTTCTGCAGTCT 
               
               
                   
                   
               
               
                   
                 TGAACTCCTGGGCTCAAGCAATCCTCCTGCCTTGG 
               
               
                   
                   
               
               
                   
                 CCTCCCAAAGTGTTGGGATACAGTCATGAGCCACT 
               
               
                   
                   
               
               
                   
                 GCATCTGGCCTAGGATCCATTTAGATTAAAATATG 
               
               
                   
                   
               
               
                   
                 CATTTTAAATTTTAAAATAATATGGCTAATTTTTA 
               
               
                   
                   
               
               
                   
                 CCTTATGTAATGTGTATACTGGCAATAAATCTAGT 
               
               
                   
                   
               
               
                   
                 TTGCTGCCTAAAGTTTAAAGTGCTTTCCAGTAAGC 
               
               
                   
                   
               
               
                   
                 TTCATGTACGTGAGGGGAGACATTTAAAGTGAAAC 
               
               
                   
                   
               
               
                   
                 AGACAGCCAGGTGTGGTGGCTCACGCCTGTAATCC 
               
               
                   
                   
               
               
                   
                 CAGCACTCTGGGAGGCTGAGGTGGGTGGATCGCTT 
               
               
                   
                   
               
               
                   
                 GAGCCCTGGAGTTCAAGACCAGCCTGAGCAACATG 
               
               
                   
                   
               
               
                   
                 GCAAAACGCTGTTTCTATAACAAAAATTAGCCGGG 
               
               
                   
                   
               
               
                   
                 CATGGTGGCATGTGCCTGTGGTCCCAGCTACTAGG 
               
               
                   
                   
               
               
                   
                 GGGCTGAGGCAGGAGAATCGTTGGAGCCCAGGAGG 
               
               
                   
                   
               
               
                   
                 TCAAGGCTGCACTGAGCAGTGCTTGCGCCACTGCA 
               
               
                   
                   
               
               
                   
                 CTCCAGCCTGGGTGACAGGACCAGACCTTGCCTCA 
               
               
                   
                   
               
               
                   
                 AAAAAATAAGAAGAAAAATTAAAAATAAATGGAAA 
               
               
                   
                   
               
               
                   
                 CAACTACAAAGAGCTGTTGTCCTAGATGAGCTACT 
               
               
                   
                   
               
               
                   
                 TAGTTAGGCTGATATTTTGGTATTTAACTTTTAAA 
               
               
                   
                   
               
               
                   
                 GTCAGGGTCTGTCACCTGCACTACATTATTAAAAT 
               
               
                   
                   
               
               
                   
                 ATCAATTCTCAATGTATATCCACACAAAGACTGGT 
               
               
                   
                   
               
               
                   
                 ACGTGAATGTTCATAGTACCTTTATTCACAAAACC 
               
               
                   
                   
               
               
                   
                 CCAAAGTAGAGACTATCCAAATATCCATCAACAAG 
               
               
                   
                   
               
               
                   
                 TGAACAAATAAACAAAATGTGCTATATCCATGCAA 
               
               
                   
                   
               
               
                   
                 TGGAATACCACCCTGCAGTACAAAGAAGCTACTTG 
               
               
                   
                   
               
               
                   
                 GGGATGAATCCCAAAGTCATGACGCTAAATGAAAG 
               
               
                   
                   
               
               
                   
                 AGTCAGACATGAAGGAGGAGATAATGTATGCCATA 
               
               
                   
                   
               
               
                   
                 CGAAATTCTAGAAAATGAAAGTAACTTATAGTTAC 
               
               
                   
                   
               
               
                   
                 AGAAAGCAAATCAGGGCAGGCATAGAGGCTCACAC 
               
               
                   
                   
               
               
                   
                 CTGTAATCCCAGCACTTTGAGAGGCCACGTGGGAA 
               
               
                   
                   
               
               
                   
                 GATTGCTAGAACTCAGGAGTTCAAGACCAGCCTGG 
               
               
                   
                   
               
               
                   
                 GCAACACAGTGAAACTCCATTCTCCACAAAAATGG 
               
               
                   
                   
               
               
                   
                 GAAAAAAAGAAAGCAAATCAGTGGTTGTCCTGTGG 
               
               
                   
                   
               
               
                   
                 GGAGGGGAAGGACTGCAAAGAGGGAAGAAGCTCTG 
               
               
                   
                   
               
               
                   
                 GTGGGGTGAGGGTGGTGATTCAGGTTCTGTATCCT 
               
               
                   
                   
               
               
                   
                 GACTGTGGTAGCAGTTTGGGGTGTTTACATCCAAA 
               
               
                   
                   
               
               
                   
                 AATATTCGTAGAATTATGCATCTTAAATGGGTGGA 
               
               
                   
                   
               
               
                   
                 GTTTACTGTATGTAAATTATACCTCAATGTAAGAA 
               
               
                   
                   
               
               
                   
                 AAAATAATGTGTAAGAAAACTTTCAATTCTCTTGC 
               
               
                   
                   
               
               
                   
                 CAGCAAACGTTATTCAAATTCCTGAGCCCTTTACT 
               
               
                   
                   
               
               
                   
                 TCGCAAATTCTCTGCACTTCTGCCCCGTACCATTA 
               
               
                   
                   
               
               
                   
                 GGTGACAGCACTAGCTCCACAAATTGGATAAATGC 
               
               
                   
                   
               
               
                   
                 ATTTCTGGAAAAGACTAGGGACAAAATCCAGGCAT 
               
               
                   
                   
               
               
                   
                 CACTTGTGCTTTCATATCAACCATGCTGTACAGCT 
               
               
                   
                   
               
               
                   
                 TGTGTTGCTGTCTGCAGCTGCAATGGGGACTCTTG 
               
               
                   
                   
               
               
                   
                 ATTTCTTTAAGGAAACTTGGGTTACCAGAGTATTT 
               
               
                   
                   
               
               
                   
                 CCACAAATGCTATTCAAATTAGTGCTTATGATATG 
               
               
                   
                   
               
               
                   
                 CAAGACACTGTGCTAGGAGCCAGAAAACAAAGAGG 
               
               
                   
                   
               
               
                   
                 AGGAGAAATCAGTCATTATGTGGGAACAACATAGC 
               
               
                   
                   
               
               
                   
                 AAGATATTTAGATCATTTTGACTAGTTAAAAAAGC 
               
               
                   
                   
               
               
                   
                 AGCAGAGTACAAAATCACACATGCAATCAGTATAA 
               
               
                   
                   
               
               
                   
                 TCCAAATCATGTAAATATGTGCCTGTAGAAAGACT 
               
               
                   
                   
               
               
                   
                 AGAGGAATAAACACAAGAATCTTAACAGTCATTGT 
               
               
                   
                   
               
               
                   
                 CATTAGACACTAAGTCTAATTATTATTATTAGACA 
               
               
                   
                   
               
               
                   
                 CTATGATATTTGAGATTTAAAAAATCTTTAATATT 
               
               
                   
                   
               
               
                   
                 TTAAAATTTAGAGCTCTTCTATTTTTCCATAGTAT 
               
               
                   
                   
               
               
                   
                 TCAAGTTTGACAATGATCAAGTATTACTCTTTCTT 
               
               
                   
                   
               
               
                   
                 TTTTTTTTTTTTTTTTTTTTTTTGAGATGGAGTTT 
               
               
                   
                   
               
               
                   
                 TGGTCTTGTTGCCCATGCTGGAGTGGAATGGCATG 
               
               
                   
                   
               
               
                   
                 ACCATAGCTCACTGCAACCTCCACCTCCTGGGTTC 
               
               
                   
                   
               
               
                   
                 AAGCAAAGCTGTCGCCTCAGCCTCCCGGGTAGATG 
               
               
                   
                   
               
               
                   
                 GGATTACAGGCGCCCACCACCACACTCGGCTAATG 
               
               
                   
                   
               
               
                   
                 TTTGTATTTTTAGTAGAGATGGGGTTTCACCATGT 
               
               
                   
                   
               
               
                   
                 TGGCCAGGCTGGTCTCAAACTCCTGACCTCAGAGG 
               
               
                   
                   
               
               
                   
                 ATCCACCTGCCTCAGCCTCCCAAAGTGCTGGGATT 
               
               
                   
                   
               
               
                   
                 ACAGATGTAGGCCACTGCGCCCGGCCAAGTATTGC 
               
               
                   
                   
               
               
                   
                 TCTTATACATTAAAAAACAGGTGTGAGCCACTGCG 
               
               
                   
                   
               
               
                   
                 CCCAGCCAGGTATTGCTCTTATACATTAAAAAATA 
               
               
                   
                   
               
               
                   
                 GGCCGGTGCAGTGGCTCACGCCTGTAATCCCAGCA 
               
               
                   
                   
               
               
                   
                 CTTTGGGAAGCCAAGGCGGGCAGAACACCCGAGGT 
               
               
                   
                   
               
               
                   
                 CAGGAGTCCAAGGCCAGCCTGGCCAAGATGGTGAA 
               
               
                   
                   
               
               
                   
                 ACCCCGTCTCTATTAAAAATACAAACATTACCTGG 
               
               
                   
                   
               
               
                   
                 GCATGATGGTGGGCGCCTGTAATCCCAGCTACTCA 
               
               
                   
                   
               
               
                   
                 GGAGGCTGAGGCAGGAGGATCCGCGGAGCCTGGCA 
               
               
                   
                   
               
               
                   
                 GATCTGCCTGAGCCTGGGAGGTTGAGGCTACAGTA 
               
               
                   
                   
               
               
                   
                 AGCCAAGATCATGCCAGTATACTTCAGCCTGGGCG 
               
               
                   
                   
               
               
                   
                 ACAAAGTGAGACCGTAACAAAAAAAAAAAAATTTA 
               
               
                   
                   
               
               
                   
                 AAAAAAGAAATTTAGATCAAGATCCAACTGTAAAA 
               
               
                   
                   
               
               
                   
                 AGTGGCCTAAACACCACATTAAAGAGTTTGGAGTT 
               
               
                   
                   
               
               
                   
                 TATTCTGCAGGCAGAAGAGAACCATCAGGGGGTCT 
               
               
                   
                   
               
               
                   
                 TCAGCATGGGAATGGCATGGTGCACCTGGTTTTTG 
               
               
                   
                   
               
               
                   
                 TGAGATCATGGTGGTGACAGTGTGGGGAATGTTAT 
               
               
                   
                   
               
               
                   
                 TTTGGAGGGACTGGAGGCAGACAGACCGGTTAAAA 
               
               
                   
                   
               
               
                   
                 GGCCAGCACAACAGATAAGGAGGAAGAAGATGAGG 
               
               
                   
                   
               
               
                   
                 GCTTGGACCGAAGCAGAGAAGAGCAAACAGGGAAG 
               
               
                   
                   
               
               
                   
                 GTACAAATTCAAGAAATATTGGGGGGTTTGAATCA 
               
               
                   
                   
               
               
                   
                 ACACATTTAGATGATTAATTAAATATGAGGACTGA 
               
               
                   
                   
               
               
                   
                 GGAATAAGAAATGAGTCAAGGATGGTTCCAGGCTG 
               
               
                   
                   
               
               
                   
                 CTAGGCTGCTTACCTGAGGTGGCAAAGTCGGGAGG 
               
               
                   
                   
               
               
                   
                 AGTGGCAGTTTAGGACAGGGGGCAGTTGAGGAATA 
               
               
                   
                   
               
               
                   
                 TTGTTTTGATCATTTTGAGTTTGAGGTACAAGTTG 
               
               
                   
                   
               
               
                   
                 GACACTTAGGTAAAGACTGGAGGGGAAATCTGAAT 
               
               
                   
                   
               
               
                   
                 ATACAATTATGGGACTGAGGAACAAGTTTATTTTA 
               
               
                   
                   
               
               
                   
                 TTTTTTGTTTCGTTTTCTTGTTGAAGAACAAATTT 
               
               
                   
                   
               
               
                   
                 AATTGTAATCCCAAGTCATCAGCATCTAGAAGACA 
               
               
                   
                   
               
               
                   
                 GTGGCAGGAGGTGACTGTCTTGTGGGTAAGGGTTT 
               
               
                   
                   
               
               
                   
                 GGGGTCCTTGATGAGTATCTCTCAATTGGCCTTAA 
               
               
                   
                   
               
               
                   
                 ATATAAGCAGGAAAAGGAGTTTATGATGGATTCCA 
               
               
                   
                   
               
               
                   
                 GGCTCAGCAGGGCTCAGGAGGGCTCAGGCAGCCAG 
               
               
                   
                   
               
               
                   
                 CAGAGGAAGTCAGAGCATCTTCTTTGGTTTAGCCC 
               
               
                   
                   
               
               
                   
                 AAGTAATGACTTCCTTAAAAAGCTGAAGGAAAATC 
               
               
                   
                   
               
               
                   
                 CAGAGTGACCAGATTATAAACTGTACTCTTGCATT 
               
               
                   
                   
               
               
                   
                 TTCTCTCCCTCCTCTCACCCACAGCCTCTTGATGA 
               
               
                   
                   
               
               
                   
                 ACCGGAGGAAGTTTCTTTACCAATTCAAAAATGTC 
               
               
                   
                   
               
               
                   
                 CGCTGGGCTAAGGGTCGGCGTGAGACCTACCTGTG 
               
               
                   
                   
               
               
                   
                 CTACGTAGTGAAGAGGCGTGACAGTGCTACATCCT 
               
               
                   
                   
               
               
                   
                 TTTCACTGGACTTTGGTTATCTTCGCAATAAGGTA 
               
               
                   
                   
               
               
                   
                 TCAATTAAAGTCGGCTTTGCAAGCAGTTTAATGGT 
               
               
                   
                   
               
               
                   
                 CAACTGTGAGTGCTTTTAGAGCCACCTGCTGATGG 
               
               
                   
                   
               
               
                   
                 TATTACTTCCATCCTTTTTTGGCATTTGTGTCTCT 
               
               
                   
                   
               
               
                   
                 ATCACATTCCTCAAATCCTTTTTTTTATTTCTTTT 
               
               
                   
                   
               
               
                   
                 TCCATGTCCATGCACCCATATTAGACATGGCCCAA 
               
               
                   
                   
               
               
                   
                 AATATGTGATTTAATTCCTCCCCAGTAATGCTGGG 
               
               
                   
                   
               
               
                   
                 CACCCTAATACCACTCCTTCCTTCAGTGCCAAGAA 
               
               
                   
                   
               
               
                   
                 CAACTGCTCCCAAACTGTTTACCAGCTTTCCTCAG 
               
               
                   
                   
               
               
                   
                 CATCTGAATTGCCTTTGAGATTAATTAAGCTAAAA 
               
               
                   
                   
               
               
                   
                 GCATTTTTATATGGGAGAATATTATCAGCTTGTCC 
               
               
                   
                   
               
               
                   
                 AAGCAAAAATTTTAAATGTGAAAAACAAATTGTGT 
               
               
                   
                   
               
               
                   
                 CTTAAGCATTTTTGAAAATTAAGGAAGAAGAATTT 
               
               
                   
                   
               
               
                   
                 GGGAAAAAATTAACGGTGGCTCAATTCTGTCTTCC 
               
               
                   
                   
               
               
                   
                 AAATGATTTCTTTTCCCTCCTACTCACATGGGTCG 
               
               
                   
                   
               
               
                   
                 TAGGCCAGTGAATACATTCAACATGGTGATCCCCA 
               
               
                   
                   
               
               
                   
                 GAAAACTCAGAGAAGCCTCGGCTGATGATTAATTA 
               
               
                   
                   
               
               
                   
                 AATTGATCTTTCGGCTACCCGAGAGAATTACATTT 
               
               
                   
                   
               
               
                   
                 CCAAGAGACTTCTTCACCAAAATCCAGATGGGTTT 
               
               
                   
                   
               
               
                   
                 ACATAAACTTCTGCCCACGGGTATCTCCTCTCTCC 
               
               
                   
                   
               
               
                   
                 TAACACGCTGTGACGTCTGGGCTTGGTGGAATCTC 
               
               
                   
                   
               
               
                   
                 AGGGAAGCATCCGTGGGGTGGAAGGTCATCGTCTG 
               
               
                   
                   
               
               
                   
                 GCTCGTTGTTTGATGGTTATATTACCATGCAATTT 
               
               
                   
                   
               
               
                   
                 TCTTTGCCTACATTTGTATTGAATACATCCCAATC 
               
               
                   
                   
               
               
                   
                 TCCTTCCTATTCGGTGACATGACACATTCTATTTC 
               
               
                   
                   
               
               
                   
                 AGAAGGCTTTGATTTTATCAAGCACTTTCATTTAC 
               
               
                   
                   
               
               
                   
                 TTCTCATGGCAGTGCCTATTACTTCTCTTACAATA 
               
               
                   
                   
               
               
                   
                 CCCATCTGTCTGCTTTACCAAAATCTATTTCCCCT 
               
               
                   
                   
               
               
                   
                 TTTCAGATCCTCCCAAATGGTCCTCATAAACTGTC 
               
               
                   
                   
               
               
                   
                 CTGCCTCCACCTAGTGGTCCAGGTATATTTCCACA 
               
               
                   
                   
               
               
                   
                 ATGTTACATCAACAGGCACTTCTAGCCATTTTCCT 
               
               
                   
                   
               
               
                   
                 TCTCAAAAGGTGCAAAAAGCAACTTCATAAACACA 
               
               
                   
                   
               
               
                   
                 AATTAAATCTTCGGTGAGGTAGTGTGATGCTGCTT 
               
               
                   
                   
               
               
                   
                 CCTCCCAACTCAGCGCACTTCGTCTTCCTCATTCC 
               
               
                   
                   
               
               
                   
                 ACAAAAACCCATAGCCTTCCTTCACTCTGCAGGAC 
               
               
                   
                   
               
               
                   
                 TAGTGCTGCCAAGGGTTCAGCTCTACCTACTGGTG 
               
               
                   
                   
               
               
                   
                 TGCTCTTTTGAGCAAGTTGCTTAGCCTCTCTGTAA 
               
               
                   
                   
               
               
                   
                 CACAAGGACAATAGCTGCAAGCATCCCCAAAGATC 
               
               
                   
                   
               
               
                   
                 ATTGCAGGAGACAATGACTAAGGCTACCAGAGCCG 
               
               
                   
                   
               
               
                   
                 CAATAAAAGTCAGTGAATTTTAGCGTGGTCCTCTC 
               
               
                   
                   
               
               
                   
                 TGTCTCTCCAGAACGGCTGCCACGTGGAATTGCTC 
               
               
                   
                   
               
               
                   
                 TTCCTCCGCTACATCTCGGACTGGGACCTAGACCC 
               
               
                   
                   
               
               
                   
                 TGGCCGCTGCTACCGCGTCACCTGGTTCACCTCCT 
               
               
                   
                   
               
               
                   
                 GGAGCCCCTGCTACGACTGTGCCCGACATGTGGCC 
               
               
                   
                   
               
               
                   
                 GACTTTCTGCGAGGGAACCCCAACCTCAGTCTGAG 
               
               
                   
                   
               
               
                   
                 GATCTTCACCGCGCGCCTCTACTTCTGTGAGGACC 
               
               
                   
                   
               
               
                   
                 GCAAGGCTGAGCCCGAGGGGCTGCGGCGGCTGCAC 
               
               
                   
                   
               
               
                   
                 CGCGCCGGGGTGCAAATAGCCATCATGACCTTCAA 
               
               
                   
                   
               
               
                   
                 AGGTGCGAAAGGGCCTTCCGCGCAGGCGCAGTGCA 
               
               
                   
                   
               
               
                   
                 GCAGCCCGCATTCGGGATTGCGATGCGGAATGAAT 
               
               
                   
                   
               
               
                   
                 GAGTTAGTGGGGAAGCTCGAGGGGAAGAAGTGGGC 
               
               
                   
                   
               
               
                   
                 GGGGATTCTGGTTCACCTCTGGAGCCGAAATTAAA 
               
               
                   
                   
               
               
                   
                 GATTAGAAGCAGAGAAAAGAGTGAATGGCTCAGAG 
               
               
                   
                   
               
               
                   
                 ACAAGGCCCCGAGGAAATGAGAAAATGGGGCCAGG 
               
               
                   
                   
               
               
                   
                 GTTGCTTCTTTCCCCTCGATTTGGAACCTGAACTG 
               
               
                   
                   
               
               
                   
                 TCTTCTACCCCCATATCCCCGCCTTTTTTTCCTTT 
               
               
                   
                   
               
               
                   
                 TTTTTTTTTTGAAGATTATTTTTACTGCTGGAATA 
               
               
                   
                   
               
               
                   
                 CTTTTGTAGAAAACCACGAAAGAACTTTCAAAGCC 
               
               
                   
                   
               
               
                   
                 TGGGAAGGGCTGCATGAAAATTCAGTTCGTCTCTC 
               
               
                   
                   
               
               
                   
                 CAGACAGCTTCGGCGCATCCTTTTGGTAAGGGGCT 
               
               
                   
                   
               
               
                   
                 TCCTCGCTTTTTAAATTTTCTTTCTTTCTCTACAG 
               
               
                   
                   
               
               
                   
                 TCTTTTTTGGAGTTTCGTATATTTCTTATATTTTC 
               
               
                   
                   
               
               
                   
                 TTATTGTTCAATCACTCTCAGTTTTCATCTGATGA 
               
               
                   
                   
               
               
                   
                 AAACTTTATTTCTCCTCCACATCAGCTTTTTCTTC 
               
               
                   
                   
               
               
                   
                 TGCTGTTTCACCATTCAGAGCCCTCTGCTAAGGTT 
               
               
                   
                   
               
               
                   
                 CCTTTTCCCTCCCTTTTCTTTCTTTTGTTGTTTCA 
               
               
                   
                   
               
               
                   
                 CATCTTTAAATTTCTGTCTCTCCCCAGGGTTGCGT 
               
               
                   
                   
               
               
                   
                 TTCCTTCCTGGTCAGAATTCTTTTCTCCTTTTTTT 
               
               
                   
                   
               
               
                   
                 TTTTTTTTTTTTTTTTTTTTAAACAAACAAACAAA 
               
               
                   
                   
               
               
                   
                 AAACCCAAAAAAACTCTTTCCCAATTTACTTTCTT 
               
               
                   
                   
               
               
                   
                 CCAACATGTTACAAAGCCATCCACTCAGTTTAGAA 
               
               
                   
                   
               
               
                   
                 GACTCTCCGGCCCCACCGACCCCCAACCTCGTTTT 
               
               
                   
                   
               
               
                   
                 GAAGCCATTCACTCAATTTGCTTCTCTCTTTCTCT 
               
               
                   
                   
               
               
                   
                 ACAGCCCCTGTATGAGGTTGATGACTTACGAGACG 
               
               
                   
                   
               
               
                   
                 CATTTCGTACTTTGGGACTTTGATAGCAACTTCCA 
               
               
                   
                   
               
               
                   
                 GGAATGTCACACACGATGAAATATCTCTGCTGAAG 
               
               
                   
                   
               
               
                   
                 ACAGTGGATAAAAAACAGTCCTTCAAGTCTTCTCT 
               
               
                   
                   
               
               
                   
                 GTTTTTATTCTTCAACTCTCACTTTCTTAGAGTTT 
               
               
                   
                   
               
               
                   
                 ACAGAAAAAATATTTATATACGACTCTTTAAAAAG 
               
               
                   
                   
               
               
                   
                 ATCTATGTCTTGAAAATAGAGAAGGAACACAGGTC 
               
               
                   
                   
               
               
                   
                 TGGCCAGGGACGTGCTGCAATTGGTGCAGTTTTGA 
               
               
                   
                   
               
               
                   
                 ATGCAACATTGTCCCCTACTGGGAATAACAGAACT 
               
               
                   
                   
               
               
                   
                 GCAGGACCTGGGAGCATCCTAAAGTGTCAACGTTT 
               
               
                   
                   
               
               
                   
                 TTCTATGACTTTTAGGTAGGATGAGAGCAGAAGGT 
               
               
                   
                   
               
               
                   
                 AGATCCTAAAAAGCATGGTGAGAGGATCAAATGTT 
               
               
                   
                   
               
               
                   
                 TTTATATCAACATCCTTTATTATTTGATTCATTTG 
               
               
                   
                   
               
               
                   
                 AGTTAACAGTGGTGTTAGTGATAGATTTTTCTATT 
               
               
                   
                   
               
               
                   
                 CTTTTCCCTTGACGTTTACTTTCAAGTAACACAAA 
               
               
                   
                   
               
               
                   
                 CTCTTCCATCAGGCCATGATCTATAGGACCTCCTA 
               
               
                   
                   
               
               
                   
                 ATGAGAGTATCTGGGTGATTGTGACCCCAAACCAT 
               
               
                   
                   
               
               
                   
                 CTCTCCAAAGCATTAATATCCAATCATGCGCTGTA 
               
               
                   
                   
               
               
                   
                 TGTTTTAATCAGCAGAAGCATGTTTTTATGTTTGT 
               
               
                   
                   
               
               
                   
                 ACAAAAGAAGATTGTTATGGGTGGGGATGGAGGTA 
               
               
                   
                   
               
               
                   
                 TAGACCATGCATGGTCACCTTCAAGCTACTTTAAT 
               
               
                   
                   
               
               
                   
                 AAAGGATCTTAAAATGGGCAGGAGGACTGTGAACA 
               
               
                   
                   
               
               
                   
                 AGACACCCTAATAATGGGTTGATGTCTGAAGTAGC 
               
               
                   
                   
               
               
                   
                 AAATCTTCTGGAAACGCAAACTCTTTTAAGGAAGT 
               
               
                   
                   
               
               
                   
                 CCCTAATTTAGAAACACCCACAAACTTCACATATC 
               
               
                   
                   
               
               
                   
                 ATAATTAGCAAACAATTGGAAGGAAGTTGCTTGAA 
               
               
                   
                   
               
               
                   
                 TGTTGGGGAGAGGAAAATCTATTGGCTCTCGTGGG 
               
               
                   
                   
               
               
                   
                 TCTCTTCATCTCAGAAATGCCAATCAGGTCAAGGT 
               
               
                   
                   
               
               
                   
                 TTGCTACATTTTGTATGTGTGTGATGCTTCTCCCA 
               
               
                   
                   
               
               
                   
                 AAGGTATATTAACTATATAAGAGAGTTGTGACAAA 
               
               
                   
                   
               
               
                   
                 ACAGAATGATAAAGCTGCGAACCGTGGCACACGCT 
               
               
                   
                   
               
               
                   
                 CATAGTTCTAGCTGCTTGGGAGGTTGAGGAGGGAG 
               
               
                   
                   
               
               
                   
                 GATGGCTTGAACACAGGTGTTCAAGGCCAGCCTGG 
               
               
                   
                   
               
               
                   
                 GCAACATAACAAGATCCTGTCTCTCAAAAAAAAAA 
               
               
                   
                   
               
               
                   
                 AAAAAAAAAAGAAAGAGAGAGGGCCGGGCGTGGTG 
               
               
                   
                   
               
               
                   
                 GCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCG 
               
               
                   
                   
               
               
                   
                 AGCCGGGCGGATCACCTGTGGTCAGGAGTTTGAGA 
               
               
                   
                   
               
               
                   
                 CCAGCCTGGCCAACATGGCAAAACCCCGTCTGTAC 
               
               
                   
                   
               
               
                   
                 TCAAAATGCAAAAATTAGCCAGGCGTGGTAGCAGG 
               
               
                   
                   
               
               
                   
                 CACCTGTAATCCCAGCTACTTGGGAGGCTGAGGCA 
               
               
                   
                   
               
               
                   
                 GGAGAATCGCTTGAACCCAGGAGGTGGAGGTTGCA 
               
               
                   
                   
               
               
                   
                 GTAAGCTGAGATCGTGCCGTTGCACTCCAGCCTGG 
               
               
                   
                   
               
               
                   
                 GCGACAAGAGCAAGACTCTGTCTCAGAAAAAAAAA 
               
               
                   
                   
               
               
                   
                 AAAAAAAGAGAGAGAGAGAGAAAGAGAACAATATT 
               
               
                   
                   
               
               
                   
                 TGGGAGAGAAGGATGGGGAAGCATTGCAAGGAAAT 
               
               
                   
                   
               
               
                   
                 TGTGCTTTATCCAACAAAATGTAAGGAGCCAATAA 
               
               
                   
                   
               
               
                   
                 GGGATCCCTATTTGTCTCTTTTGGTGTCTATTTGT 
               
               
                   
                   
               
               
                   
                 CCCTAACAACTGTCTTTGACAGTGAGAAAAATATT 
               
               
                   
                   
               
               
                   
                 CAGAATAACCATATCCCTGTGCCGTTATTACCTAG 
               
               
                   
                   
               
               
                   
                 CAACCCTTGCAATGAAGATGAGCAGATCCACAGGA 
               
               
                   
                   
               
               
                   
                 AAACTTGAATGCACAACTGTCTTATTTTAATCTTA 
               
               
                   
                   
               
               
                   
                 TTGTACATAAGTTTGTAAAAGAGTTAAAAATTGTT 
               
               
                   
                   
               
               
                   
                 ACTTCATGTATTCATTTATATTTTATATTATTTTG 
               
               
                   
                   
               
               
                   
                 CGTCTAATGATTTTTTATTAACATGATTTCCTTTT 
               
               
                   
                   
               
               
                   
                 CTGATATATTGAAATGGAGTCTCAAAGCTTCATAA 
               
               
                   
                   
               
               
                   
                 ATTTATAACTTTAGAAATGATTCTAATAACAACGT 
               
               
                   
                   
               
               
                   
                 ATGTAATTGTAACATTGCAGTAATGGTGCTACGAA 
               
               
                   
                   
               
               
                   
                 GCCATTTCTCTTGATTTTTAGTAAACTTTTATGAC 
               
               
                   
                   
               
               
                   
                 AGCAAATTTGCTTCTGGCTCACTTTCAATCAGTTA 
               
               
                   
                   
               
               
                   
                 AATAAATGATAAATAATTTTGGAAGCTGTGAAGAT 
               
               
                   
                   
               
               
                   
                 AAAATACCAAATAAAATAATATAAAAGTGATTTAT 
               
               
                   
                   
               
               
                   
                 ATGAAGTTAAAATAAAAAATCAGTATGATGGAATA 
               
               
                   
                   
               
               
                   
                 AACTTG 
               
            
           
         
       
     
     Other cytidine deaminases useful in the methods of the invention are provided below. 
     
       
         
           
               
               
            
               
                 rAPOBEC-1  Rattus norvegicus   
                   
               
               
                 (SEQ ID NO: 204) 
                   
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNT 
                   
               
               
                   
               
               
                 NKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIAR 
               
               
                   
               
               
                 LYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLW 
               
               
                   
               
               
                 VRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK 
               
               
                   
               
               
                 mAPOBEC-1  Mus musculus   
               
               
                 (SEQ ID NO: 205) 
                   
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSVWRHTSQNTSN 
                   
               
               
                   
               
               
                 HVEVNFLEKFTTERYFRPNTRCSITWFLSWSPCGECSRAITEFLSRHPYVTLFIYIARLY 
               
               
                   
               
               
                 HHTDQRNRQGLRDLISSGVTIQIMTEQEYCYCWRNFVNYPPSNEAYWPRYPHLWVK 
               
               
                   
               
               
                 LYVLELYCIILGLPPCLKILRRKQPQLTFFTITLQTCHYQRIPPHLLWATGLK 
               
               
                   
               
               
                 maAPOBEC-1  Mesocricetus auratus   
               
               
                 (SEQ ID NO: 206) 
                   
               
               
                 MSSETGPVVVDPTLRRRIEPHEFDAFFDQGELRKETCLLYEIRWGGRHNIWRHTGQN 
                   
               
               
                   
               
               
                 TSRHVEINFIEKFTSERYFYPSTRCSIVWFLSWSPCGECSKAITEFLSGHPNVTLFIYA 
               
               
                   
               
               
                 ARLYHHTDQRNRQGLRDLISRGVTIRIMTEQEYCYCWRNFVNYPPSNEVYWPRYPNLWMR 
               
               
                   
               
               
                 LYALELYCIHLGLPPCLKIKRRHQYPLTFFRLNLQSCHYQRIPPHILWATGFI 
               
               
                   
               
               
                 hAPOBEC-1  Homo sapiens   
               
               
                 (SEQ ID NO: 207) 
                   
               
               
                 MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKN 
                   
               
               
                   
               
               
                 TTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYV 
               
               
                   
               
               
                 ARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLW 
               
               
                   
               
               
                 MMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR 
               
               
                   
               
               
                 ppAPOBEC-1  Pongo pygmaeus   
               
               
                 (SEQ ID NO: 208) 
                   
               
               
                 MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRSSGKN 
                   
               
               
                   
               
               
                 TTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYV 
               
               
                   
               
               
                 ARLFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLW 
               
               
                   
               
               
                 MMLYALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSV 
               
               
                   
               
               
                 TWR 
               
               
                   
               
               
                 ocAPOBECI  Oryctolagus cuniculus   
               
               
                 (SEQ ID NO: 209) 
                   
               
               
                 MASEKGPSNKDYTLRRRIEPWEFEVFFDPQELRKEACLLYEIKWGASSKTWRSSGKNT 
                   
               
               
                   
               
               
                 TNHVEVNFLEKLTSEGRLGPSTCCSITWFLSWSPCWECSMAIREFLSQHPGVTLIIFV 
               
               
                   
               
               
                 ARLFQHMDRRNRQGLKDLVTSGVTVRVMSVSEYCYCWENFVNYPPGKAAQWPRYPPRW 
               
               
                   
               
               
                 MLMYALELYCIILGLPPCLKISRRHQKQLTFFSLTPQYCHYKMIPPYILLATGLLQPS 
               
               
                   
               
               
                 VPWR 
               
               
                   
               
               
                 mdAPOBEC-1  Monodelphis domestica   
               
               
                 (SEQ ID NO: 210) 
                   
               
               
                 MNSKTGPSVGDATLRRRIKPWEFVAFFNPQELRKETCLLYEIKWGNQNIWRHSNQN 
                   
               
               
                   
               
               
                 TSQHAEINFMEKFTAERHFNSSVRCSITWFLSWSPCWECSKAIRKFLDHYPNVTLAI 
               
               
                   
               
               
                 FISRLYWHMDQQHRQGLKELVHSGVTIQIMSYSEYHYCWRNFVDYPQGEEDYWPKYP 
               
               
                   
               
               
                 YLWIMLYVLELHCIILGLPPCLKISGSHSNQLALFSLDLQDCHYQKIPYNVLVATGL 
               
               
                   
               
               
                 VQPFVTWR 
               
               
                   
               
               
                 mAPOBEC-2  Mus musculus   
               
               
                 (SEQ ID NO: 211) 
                   
               
               
                 MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFR 
                   
               
               
                   
               
               
                 NVEYSSGRNKTFLCYVVEVQSKGGQAQATQGYLEDEHAGAHAEEAFFNTILPAFDP 
               
               
                   
               
               
                 ALKYNVTWYVSSSPCAACADRILKTLSKTKNLRLLILVSRLFMWEEPEVQAALKKL 
               
               
                   
               
               
                 KEAGCKLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK 
               
               
                   
               
               
                 hAPOBEC-2  Homo sapiens   
               
               
                 (SEQ ID NO: 212) 
                   
               
               
                 MAQKEEAAVATEAASQNGEDLENLDDPEKLKELIELPPFEIVTGERLPANFFKFQFRN 
                   
               
               
                   
               
               
                 VEYSSGRNKTFLCYVVEAQGKGGQVQASRGYLEDEHAAAHAEEAFFNTILPAFDPALR 
               
               
                   
               
               
                 YNVTWYVSSSPCAACADRIIKTLSKTKNLRLLILVGRLFMWEEPEIQAALKKLKEAG 
               
               
                   
               
               
                 CKLRIMKPQDFEYVWQNFVEQEEGESKAFQPWEDIQENFLYYEEKLADILK 
               
               
                   
               
               
                 ppAPOBEC-2  Pongo pygmaeus   
               
               
                 (SEQ ID NO: 213) 
                   
               
               
                 MAQKEEAAAATEAASQNGEDLENLDDPEKLKELIELPPFEIVTGERLPANFFKFQFRN 
                   
               
               
                   
               
               
                 VEYSSGRNKTFLCYVVEAQGKGGQVQASRGYLEDEHAAAHAEEAFFNTILPAFDPALR 
               
               
                   
               
               
                 YNVTWYVSSSPCAACADRIIKTLSKTKNLRLLILVGRLFMWEELEIQDALKKLKEAG 
               
               
                   
               
               
                 CKLRIMKPQDFEYVWQNFVEQEEGESKAFQPWEDIQENFLYYEEKLADILK 
               
               
                   
               
               
                 btAPOBEC-2  Bos Taurus   
               
               
                 (SEQ ID NO: 214) 
                   
               
               
                 MAQKEEAAAAAEPASQNGEEVENLEDPEKLKELIELPPFEIVTGERLPAHYFKFQFRN 
                   
               
               
                   
               
               
                 VEYSSGRNKTFLCYVVEAQSKGGQVQASRGYLEDEHATNHAEEAFFNSIMPTFDPALR 
               
               
                   
               
               
                 YMVTWYVSSSPCAACADRIVKTLNKTKNLRLLILVGRLFMWEEPEIQAALRKLKEA 
               
               
                   
               
               
                 GCRLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK 
               
               
                   
               
               
                 mAPOBEC-3  Mus musculus   
               
               
                 (SEQ ID NO: 215) 
                   
               
               
                 MQPQRLGPRAGMGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLGYAKGRKDTFLCY 
                   
               
               
                   
               
               
                 EVTRKDCDSPVSLHHGVFKNKDNIHAEICFLYWFHDKVLKVLSPREEFKITWYMSW 
               
               
                   
               
               
                 SPCFECAEQIVRFLATHHNLSLDIFSSRLYNVQDPETQQNLCRLVQEGAQVAAMDLY 
               
               
                   
               
               
                 EFKKCWKKFVDNGGRRFRPWKRLLTNFRYQDSKLQEILRPCYISVPSSSSSTLSNICL 
               
               
                   
               
               
                 TKGLPETRFWVEGRRMDPLSEEEFYSQFYNQRVKHLCYYHRMKPYLCYQLEQFNG 
               
               
                   
               
               
                 QAPLKGCLLSEKGKQHAEILFLDKIRSMELSQVTITCYLTWSPCPNCAWQLAAFKRD 
               
               
                   
               
               
                 RPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPF 
               
               
                   
               
               
                 WPWKGLEIISRRTQRRLRRIKESWGLQDLVNDFGNLQLGPPMS 
               
               
                   
               
               
                 hAPOBEC-3A  Homo sapiens   
               
               
                 (SEQ ID NO: 216) 
                   
               
               
                 MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLH 
                   
               
               
                   
               
               
                 NQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAF 
               
               
                   
               
               
                 LQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQ 
               
               
                   
               
               
                 GCPFQPWDGLDEHSQALSGRLRAILQNQGN 
               
               
                   
               
               
                 hAPOBEC-3B  Homo sapiens   
               
               
                 (SEQ ID NO: 217) 
                   
               
               
                 MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFR 
                   
               
               
                   
               
               
                 GQVYFKPQYHAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLSEHP 
               
               
                   
               
               
                 NVTLTISAARLYYYWERDYRRALCRLSQAGARVTIMDYEEFAYCWENFVYNEGQQ 
               
               
                   
               
               
                 FMPWYKFDENYAFLHRTLKEILRYLMDPDTFTFNFNNDPLVLRRRQTYLCYEVERL 
               
               
                   
               
               
                 DNGTWVLMDQHMGFLCNEAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFI 
               
               
                   
               
               
                 SWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSI 
               
               
                   
               
               
                 MTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQNQGN 
               
               
                   
               
               
                 hAPOBEC-3C  Homo sapiens   
               
               
                 (SEQ ID NO: 218) 
                   
               
               
                 MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVF 
                   
               
               
                   
               
               
                 RNQVDSETHCHAERCFLSWFCDDILSPNTKYQVTWYTSWSPCPDCAGEVAEFLARH 
               
               
                   
               
               
                 SNVNLTIFTARLYYFQYPCYQEGLRSLSQEGVAVEIMDYEDFKYCWENFVYNDNEPF 
               
               
                   
               
               
                 KPWKGLKTNFRLLKRRLRESLQ 
               
               
                   
               
               
                 hAPOBEC-3D  Homo sapiens   
               
               
                 (SEQ ID NO: 219) 
                   
               
               
                 MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFR 
                   
               
               
                   
               
               
                 GPVLPKRQSNHRQEVYFRFENHAEMCFLSWFCGNRLPANRRFQITWFVSWNPCLPC 
               
               
                   
               
               
                 VVKVTKFLAEHPNVTLTISAARLYYYRDRDWRWVLLRLHKAGARVKIMDYEDFAY 
               
               
                   
               
               
                 CWENFVCNEGQPFMPWYKFDDNYASLHRTLKEILRNPMEAMYPHIFYFHFKNLLKA 
               
               
                   
               
               
                 CGRNESWLCFTMEVTKHHSAVFRKRGVFRNQVDPETHCHAERCFLSWFCDDILSPN 
               
               
                   
               
               
                 TNYEVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLCYFWDTDYQEGLCSLS 
               
               
                   
               
               
                 QEGASVKIMGYKDFVSCWKNFVYSDDEPFKPWKGLQTNFRLLKRRLREILQ 
               
               
                   
               
               
                 hAPOBEC-3F  Homo sapiens   
               
               
                 (SEQ ID NO: 220) 
                   
               
               
                 MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPRLDAKIFRGQ 
                   
               
               
                   
               
               
                 VYSQPEHHAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLAEHPNV 
               
               
                   
               
               
                 TLTISAARLYYYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYSEGQPFMP 
               
               
                   
               
               
                 WYKFDDNYAFLHRTLKEILRNPMEAMYPHIFYFHFKNLRKAYGRNESWLCFTMEV 
               
               
                   
               
               
                 VKHHSPVSWKRGVFRNQVDPETHCHAERCFLSWFCDDILSPNTNYEVTWYTSWSPC 
               
               
                   
               
               
                 PECAGEVAEFLARHSNVNLTIFTARLYYFWDTDYQEGLRSLSQEGASVEIMGYKDFK 
               
               
                   
               
               
                 YCWENFVYNDDEPFKPWKGLKYNFLFLDSKLQEILE 
               
               
                   
               
               
                 hAP0BEC-3G  Homo sapiens   
               
               
                 (SEQ ID NO: 221) 
                   
               
               
                 MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRG 
                   
               
               
                   
               
               
                 QVYSELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDP 
               
               
                   
               
               
                 KVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQ 
               
               
                   
               
               
                 RELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEV 
               
               
                   
               
               
                 ERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVT 
               
               
                   
               
               
                 CFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKIS 
               
               
                   
               
               
                 IMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN 
               
               
                   
               
               
                 hAPOBEC-4  Homo sapiens   
               
               
                 (SEQ ID NO: 222) 
                   
               
               
                 MEPIYEEYLANHGTIVKPYYWLSFSLDCSNCPYHIRTGEEARVSLTEFCQIFGFPYGTTF 
                   
               
               
                   
               
               
                 PQTKHLTFYELKTSSGSLVQKGHASSCTGNYIHPESMLFEMNGYLDSAIYNNDSIRHIIL 
               
               
                   
               
               
                 YSNNSPCNEANHCCISKMYNFLITYPGITLSIYFSQLYHTEMDFPASAWNREALRSLASL 
               
               
                   
               
               
                 WPRVVLSPISGGIWHSVLHSFISGVSGSHVFQPILTGRALADRHNAYEINAITGVKPYFT 
               
               
                   
               
               
                 DVLLQTKRNPNTKAQEALESYPLNNAFPGQFFQMPSGQLQPNLPPDLRAPVVFVLVP 
               
               
                   
               
               
                 LRDLPPMHMGQNPNKPRNIVRHLNMPQMSFQETKDLGRLPTGRSVEIVEITEQFASS 
               
               
                   
               
               
                 KEADEKKKKKGKK 
               
               
                   
               
               
                 mAPOBEC-4  Mus musculus   
               
               
                 (SEQ ID NO: 223) 
                   
               
               
                 MDSLLMKQKKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSCSLDFGHLRNKSGC 
                   
               
               
                   
               
               
                 HVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVAEFLRWNPNLSLRIFTAR 
               
               
                   
               
               
                 LYFCEDRKAEPEGLRRLHRAGVQIGIMTFKDYFYCWNTFVENRERTFKAWEGLHEN 
               
               
                   
               
               
                 SVRLTRQLRRILLPLYEVDDLRDAFRMLGF 
               
               
                   
               
               
                 rAPOBEC-4  Rattus norvegicus   
               
               
                 (SEQ ID NO: 224) 
                   
               
               
                 MEPLYEEYLTHSGTIVKPYYWLSVSLNCTNCPYHIRTGEEARVPYTEFHQTFGFPWSTYP 
                   
               
               
                   
               
               
                 QTKHLTFYELRSSSGNLIQKGLASNCTGSHTHPESMLFERDGYLDSLIFHDSNIRHIILY 
               
               
                   
               
               
                 SNNSPCDEANHCCISKMYNFLMNYPEVTLSVFFSQLYHTENQFPTSAWNREALRGLASLW 
               
               
                   
               
               
                 PQVTLSAISGGIWQSILETFVSGISEGLTAVRPFTAGRTLTDRYNAYEINCITEVKPYFT 
               
               
                   
               
               
                 DALHSWQKENQDQKVWAASENQPLHNTTPAQWQPDMSQDCRTPAVFMLVPYRDLPPIHVN 
               
               
                   
               
               
                 PSPQKPRTVVRHLNTLQLSASKVKALRKSPSGRPVKKEEARKGSTRSQEAN 
               
               
                   
               
               
                 ETNKSKWKKQTLFIKSNICHLLEREQKKIGILSSWSV 
               
               
                   
               
               
                 mfAPOBEC-4  Macaca fascicularis   
               
               
                 (SEQ ID NO: 225) 
                   
               
               
                 MEPTYEEYLANHGTIVKPYYWLSFSLDCSNCPYHIRTGEEARVSLTEFCQIFGFPYGTTY 
                   
               
               
                   
               
               
                 PQTKHLTFYELKTSSGSLVQKGHASSCTGNYIHPESMLFEMNGYLDSAIYNNDS1RFIII 
               
               
                   
               
               
                 LYCNNSPCNEANHCCISKVYNFLITYPG1TLSIYFSQLYHTEMDFPASAWNREALRSLAS 
               
               
                   
               
               
                 LWPRVVLSPISGGIWHSVLHSFVSGVSGSHVFQPILTGRALTDRYNAYEINAITGVKPFF 
               
               
                   
               
               
                 TDVLLHTKRNPNTKAQMALESYPLNNAFPGQSFQMTSGIPPDLRAPVVFVLLPLRDLP 
               
               
                   
               
               
                 PMHMGQDPNKPRNIIRHLNMPQMSFQETKDLERLPTRRSVETVEITERFASSKQAEEE 
               
               
                   
               
               
                 KTKKKKGKK 
               
               
                   
               
               
                 hAID  Homo sapiens   
               
               
                 (SEQ ID NO: 226) 
                   
               
               
                 MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGC 
                   
               
               
                   
               
               
                 HVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTAR 
               
               
                   
               
               
                 LYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHEN 
               
               
                   
               
               
                 SVRLSRQLRRILLPLYEVDDLRDAFRTLGL 
               
               
                   
               
               
                 clAID  Canis lupus familiaris   
               
               
                 (SEQ ID NO: 227) 
                   
               
               
                 MDSLLMKQRKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGHLRNKSGC 
                   
               
               
                   
               
               
                 HVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFAAR 
               
               
                   
               
               
                 LYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENREKTFKAWEGLHEN 
               
               
                   
               
               
                 SVRLSRQLRRILLPLYEVDDLRDAFRTLGL 
               
               
                   
               
               
                 btAID  Bos Taurus   
               
               
                 (SEQ ID NO: 228) 
                   
               
               
                 MDSLLKKQRQFLYQFKNVRWAKGRHETYLCYVVKRRDSPTSFSLDFGHLRNKAGC 
                   
               
               
                   
               
               
                 HVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFTAR 
               
               
                   
               
               
                 LYFCDKERKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHE 
               
               
                   
               
               
                 NSVRLSRQLRRILLPLYEVDDLRDAFRTLGL 
               
               
                   
               
               
                 mAID  Mus musculus   
               
               
                 (SEQ ID NO: 229) 
                   
               
               
                 MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGC 
                   
               
               
                   
               
               
                 HVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTAR 
               
               
                   
               
               
                 LYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHEN 
               
               
                   
               
               
                 SVRLSRQLRRILLPLYEVDDLRDAFRTLGL 
               
               
                   
               
               
                 pmCDA-1  Petromyzon marinus   
               
               
                 (SEQ ID NO: 230) 
                   
               
               
                 MAGYECVRVSEKLDFDTFEFQFENLHYATERHRTYVIFDVKPQSAGGRSRRLWGYII 
                   
               
               
                   
               
               
                 NNPNVCHAELILMSMIDRHLESNPGVYAMTWYMSWSPCANCSSKLNPWLKNLLEE 
               
               
                   
               
               
                 QGHTLTMHFSRIYDRDREGDHRGLRGLKHVSNSFRMGVVGRAEVKECLAEYVEASR 
               
               
                   
               
               
                 RTLTWLDTTESMAAKMRRKLFCILVRCAGMRESGIPLHLFTLQTPLLSGRVVWWRV 
               
               
                   
               
               
                 pmCDA-2  Petromyzon marinus   
               
               
                 (SEQ ID NO: 231) 
                   
               
               
                 MELREVVDCALASCVRHEPLSRVAFLRCFAAPSQKPRGTVILFYVEGAGRGVTGGH 
                   
               
               
                   
               
               
                 AVNYNKQGTSIHAEVLLLSAVRAALLRRRRCEDGEEATRGCTLHCYSTYSPCRDCV 
               
               
                   
               
               
                 EYIQEFGASTGVRVVIHCCRLYELDVNRRRSEAEGVLRSLSRLGRDFRLMGPRDAIA 
               
               
                   
               
               
                 LLLGGRLANTADGESGASGNAWVTETNVVEPLVDMTGFGDEDLHAQVQRNKQIREAY 
               
               
                   
               
               
                 ANYASAVSLMLGELHVDPDKFPFLAEFLAQTSVEPSGTPRETRGRPRGASSRGPEI 
               
               
                   
               
               
                 GRQRPADFERALGAYGLFLHPRIVSREADREEIKRDLIVVMRKHNYQGP 
               
               
                   
               
               
                 pmCDA-5  Petromyzon marinus   
               
               
                 (SEQ ID NO: 232) 
                   
               
               
                 MAGDENVRVSEKLDFDTFEFQFENLHYATERHRTYVIFDVKPQSAGGRSRRLWGYI 
                   
               
               
                   
               
               
                 INNPNVCHAELILMSMIDRHLESNPGVYAMTWYMSWSPCANCSSKLNPWLKNLLEE 
               
               
                   
               
               
                 QGHTLMMHFSRIYDRDREGDHRGLRGLKHVSNSFRMGVVGRAEVKECLAEYVEAS 
               
               
                   
               
               
                 RRTLTWLDTTESMAAKMRRKLFCILVRCAGMRESGMPLHLFT 
               
               
                   
               
               
                 yCD  Saccharomyces cerevisiae   
               
               
                 (SEQ ID NO: 233) 
                   
               
               
                 MVTGGMASKWDQKGMDIAYEEAALGYKEGGVPIGGCLINNKDGSVLGRGHNMRF 
                   
               
               
                   
               
               
                 QKGSATLHGEISTLENCGRLEGKVYKDTTLYTTLSPCDMCTGAIIMYGIPRCVV 
               
               
                   
               
               
                 GENVNFKSKGEKYLQTRGHEVVVVDDERCKKIMKQFIDERPQDWFEDIGE 
               
               
                   
               
               
                 rAPOBEC-1 (delta 177-186) 
               
               
                 (SEQ ID NO: 234) 
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNT 
               
               
                   
               
               
                 NKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYI 
               
               
                   
               
               
                 ARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLW 
               
               
                   
               
               
                 VRGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK 
               
               
                   
               
               
                 rAPOBEC-1 (delta 202-213) 
               
               
                 (SEQ ID NO: 235) 
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNT 
               
               
                   
               
               
                 NKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYI 
               
               
                   
               
               
                 ARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLW 
               
               
                   
               
               
                 VRLYVLELYCIILGLPPCLNILRRKQPQHYQRLPPHILWATGLK 
               
               
                   
               
               
                 Human AID: 
               
               
                 (SEQ ID NO: 226) 
                   
               
               
                   MDSLLMNRRKFLYQFKNVRWAKGRRETYLC YVVKRRDSATSFSLDFGYLRNKNGCHVELLFL 
                   
               
               
                   
               
               
                 RYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPE 
               
               
                   
               
               
                 GLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLP  
               
               
                   
               
               
                 
                   LYEVDDLRDAFRTLGL 
                 
               
               
                 (underline: nuclear localization sequence; 
               
               
                 double underline: nuclear export signal) 
               
               
                   
               
               
                 Mouse AID: 
               
               
                 (SEQ ID NO: 223) 
                   
               
               
                   MDSLLMKQKKFLYHFKNVRWAKGRHETYLC YVVKRRDSATSCSLDFGHLRNKSGCHVELLFL 
                   
               
               
                   
               
               
                 RYISDWDLDPGRCYRVTWFTSWSPCYDCARHVAEFLRWNPNLSLRIFTARLYFCEDRKAEPE 
               
               
                   
               
               
                 GLRRLHRAGVQIGIMTFKDYFYCWNTFVENRERTFKAWEGLHENSVRLTRQLRRILLP 
               
               
                   
               
               
                 
                   LYEVDDLRDAFRMLGF 
                 
               
               
                 (underline: nuclear localization sequence; 
               
               
                 double underline: nuclear export signal) 
               
               
                   
               
               
                 Canine AID: 
               
               
                 (SEQ ID NO: 227) 
                   
               
               
                   MDSLLMKQRKFLYHFKNVRWAKGRHETYLC YVVKRRDSATSFSLDFGHLRNKSGCHVELLFL 
                   
               
               
                   
               
               
                 RYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFAARLYFCEDRKAEPE 
               
               
                   
               
               
                 GLRRLHRAGVQIAIMTFKDYFYCWNTFVENREKTFKAWEGLHENSVRLSRQLRRILLP 
               
               
                   
               
               
                 
                   LYEVDDLRDAFRTLGL 
                 
               
               
                 (underline: nuclear localization sequence; 
               
               
                 double underline: nuclear export signal) 
               
               
                   
               
               
                 Bovine AID: 
               
               
                 (SEQ ID NO: 228) 
                   
               
               
                   MDSLLKKQRQFLYQFKNVRWAKGRHETYLC YVVKRRDSPTSFSLDFGHLRNKAGCHVELLFL 
                   
               
               
                   
               
               
                 RYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFTARLYFCDKERKAEP 
               
               
                   
               
               
                 EGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLP 
               
               
                   
               
               
                 
                   LYEVDDLRDAFRTLGL 
                 
               
               
                   
               
               
                 (underline: nuclear localization sequence; 
               
               
                 double underline: nuclear export signal) 
               
               
                   
               
               
                 Rat AID 
               
               
                 (SEQ ID NO: 236) 
                   
               
               
                   MAVGSKPKAALVGPHWERERIWCFLC STGLGTQQTGQTSRWLRPAATQDPVSPPRSLLMKQR 
                   
               
               
                   
               
               
                 KFLYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGYLRNKSGCHVELLFLRYISDWDLD 
               
               
                   
               
               
                 PGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLTGWGALPAGLMSPARPSDYF 
               
               
                   
               
               
                 YCWNTFV ENHERTFKAWEGLHENSVRLSRRLRRILLPLYEVDDLRDAFRTLGL   
               
               
                   
               
               
                 (underline: nuclear localization sequence; 
               
               
                 double underline: nuclear export signal) 
               
               
                   
               
               
                 Mouse APOBEC-3 
               
               
                 (SEQ ID NO: 237) 
                   
               
               
                 MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLGYAKGRKDTFLCYEVTRKDCDSPVSLHHG 
                   
               
               
                   
               
               
                 VFKNRDNI HAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFEC AEQIVRFLNTRWLSL 
               
               
                   
               
               
                 DIFSSRLYNVQDPETQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKRLLTNF 
               
               
                   
               
               
                 RYQDSKLQEILRPCYIPVPSSSSSTLSNICLTKGLPETRFCVEGRRMDPLSEEEFYSQFYNQ 
               
               
                   
               
               
                 RVKHLCYYHRMKPYLCYQLEQFNGQAPLKGCLLSEKGKQ HAEILFLDKIRSMELSOVTITCY   
               
               
                   
               
               
                   AWSPCPNC AWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQF 
               
               
                   
               
               
                 TDCWTNFVNPKRPFWPWKGLEIISRRTQRRLRRIKESWGLQDLVNDFGNLQLGPPMS 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Rat APOBEC-3: 
               
               
                 (SEQ ID NO: 238) 
                   
               
               
                 MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNRLRYAIDRKDTELCYEVTRKDCDSPVSLHH 
                   
               
               
                   
               
               
                 GVFKNKDNI HAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFEC AEQVLRFLATHHNLS 
               
               
                   
               
               
                 LDIFSSRLYNIRDPENQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKKLLTN 
               
               
                   
               
               
                 FRYQDSKLQEILRPCYIPVPSSSSSTLSNICLTKGLPETRFCVERRRVHLLSEEEFYSQFYN 
               
               
                   
               
               
                 QRVKHLCYYHGVKPYLCYQLEQFNGQAPLKGCLLSEKGKQ HAEILFLDKIRSMELSOVIITC   
               
               
                   
               
               
                   YLTWSPCPNC AWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQ 
               
               
                   
               
               
                 FTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLHRIKESWGLQDLVNDFGNLQLGPPMS 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Rhesus macaque APOBEC-3G: 
               
               
                 (SEQ ID NO: 239) 
                   
               
               
                 
                   MVEPMDPRTFVSNFNNRPILSGLNTVWLCCEVKTKDPSGPPLDAKIFQGKVYSKAKY HPEMR   
                 
                   
               
               
                   
               
               
                   FLRWFHKWROLHHDOEYKVTWYVSWSPCTRC ANSVATFLAKDPKVTLTIFVARLYYFWKPDY 
               
               
                   
               
               
                 QQALRILCQKRGGPHATMKIMNYNEFQDCWNKFVDGRGKPFKPRNNLPKHYTLLQATLGELL 
               
               
                   
               
               
                 RHLMDPGTFTSNFNNKPWVSGQHETYLCYKVERLHNDTWVPLNQHRGFLRNQAPNIHGFPKG 
               
               
                   
               
               
                   RHAELCFLDLIPFWKLDGQQYRVTCFTSWS PCFSCAQEMARFISNNEHVSLCIFAARIYDDQ 
               
               
                   
               
               
                 GRYQEGLRALHRDGAKIAMMNYSEFEYCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAI 
               
               
                   
               
               
                 (italic: nucleic acid editing domain; 
               
               
                 underline: cytoplasmic localization signal) 
               
               
                   
               
               
                 Chimpanzee APOBEC-3G: 
               
               
                 (SEQ ID NO: 240) 
                   
               
               
                   MKPHFRNPVERMYQDTFSDNFYNRPILSHRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYS KL 
                   
               
               
                   
               
               
                 KY HPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKC TRDVAYFLAEDPKVTLTIFVARLY 
               
               
                   
               
               
                 YFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLH 
               
               
                   
               
               
                 IMLGEILRHSMDPPTFTSNFNNELWVRGRHETYLCYEVERLHNDTWVLLNQRRGFLCNQAPH 
               
               
                   
               
               
                 RRGFLEGR HAELCFLDVIPFWKLDLHQDYRVTCFTSWSPCFSC AQEMARFISRRRHVSLCIF 
               
               
                   
               
               
                 AARIYDDQGRCQEGLRTLAKAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLEEHSQALS 
               
               
                   
               
               
                 GRLRAILQNQGN 
               
               
                 (italic: nucleic acid editing domain; 
               
               
                 underline: cytoplasmic localization signal) 
               
               
                   
               
               
                 Green monkey APOBEC-3G: 
               
               
                 (SEQ ID NO: 241) 
                   
               
               
                   MNPQIRNMVEQMEPDIFVYYFNNRPILSGRNTVWLCYEVKTKDPSGPPLDANIFQGKLYP EA 
                   
               
               
                   
               
               
                 KD HPEMKFLHWFRKWRQLHRDQEYEVTWYVSWSPCTRC ANSVATFLAEDPKVTLTIFVARLY 
               
               
                   
               
               
                 YFWKPDYQQALRILCQERGGPHATMKIMNYNEFQHCWNEFVDGQGKPFKPRKNLPKHYTLLH 
               
               
                   
               
               
                 ATLGELLRHVMDPGTFTSNFNNKPWVSGQRETYLCYKVERSHNDTWVLLNQHRGFLRNQAPD 
               
               
                   
               
               
                 RHGFPKGR HAELCELDLTPEWKLDDQOYRVTCFTSWSPCFSC AQKMAKFISNNKHVSLCIFA 
               
               
                   
               
               
                 ARIYDDQGRCQEGLRTLHRDGAKIAVMNYSEFEYCWDTFVDRQGRPFQPWDGLDEHSQALSG 
               
               
                   
               
               
                 RLRAI 
               
               
                 (italic: nucleic acid editing domain; 
               
               
                 underline: cytoplasmic localization signal) 
               
               
                   
               
               
                 Human APOBEC-3G: 
               
               
                 (SEQ ID NO: 221) 
                   
               
               
                   MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYS EL 
                   
               
               
                   
               
               
                 KY HPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKC TRDMATFLAEDPKVTLTIFVARLY 
               
               
                   
               
               
                 YFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLH 
               
               
                   
               
               
                 IMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPH 
               
               
                   
               
               
                 KHGFLEGR HAELCFLDVIPFWKLDLDODYRVTCFTSWSPCFSC AQEMAKFISKNKHVSLCIF 
               
               
                   
               
               
                 TARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLS 
               
               
                   
               
               
                 GRLRAILQNQEN 
               
               
                 (italic: nucleic acid editing domain; 
               
               
                 underline: cytoplasmic localization signal) 
               
               
                   
               
               
                 Human APOBEC-3F: 
               
               
                 (SEQ ID NO: 220) 
                   
               
               
                 MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPRLDAKIFRGQVYSQP 
                   
               
               
                   
               
               
                 ER HAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDC VAKLAEFLAEHPNVTLTISAARLYY 
               
               
                   
               
               
                 YWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYSEGQPFMPWYKFDDNYAFLHRTLKE 
               
               
                   
               
               
                 ILRNPMEAMYPHIFYFHFKNLRKAYGRNESWLCFTMEVVKHHSPVSWKRGVFRNQVDPETHC 
               
               
                   
               
               
                   HAERCFLSWECDDILSPNTNYEVTWYTSWSPCPEC AGEVAEFLARHSNVNLTIFTARLYYFW 
               
               
                   
               
               
                 DTDYQEGLRSLSQEGASVEIMGYKDFKYCWENFVYNDDEPFKPWKGLKYNFLFLDSKLQEIL 
               
               
                   
               
               
                 E 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Human APOBEC-3B: 
               
               
                 (SEQ ID NO: 217) 
                   
               
               
                 MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGQVYFK 
                   
               
               
                   
               
               
                 PQY HAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDC VAKLAEFLSEHPNVYLYISAARLY 
               
               
                   
               
               
                 YYWERDYRRALCRLSQAGARVTIMDYEEFAYCWENFVYNEGQQFMPWYKFDENYAFLHRTLK 
               
               
                   
               
               
                 EILRYLMDPDTFTFNFNNDPLVLRRRQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNLLCG 
               
               
                   
               
               
                 FY GRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSW GCAGEVRAFLQENTHVRLRIFAA 
               
               
                   
               
               
                 RIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGR 
               
               
                   
               
               
                 LRAILQNQGN 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Rat APOBEC-3B: 
               
               
                 (SEQ ID NO: 242) 
                   
               
               
                 MQPQGLGPNAGMGPVCLGCSHRRPYSPIRNPLKKLYQQTFYFHFKNVRYAWGRKNNFLCYEV 
                   
               
               
                   
               
               
                 NGMDCALPVPLRQGVFRKQGHIHAELCFIYWFHDKVLRVLSPMEEFKVTWYMSWSPCSKCAE 
               
               
                   
               
               
                 QVARFLAAHRNLSLAIFSSRLYYYLRNPNYQQKLCRLIQEGVHVAAMDLPEFKKCWNKFVDN 
               
               
                   
               
               
                 DGQPFRPWMRLRINFSFYDCKLQEIFSRMNLLREDVFYLQFNNSHRVKPVQNRYYRRKSYLC 
               
               
                   
               
               
                 YQLERANGQEPLKGYLLYKKGEQHVEILFLEKMRSMELSQVRITCYLTWSPCPNCARQLAAF 
               
               
                   
               
               
                 KKDHPDLILRIYTSRLYFWRKKFQKGLCTLWRSGIHVDVMDLPQFADCWTNFVNPQRPFRPW 
               
               
                   
               
               
                 NELEKNSWRIQRRLRRIKESWGL 
               
               
                   
               
               
                 Bovine APOBEC-3B: 
               
               
                 (SEQ ID NO: 243) 
                   
               
               
                 DGWEVAFRSGTVLKAGVLGVSMTEGWAGSGHPGQGACVWTPGTRNTMNLLREVLFKQQFGNQ 
                   
               
               
                   
               
               
                 PRVPAPYYRRKTYLCYQLKQRNDLTLDRGCFRNKKQRHAERFIDKINSLDLNPSQSYKIICY 
               
               
                   
               
               
                 ITWSPCPNCANELVNFITRNNHLKLEIFASRLYFHWIKSFKMGLQDLQNAGISVAVMTHTEF 
               
               
                   
               
               
                 EDCWEQFVDNQSRPFQPWDKLEQYSASIRRRLQRILTAPI 
               
               
                   
               
               
                 Chimpanzee APOBEC-3B: 
               
               
                 (SEQ ID NO: 244) 
                   
               
               
                 MNPQIRNPMEWMYQRTFYYNFENEPILYGRSYTWLCYEVKIRRGHSNLLWDTGVFRGQMYSQ 
                   
               
               
                   
               
               
                 PEHHAEMCFLSWFCGNQLSAYKCFQITWFVSWTPCPDCVAKLAKFLAEHPNVTLTISAARLY 
               
               
                   
               
               
                 YYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYNEGQPFMPWYKFDDNYAFLHRTLK 
               
               
                   
               
               
                 EIIRHLMDPDTFTFNFNNDPLVLRRHQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNLLCG 
               
               
                   
               
               
                 FYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGQVRAFLQENTHVRLRIFAA 
               
               
                   
               
               
                 RIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGR 
               
               
                   
               
               
                 LRAILQVRASSLCMVPHRPPPPPQSPGPCLPLCSEPPLGSLLPTGRPAPSLPFLLTASFSFP 
               
               
                   
               
               
                 PPASLPPLPSLSLSPGHLPVPSFHSLTSCSIQPPCSSRIRETEGWASVSKEGRDLG 
               
               
                   
               
               
                 Human APOBEC-3C: 
               
               
                 (SEQ ID NO: 218) 
                   
               
               
                 MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVFRNQVDSE 
                   
               
               
                   
               
               
                 YRC HAERCFLSWFCDDILSPNTKYQVTWYTSWSPCPD CAGEVAEFLARHSNVNLTIFTARLY 
               
               
                   
               
               
                 YFQYPCYQEGLRSLSQEGVAVEIMDYEDFKYCWENFVYNDNEPFKPWKGLKTNFRLLKRRLR 
               
               
                   
               
               
                 ESLQ 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Gorilla APOBEC-3C3C 
               
               
                 (SEQ ID NO: 245) 
                   
               
               
                 MNPQIRNPMKAMYPGTEYFQFKNLWEANDRNETWLCETVEGIKRRSVVSWKTGVFRNQVDSE 
                   
               
               
                   
               
               
                 THC HAERCFLSWECDDILSPNTNYOVTWYTSWSPCPEC AGEVAEFLARHSNVNLTIFTARLY 
               
               
                   
               
               
                 YFQDTDYQEGLRSLSQEGVAVKIMDYKDFKYCWENFVYNDDEPFKPWKGLKYNFRFLKRRLQ 
               
               
                   
               
               
                 EILE 
               
               
                   
               
               
                 Human APOBEC-3A: 
               
               
                 (SEQ ID NO: 216) 
                   
               
               
                 MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNL 
                   
               
               
                   
               
               
                 LCGFYGR HAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGC AGEVRAFLQENTHVRLRI 
               
               
                   
               
               
                 FAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQAL 
               
               
                   
               
               
                 SGRLRAILQNQGN 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Rhesus macaque APOBEC-3A: 
               
               
                 (SEQ ID NO: 246) 
                   
               
               
                 MDGSPASRPRHLMDPNTFTFNFNNDLSVRGRHQTYLCYEVERLDNGTWVPMDERRGFLCNKA 
                   
               
               
                   
               
               
                 KNVPCGDYGC HVELRFLCEVPSWQLDPAQTYRVTWFISWSPC FRRGCAGQVRVFLQENRHVR 
               
               
                   
               
               
                 LRIFAARIYDYDPLYQEALRTLRDAGAQVSIMTYEEFKHCWDTFVDRQGRPFQPWDGLDEHS 
               
               
                   
               
               
                 QALSGRLRAILQNQGN 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Bovine APOBEC-3A3A: 
               
               
                 (SEQ ID NO: 247) 
                   
               
               
                 MDEYTFTENFNNQGWPSKTYLCYEMERLDGDATIPLDEYKGFVRNKGLDQPEKPC HAEAYFL   
                   
               
               
                   
               
               
                   GKIHSWNLDRNQHYRLTCFISWSPC YDCAQRLTTFLKENHHISLHILASRIYTHNRFGCHQS 
               
               
                   
               
               
                 GLCELQAAGARITIMTFEDFKHCWETFVDHKGKPFQPWEGLNVKSQALCTELQAILKTQQN 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Human APOBEC-3H: 
               
               
                 (SEQ ID NO: 248) 
                   
               
               
                 MALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTPQNGSTPTRGYFENKKKC HAEICFINE   
                   
               
               
                   
               
               
                   IKSMGLDETQCYQVTCYLTWSPCSSC AWELVDFIKAHDHLNLGIFASRLYYHWCKPQQKGLR 
               
               
                   
               
               
                 LLCGSQVPVEVMGFPKFADCWENFVDHEKPLSFNPYKMLEELDKNSRAIKRRLERIKIPGVR 
               
               
                   
               
               
                 AQGRYMDILCDAEV 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Rhesus macaque APOBEC-3H: 
               
               
                 (SEQ ID NO: 249) 
                   
               
               
                 MALLTAKTFSLQFNNKRRVNKPYYPRKALLCYQLTPQNGSTPTRGHLKNKKKDHAEIRFINK 
                   
               
               
                   
               
               
                 IKSMGLDETQCYQVTCYLTWSPCPSCAGELVDFIKAHRHLNLRIFASRLYYHWRPNYQEGLL 
               
               
                   
               
               
                 LLCGSQVPVEVMGLPEETDCWENEVDHKEPPSFNPSEKLEELDKNSQAIKRRLERIKSRSVD 
               
               
                   
               
               
                 VLENGLRSLQLGPVTPSSSIRNSR 
               
               
                   
               
               
                 Human APOBEC-3D: 
               
               
                 (SEQ ID NO: 219) 
                   
               
               
                 MNPQIRNPMERMYRDTFYDNFENEP1LYGRSYTWLCYEVKIKRGRSNLLWDTGVFR 
                   
               
               
                   
               
               
                 GPVLPKRQSNHRQEVYFRFENHAEMCFLSWFCGNRLPANRRFQITWFVSWNPCLPC 
               
               
                   
               
               
                 VVKVTKFLAEHPNVTLTISAARLYYYRDRDWRWVLLRLHKAGARVK1MDYEDFAY 
               
               
                   
               
               
                 CWENFVCNEGQPFMPWYKFDDNYASLHRTLKEILRNPMEAMYPHIFYFHFKNLLKA 
               
               
                   
               
               
                 CGRNESWLCFTMEVTKHHSAVFRKRGVFRNQVDPETHCHAERCFLSWFCDDILSPN 
               
               
                   
               
               
                 TNYEVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLCYFWDTDYQEGLCSLS 
               
               
                   
               
               
                 QEGASVKIMGYKDFVSCWKNFVYSDDEPFKPWKGLQTNFRLLKRRLREILQ 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Human APOBEC-1: 
               
               
                 (SEQ ID NO: 207) 
                   
               
               
                 MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHV 
                   
               
               
                   
               
               
                 EVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMD 
               
               
                   
               
               
                 QQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCI1 
               
               
                   
               
               
                 LSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR 
               
               
                   
               
               
                 Mouse APOBEC-1: 
               
               
                 (SEQ ID NO: 205) 
                   
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSVWRHTSQNTSNHV 
                   
               
               
                   
               
               
                 EVNFLEKFTTERYFRPNTRCSITWFLSWSPCGECSRAITEFLSRHPYVTLFIYIARLYHHTD 
               
               
                   
               
               
                 QRNRQGLRDLISSGVTIQIMTEQEYCYCWRNFVNYPPSNEAYWPRYPHLWVKLYVLELYCI1 
               
               
                   
               
               
                 LGLPPCLKILRRKQPQLTFFTITLQTCHYQRIPPHLLWATGLK 
               
               
                   
               
               
                 Rat APOBEC-1: 
               
               
                 (SEQ ID NO: 204) 
                   
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHV 
                   
               
               
                   
               
               
                 EVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHAD 
               
               
                   
               
               
                 PRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCII 
               
               
                   
               
               
                 LGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK 
               
               
                   
               
               
                 Human APOBEC-2: 
               
               
                 (SEQ ID NO: 212) 
                   
               
               
                 MAQKEEAAVATEAASQNGEDLENLDDPEKLKELIELPPFEIVTGERLPANFFKFQFRNVEYS 
                   
               
               
                   
               
               
                 SGRNKTFLCYVVEAQGKGGQVOASRGYLEDEHAAAHAEEAFFNTILPAFDPALRYNVTWYVS 
               
               
                   
               
               
                 SSPCAACADRIIKTLSKTKNLRLLILVGRLFMWEEPEIQAALKKLKEAGCKLRIMKPQDFEY 
               
               
                   
               
               
                 VWQNFVEQEEGESKAFQPWEDIQENFLYYEEKLADILK 
               
               
                   
               
               
                 Mouse APOBEC-2: 
               
               
                 (SEQ ID NO: 211) 
                   
               
               
                 MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFRNVEYS 
                   
               
               
                   
               
               
                 SGRNKTFLCYWEVQSKGGQAQATQGYLEDEHAGAHAEEAFFNTILPAFDPALKYNVTWYVS 
               
               
                   
               
               
                 SSPCAACADRILKTLSKTKNLRLLILVSRLFMWEEPEVQAALKKLKEAGCKLRIMKPQDFEY 
               
               
                   
               
               
                 IWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK 
               
               
                   
               
               
                 Rat APOBEC-2: 
               
               
                 (SEQ ID NO: 250) 
                   
               
               
                 MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFRNVEYS 
                   
               
               
                   
               
               
                 SGRNKTFLCYVVEAQSKGGQVQATQGYLEDEHAGAHAEEAFFNTILPAFDPALKYNVTWYVS 
               
               
                   
               
               
                 SSPCAACADRILKTLSKTKNLRLLILVSRLFMWEEPEVQAALKKLKEAGCKLRIMKPQDFEY 
               
               
                   
               
               
                 LWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK 
               
               
                   
               
               
                 Bovine APOBEC-2: 
               
               
                 (SEQ ID NO: 214) 
                   
               
               
                 MAQKEEAAAAAEPASQNGEEVENLEDPEKLKELIELPPFEIVTGERLPAHYFKFQFRNVEYS 
                   
               
               
                   
               
               
                 SGRNKTFLCYVVEAQSKGGQVQASRGYLEDEHATNHAEEAFFNSIMPTFDPALRYMVTWYVS 
               
               
                   
               
               
                 SSPCAACADRIVKTLNKTKNLRLLILVGRLFMWEEPEIQAALRKLKEAGCRLRIMKPQDFEY 
               
               
                   
               
               
                 IWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK 
               
               
                   
               
               
                   Petromyzon marinus CDA1 
               
               
                 (pmCDAl) 
               
               
                   
               
               
                 (SEQ ID NO: 251) 
                   
               
               
                 MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTE 
                   
               
               
                   
               
               
                 RGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWAC 
               
               
                   
               
               
                 KLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEK 
               
               
                   
               
               
                 RRSELSFMIQVKILHTTKSPAV 
               
               
                   
               
               
                 Human APOBEC3G chain A 
               
               
                 (SEQ ID NO: 252) 
                   
               
               
                 MDPPTFTFNFNNEPWWGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAE 
                   
               
               
                   
               
               
                 LCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRC 
               
               
                   
               
               
                 QEGLRTLAEAGAKISFTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQ 
               
            
           
         
       
     
     Other exemplary deaminases that can be internally fused within the amino acid sequence of a Cas12 according to aspects of this disclosure are provided below. It should be understood that, in some embodiments, the active domain of the respective sequence can be used, e.g., the domain without a localizing signal (e.g., a nuclear localization sequence, a nuclear export signal, or a cytoplasmic localizing signal). 
     Details of C to T nucleobase editing proteins are described in International PCT Application No. PCT/US2016/058344 (WO 2017/070632) and Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016), the entire contents of which are hereby incorporated by reference. 
     Cytidine Deaminase 
     In one embodiment, a fusion protein of the invention comprises a cytidine deaminase. In some embodiments, the cytidine deaminases provided herein are capable of deaminating cytosine or 5-methylcytosine to uracil or thymine. In some embodiments, the cytosine deaminases provided herein are capable of deaminating cytosine in DNA. The cytidine deaminase may be derived from any suitable organism. In some embodiments, the cytidine deaminase is from a prokaryote. In some embodiments, the cytidine deaminase is from a bacterium. In some embodiments, the cytidine deaminase is from a mammal (e.g., human). 
     In some embodiments, the cytidine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the cytidine deaminase amino acid sequences set forth herein. 
     A fusion protein of the invention comprises a nucleic acid editing domain. In some embodiments, the nucleic acid editing domain can catalyze a C to U base change. In some embodiments, the nucleic acid editing domain is a deaminase domain. In some embodiments, the deaminase is a cytidine deaminase or an adenosine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC1 deaminase. In some embodiments, the deaminase is an APOBEC2 deaminase. In some embodiments, the deaminase is an APOBEC3 deaminase. In some embodiments, the deaminase is an APOBEC3 A deaminase. In some embodiments, the deaminase is an APOBEC3B deaminase. In some embodiments, the deaminase is an APOBEC3C deaminase. In some embodiments, the deaminase is an APOBEC3D deaminase. In some embodiments, the deaminase is an APOBEC3E deaminase. In some embodiments, the deaminase is an APOBEC3F deaminase. In some embodiments, the deaminase is an APOBEC3G deaminase. In some embodiments, the deaminase is an APOBEC3H deaminase. In some embodiments, the deaminase is an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). In some embodiments, the deaminase is a vertebrate deaminase. In some embodiments, the deaminase is an invertebrate deaminase. In some embodiments, the deaminase is a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse deaminase. In some embodiments, the deaminase is a human deaminase. In some embodiments, the deaminase is a rat deaminase, e.g., rAPOBEC1. In some embodiments, the deaminase is a  Petromyzon marinus  cytidine deaminase 1 (pmCDA1). In some embodiments, the deaminase is a human APOBEC3G. In some embodiments, the deaminase is a fragment of the human APOBEC3G. In some embodiments, the nucleic acid editing domain is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or at least 99.5% identical to the deaminase domain of any deaminase described herein. 
     Additional Domains 
     A base editor described herein can include any domain which helps to facilitate the nucleobase editing, modification or altering of a nucleobase of a polynucleotide. In some embodiments, a base editor comprises a polynucleotide programmable nucleotide binding domain (e.g., Cas9), a nucleobase editing domain (e.g., deaminase domain), and one or more additional domains. In some embodiments, the additional domain can facilitate enzymatic or catalytic functions of the base editor, binding functions of the base editor, or be inhibitors of cellular machinery (e.g., enzymes) that could interfere with the desired base editing result. In some embodiments, a base editor can comprise a nuclease, a nickase, a recombinase, a deaminase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain. 
     In some embodiments, a base editor can comprise an uracil glycosylase inhibitor (UGI) domain. A UGI domain can for example improve the efficiency of base editors comprising a cytidine deaminase domain by inhibiting the conversion of a U formed by deamination of a C back to the C nucleobase. In some embodiments, cellular DNA repair response to the presence of U: G heteroduplex DNA can be responsible for a decrease in nucleobase editing efficiency in cells. In such embodiments, uracil DNA glycosylase (UDG) can catalyze removal of U from DNA in cells, which can initiate base excision repair (BER), mostly resulting in reversion of the U:G pair to a C:G pair. In such embodiments, BER can be inhibited in base editors comprising one or more domains that bind the single strand, block the edited base, inhibit UGI, inhibit BER, protect the edited base, and/or promote repairing of the non-edited strand. Thus, this disclosure contemplates a base editor fusion protein comprising a UGI domain. 
     In some embodiments, a base editor comprises as a domain all or a portion of a double-strand break (DSB) binding protein. For example, a DSB binding protein can include a Gam protein of bacteriophage Mu that can bind to the ends of DSBs and can protect them from degradation. See Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), the entire content of which is hereby incorporated by reference. 
     Additionally, in some embodiments, a Gam protein can be fused to an N terminus of a base editor. In some embodiments, a Gam protein can be fused to a C-terminus of a base editor. The Gam protein of bacteriophage Mu can bind to the ends of double strand breaks (DSBs) and protect them from degradation. In some embodiments, using Gam to bind the free ends of DSB can reduce indel formation during the process of base editing. In some embodiments, 174-residue Gam protein is fused to the N terminus of the base editors. See. Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017). In some embodiments, a mutation or mutations can change the length of a base editor domain relative to a wild-type domain. For example, a deletion of at least one amino acid in at least one domain can reduce the length of the base editor. In another case, a mutation or mutations do not change the length of a domain relative to a wild-type domain. For example, substitution(s) in any domain does/do not change the length of the base editor. 
     In some embodiments, a base editor can comprise as a domain all or a portion of a nucleic acid polymerase (NAP). For example, a base editor can comprise all or a portion of a eukaryotic NAP. In some embodiments, a NAP or portion thereof incorporated into a base editor is a DNA polymerase. In some embodiments, a NAP or portion thereof incorporated into a base editor has translesion polymerase activity. In some embodiments, a NAP or portion thereof incorporated into a base editor is a translesion DNA polymerase. In some embodiments, a NAP or portion thereof incorporated into a base editor is a Rev7, Rev1 complex, polymerase iota, polymerase kappa, or polymerase eta. In some embodiments, a NAP or portion thereof incorporated into a base editor is a eukaryotic polymerase alpha, beta, gamma, delta, epsilon, gamma, eta, iota, kappa, lambda, mu, or nu component. In some embodiments, a NAP or portion thereof incorporated into a base editor comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a nucleic acid polymerase (e.g., a translesion DNA polymerase). 
     Other Nucleobase Editors 
     The invention provides for a modular multi-effector nucleobase editor wherein virtually any nucleobase editor known in the art can be inserted into the fusion protein described herein or swapped in for a cytidine deaminase or adenosine deaminase. In one embodiment, the invention features a multi-effector nucleobase editor comprising an abasic nucleobase editor domain. Abasic nucleobase editors are known in the art and described, for example, by Kavli et al., EMBO J. 15:3442-3447, 1996, which is incorporated herein by reference. 
     In one embodiment, a multi-effector nucleobase editor comprises the following domains A-C, A-D, or A-E:
         NH 2 -[A-B-C]-COOH,   NH 2 -[A-B-C-D]-COOH, or   NH 2 -[A-B-C-D-E]-COOH
 
wherein A and C or A, C, and E, each comprises one or more of the following: an adenosine deaminase domain or an active fragment thereof, a cytidine deaminase domain or an active fragment thereof, a DNA glycosylase domain or an active fragment thereof; and where B or B and D, each comprises one or more domains having nucleic acid sequence specific binding activity.
       

     In one embodiment, a multi-effector nucleobase editor comprises NH 2 -[A n -B o -C n ]-COOH,
         NH 2 -[A n -B o -C n -D o ]-COOH, or   NH 2 -[A n -B o -C p -D o -Eq]-COOH;
 
wherein A and C or A, C, and E, each comprises one or more of the following: an adenosine deaminase domain or an active fragment thereof, a cytidine deaminase domain or an active fragment thereof, and a DNA glycosylase domain or an active fragment thereof; and where n is an integer: 1, 2, 3, 4, or 5, and where p is an integer: 0, 1, 2, 3, 4, or 5; and B or B and D each comprises a domain having nucleic acid sequence specific binding activity; and wherein o is an integer: 1, 2, 3, 4, or 5.
       

     Base Editor System 
     Use of the base editor system provided herein comprises the steps of: (a) contacting a target nucleotide sequence of a polynucleotide (e.g., double- or single stranded DNA or RNA) of a subject with a base editor system comprising a nucleobase editor (e.g., an adenosine base editor) and a guide polynucleic acid (e.g., gRNA), wherein the target nucleotide sequence comprises a targeted nucleobase pair; (b) inducing strand separation of said target region; (c) converting a first nucleobase of said target nucleobase pair in a single strand of the target region to a second nucleobase; and (d) cutting no more than one strand of said target region, where a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase. It should be appreciated that in some embodiments, step (b) is omitted. In some embodiments, said targeted nucleobase pair is a plurality of nucleobase pairs in one or more genes. In some embodiments, the base editor system provided herein is capable of multiplex editing of a plurality of nucleobase pairs in one or more genes. In some embodiments, the plurality of nucleobase pairs is located in the same gene. In some embodiments, the plurality of nucleobase pairs is located in one or more genes, wherein at least one gene is located in a different locus. 
     In some embodiments, the cut single strand (nicked strand) is hybridized to the guide nucleic acid. In some embodiments, the cut single strand is opposite to the strand comprising the first nucleobase. In some embodiments, the base editor comprises a Cas9 domain. In some embodiments, the first base is adenine, and the second base is not a G, C, A, or T. In some embodiments, the second base is inosine. 
     Base editing system as provided herein provides anew approach to genome editing that uses a fusion protein containing a catalytically defective  Streptococcus pyogenes  Cas9, a cytidine deaminase, and an inhibitor of base excision repair to induce programmable, single nucleotide (C→T or A→G) changes in DNA without generating double-strand DNA breaks, without requiring a donor DNA template, and without inducing an excess of stochastic insertions and deletions. 
     Provided herein are systems, compositions, and methods for editing a nucleobase using a base editor system. In some embodiments, the base editor system comprises (1) a base editor (BE) comprising a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain (e.g., a deaminase domain) for editing the nucleobase; and (2) a guide polynucleotide (e.g., guide RNA) in conjunction with the polynucleotide programmable nucleotide binding domain. In some embodiments, the base editor system comprises an adenosine base editor (ABE). In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the nucleobase editing domain is a deaminase domain. In some embodiments, a deaminase domain can be an adenine deaminase or an adenosine deaminase. In some embodiments, the adenosine base editor can deaminate adenine in DNA. In some embodiments, ABE comprises an evolved TadA variant. 
     Details of nucleobase editing proteins are described in International PCT Application Nos. PCT/2017/045381 (WO2018/027078) and PCT/US2016/058344 (WO2017/070632), each of which is incorporated herein by reference for its entirety. Also see Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), the entire contents of which are hereby incorporated by reference. 
     In some embodiments, a single guide polynucleotide may be utilized to target a deaminase to a target nucleic acid sequence. In some embodiments, a single pair of guide polynucleotides may be utilized to target different deaminases to a target nucleic acid sequence. 
     The nucleobase components and the polynucleotide programmable nucleotide binding component of a base editor system may be associated with each other covalently or non-covalently. For example, in some embodiments, the deaminase domain can be targeted to a target nucleotide sequence by a polynucleotide programmable nucleotide binding domain. In some embodiments, a polynucleotide programmable nucleotide binding domain can be fused or linked to a deaminase domain. In some embodiments, a polynucleotide programmable nucleotide binding domain can target a deaminase domain to a target nucleotide sequence by non-covalently interacting with or associating with the deaminase domain. For example, in some embodiments, the nucleobase editing component, e.g., the deaminase component can comprise an additional heterologous portion or domain that is capable of interacting with, associating with, or capable of forming a complex with an additional heterologous portion or domain that is part of a polynucleotide programmable nucleotide binding domain. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous portion may be a protein domain. In some embodiments, the additional heterologous portion may be a K Homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a steril alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif. 
     A base editor system may further comprise a guide polynucleotide component. It should be appreciated that components of the base editor system may be associated with each other via covalent bonds, noncovalent interactions, or any combination of associations and interactions thereof. In some embodiments, a deaminase domain can be targeted to a target nucleotide sequence by a guide polynucleotide. For example, in some embodiments, the nucleobase editing component of the base editor system, e.g., the deaminase component, can comprise an additional heterologous portion or domain (e.g., polynucleotide binding domain such as an RNA or DNA binding protein) that is capable of interacting with, associating with, or capable of forming a complex with a portion or segment (e.g., a polynucleotide motif) of a guide polynucleotide. In some embodiments, the additional heterologous portion or domain (e.g., polynucleotide binding domain such as an RNA or DNA binding protein) can be fused or linked to the deaminase domain. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous portion may be a protein domain. In some embodiments, the additional heterologous portion may be a K Homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif. 
     In some embodiments, a base editor system can further comprise an inhibitor of base excision repair (BER) component. It should be appreciated that components of the base editor system may be associated with each other via covalent bonds, noncovalent interactions, or any combination of associations and interactions thereof. The inhibitor of BER component may comprise a base excision repair inhibitor. In some embodiments, the inhibitor of base excision repair can be a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the inhibitor of base excision repair can be an inosine base excision repair inhibitor. In some embodiments, the inhibitor of base excision repair can be targeted to the target nucleotide sequence by the polynucleotide programmable nucleotide binding domain. In some embodiments, a polynucleotide programmable nucleotide binding domain can be fused or linked to an inhibitor of base excision repair. In some embodiments, a polynucleotide programmable nucleotide binding domain can be fused or linked to a deaminase domain and an inhibitor of base excision repair. In some embodiments, a polynucleotide programmable nucleotide binding domain can target an inhibitor of base excision repair to a target nucleotide sequence by non-covalently interacting with or associating with the inhibitor of base excision repair. For example, in some embodiments, the inhibitor of base excision repair component can comprise an additional heterologous portion or domain that is capable of interacting with, associating with, or capable of forming a complex with an additional heterologous portion or domain that is part of a polynucleotide programmable nucleotide binding domain. In some embodiments, the inhibitor of base excision repair can be targeted to the target nucleotide sequence by the guide polynucleotide. For example, in some embodiments, the inhibitor of base excision repair can comprise an additional heterologous portion or domain (e.g., polynucleotide binding domain such as an RNA or DNA binding protein) that is capable of interacting with, associating with, or capable of forming a complex with a portion or segment (e.g., a polynucleotide motif) of a guide polynucleotide. In some embodiments, the additional heterologous portion or domain of the guide polynucleotide (e.g., polynucleotide binding domain such as an RNA or DNA binding protein) can be fused or linked to the inhibitor of base excision repair. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous portion may be a protein domain. In some embodiments, the additional heterologous portion may be a K Homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif. 
     In some embodiments, the base editor inhibits base excision repair (BER) of the edited strand. In some embodiments, the base editor protects or binds the non-edited strand. In some embodiments, the base editor comprises UGI activity. In some embodiments, the base editor comprises a catalytically inactive inosine-specific nuclease. In some embodiments, the base editor comprises nickase activity. In some embodiments, the intended edit of base pair is upstream of a PAM site. In some embodiments, the intended edit of base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended edit of base-pair is downstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream stream of the PAM site. 
     In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the nucleobase editor comprises a linker or a spacer. In some embodiments, the linker or spacer is 1-25 amino acids in length. In some embodiments, the linker or spacer is 5-20 amino acids in length. In some embodiments, the linker or spacer is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. 
     In some embodiments, the base editing fusion proteins provided herein need to be positioned at a precise location, for example, where a target base is placed within a defined region (e.g., a “deamination window”). In some embodiments, a target can be within a 4 base region. In some embodiments, such a defined target region can be approximately 15 bases upstream of the PAM. See Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), the entire contents of which are hereby incorporated by reference. 
     In some embodiments, the target region comprises a target window, wherein the target window comprises the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended edit of base pair is within the target window. In some embodiments, the target window comprises the intended edit of base pair. In some embodiments, the method is performed using any of the base editors provided herein. In some embodiments, a target window is a deamination window. A deamination window can be the defined region in which a base editor acts upon and deaminates a target nucleotide. In some embodiments, the deamination window is within a 2, 3, 4, 5, 6, 7, 8, 9, or 10 base regions. In some embodiments, the deamination window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases upstream of the PAM. 
     The base editors of the present disclosure can comprise any domain, feature or amino acid sequence which facilitates the editing of a target polynucleotide sequence. For example, in some embodiments, the base editor comprises a nuclear localization sequence (NLS). In some embodiments, an NLS of the base editor is localized between a deaminase domain and a polynucleotide programmable nucleotide binding domain. In some embodiments, an NLS of the base editor is localized C-terminal to a polynucleotide programmable nucleotide binding domain. 
     Other exemplary features that can be present in a base editor as disclosed herein are localization sequences, such as cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequences will be apparent to those of skill in the art. In some embodiments, the fusion protein comprises one or more His tags. 
     Non-limiting examples of protein domains which can be included in the fusion protein include deaminase domains (e.g., cytidine deaminase, adenosine deaminase), a uracil glycosylase inhibitor (UGI) domain, epitope tags, and reporter gene sequences. 
     Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). Additional protein sequences can include amino acid sequences that bind DNA molecules or bind other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. 
     Base editors or base editor systems may be any base editor as described herein. In some embodiments, the base editor comprises an adenosine deaminase monomer or an adenosine deaminase dimer as described herein. In some embodiments, the base editor comprises a Cas9 with a deaminase, e.g. an adenosine deaminase, inserted in a flexible loop of the Cas9 polypeptide. In some embodiments, the base editor is generated by expression of two polynucleotides each encoding a N-terminal fragment and a C-terminal fragment of a Cas9-split intein fusion protein. A base editor may be delivered to a host cell via RNP, vector, viral vector, or nucleic acid such as mRNA delivery. In some embodiments, a polynucleotide construct encoding the base editor comprises the structure pMRNA-Trilink-ISLAY3-monoTadA-ABE7.10(V82S)-MQKFRAER 120A Bbsl, pMRNA-Trilink-ISLAY3-ABE7.10(V82S, Y147T, Q154S)-MQKFRAER 120A Bbsl, or pMRNA-Trilink-ISLAY3-ABE7.10(V82T, Y147T, Q154S)-MQKFRAER 120A Bbsl. The guide RNA may be chemically modified. In some embodiments, the guide RNA comprises one or more chemically modified nucleobases, such as 2′-O-methyl (2′-OMe), 2′-deoxy (2′-H), 2′-O—C1-3alkyl-O—C1-3alkyl such as 2′-methoxyethyl (“2′-MOE”), 2′-fluoro (“2′-F”), 2′-amino (“2′-NH 2 ”), 2′-arabinosyl (“2′-arabino”) nucleotide, 2′-F-arabinosyl (“2′-F-arabino”) nucleotide, 2′-locked nucleic acid (“LNA”) nucleotide, 2′-unlocked nucleic acid (“ULNA”) nucleotide, a sugar in L form (“L-sugar”), 4′-thioribosyl nucleotide, or any chemical modification as described herein. In some embodiments, the guide RNA comprises an internucleotide linkage modification such as phosphorothioate “P(S)” (P(S)), phosphonocarboxylate (P(CH2)nCOOR) such as phosphonoacetate “PACE” (P(CH2COO—)), thiophosphonocarboxylate ((S)P(CH2)nCOOR) such as thiophosphonoacetate “thioPACE” ((S)P(CH2)nCOO—)), alkylphosphonate (P(C1-3alkyl) such as methylphosphonate —P(CH3), boranophosphonate (P(BH3)), and phosphorodithioate (P(S)2). In some embodiments, the guide RNA comprises a nucleobase chemical modification such as 2-thiouracil (“2-thioU”), 2-thiocytosine (“2-thioC”), 4-thiouracil (“4-thioU”), 6-thioguanine (“6-thioG”), 2-aminoadenine (“2-aminoA”), 2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-methylcytosine (“5-methylC”), 5-methyluracil (“5-methylU”), 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil, 5-propynylcytosine, 5-propynyluracil, 5-ethynylcytosine, 5-ethynyluracil, 5-allyluracil (“5-allylU”), 5-allylcytosine (“5-allylC”), 5-aminoallyluracil (“5-aminoallylU”), 5-aminoallyl-cytosine (“5-aminoallylC”), an abasic nucleotide, Z base, P base, Unstructured Nucleic Acid (“UNA”), isoguanine (“isoG”), isocytosine (“isoC”). In some embodiments, the guide RNA comprises one or more isotopic modifications on the nucleotide sugar, the nucleobase, the phosphodiester linkage and/or the nucleotide phosphates. Such modifications include nucleotides comprising one or more 15N, 13C, 14C, Deuterium, 3H, 32P, 125I, 131I atoms or other atoms or elements thereof. 
     In some embodiments, the adenosine base editor (ABE) can deaminate adenine in DNA. In some embodiments, ABE is generated by replacing APOBEC component of BE3 with natural or engineered  E. coli  TadA, human ADAR2, mouse ADA, or human ADAT2. In some embodiments, ABE comprises evolved TadA variant. In some embodiments, the ABE is ABE 1.2 (TadA*-XTEN-nCas9-NLS). In some embodiments, TadA* comprises A106V and D108N mutations. 
     In some embodiments, the ABE is a second-generation ABE. In some embodiments, the ABE is ABE2.1, which comprises additional mutations D147Y and E155V in TadA* (TadA*2.1). In some embodiments, the ABE is ABE2.2, ABE2.1 fused to catalytically inactivated version of human alkyl adenine DNA glycosylase (AAG with E125Q mutation). In some embodiments, the ABE is ABE2.3, ABE2.1 fused to catalytically inactivated version of  E. coli  Endo V (inactivated with D35A mutation). In some embodiments, the ABE is ABE2.6 which has a linker twice as long (32 amino acids, (SGGS) 2 -XTEN-(SGGS) 2  (“(SGGS) 2 ” disclosed as SEQ ID NO: 253)) as the linker in ABE2.1. In some embodiments, the ABE is ABE2.7, which is ABE2.1 tethered with an additional wild-type TadA monomer. In some embodiments, the ABE is ABE2.8, which is ABE2.1 tethered with an additional TadA*2.1 monomer. In some embodiments, the ABE is ABE2.9, which is a direct fusion of evolved TadA (TadA*2.1) to the N-terminus of ABE2.1. In some embodiments, the ABE is ABE2.10, which is a direct fusion of wild-type TadA to the N-terminus of ABE2.1. In some embodiments, the ABE is ABE2.11, which is ABE2.9 with an inactivating E59A mutation at the N-terminus of TadA* monomer. In some embodiments, the ABE is ABE2.12, which is ABE2.9 with an inactivating E59A mutation in the internal TadA* monomer. 
     In some embodiments, the ABE is a third generation ABE. In some embodiments, the ABE is ABE3.1, which is ABE2.3 with three additional TadA mutations (L84F, H123Y, and I156F). 
     In some embodiments, the ABE is a fourth generation ABE. In some embodiments, the ABE is ABE4.3, which is ABE3.1 with an additional TadA mutation A142N (TadA*4.3). 
     In some embodiments, the ABE is a fifth generation ABE. In some embodiments, the ABE is ABE5.1, which is generated by importing a consensus set of mutations from surviving clones (H36L, R51L, S146C, and K157N) into ABE3.1. In some embodiments, the ABE is ABE5.3, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to an internal evolved TadA*. In some embodiments, the ABE is ABE5.2, ABE5.4, ABE5.5, ABE5.6, ABE5.7, ABE5.8, ABE5.9, ABE5.10, ABE5.11, ABE5.12, ABE5.13, or ABE5.14, as shown in below Table 6. In some embodiments, the ABE is a sixth generation ABE. In some embodiments, the ABE is ABE6.1, ABE6.2, ABE6.3, ABE6.4, ABE6.5, or ABE6.6, as shown in below Table 6. In some embodiments, the ABE is a seventh generation ABE. In some embodiments, the ABE is ABE7.1, ABE7.2, ABE7.3, ABE7.4, ABE7.5, ABE7.6, ABE7.7, ABE7.8, ABE 7.9, or ABE7.10, as shown in Table 6 below. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Genotypes of ABEs 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 23 
                 26 
                 36 
                 37 
                 48 
                 49 
                 51 
                 72 
                 84 
                 87 
                 106 
                 108 
                 123 
                 125 
                 142 
                 146 
                 147 
                 152 
                 155 
                 156 
                 157 
                 161 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 ABE0.1 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 L 
                 S 
                 A 
                 D 
                 H 
                 G 
                 A 
                 S 
                 D 
                 R 
                 E 
                 I 
                 K 
                 K 
               
               
                 ABE0.2 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 L 
                 S 
                 A 
                 D 
                 H 
                 G 
                 A 
                 S 
                 D 
                 R 
                 E 
                 I 
                 K 
                 K 
               
               
                 ABE1.1 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 L 
                 S 
                 A 
                 N 
                 H 
                 G 
                 A 
                 S 
                 D 
                 R 
                 E 
                 I 
                 K 
                 K 
               
               
                 ABE1.2 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 L 
                 S 
                 V 
                 N 
                 H 
                 G 
                 A 
                 S 
                 D 
                 R 
                 E 
                 I 
                 K 
                 K 
               
               
                 ABE2.1 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 L 
                 S 
                 V 
                 N 
                 H 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 I 
                 K 
                 K 
               
               
                 ABE2.2 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 L 
                 S 
                 V 
                 N 
                 H 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 I 
                 K 
                 K 
               
               
                 ABE2.3 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 L 
                 S 
                 V 
                 N 
                 H 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 I 
                 K 
                 K 
               
               
                 ABE2.4 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 L 
                 S 
                 V 
                 N 
                 H 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 I 
                 K 
                 K 
               
               
                 ABE2.5 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 L 
                 S 
                 V 
                 N 
                 H 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 I 
                 K 
                 K 
               
               
                 ABE2.6 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 L 
                 S 
                 V 
                 N 
                 H 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 I 
                 K 
                 K 
               
               
                 ABE2.7 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 L 
                 S 
                 V 
                 N 
                 H 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 I 
                 K 
                 K 
               
               
                 ABE2.8 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 L 
                 S 
                 V 
                 N 
                 H 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 I 
                 K 
                 K 
               
               
                 ABE2.9 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 L 
                 S 
                 V 
                 N 
                 H 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 I 
                 K 
                 K 
               
               
                 ABE2.10 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 L 
                 S 
                 V 
                 N 
                 H 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 I 
                 K 
                 K 
               
               
                 ABE2.11 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 L 
                 S 
                 V 
                 N 
                 H 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 I 
                 K 
                 K 
               
               
                 ABE2.12 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 L 
                 S 
                 V 
                 N 
                 H 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 I 
                 K 
                 K 
               
               
                 ABE3.1 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 F 
                 K 
                 K 
               
               
                 ABE3.2 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 F 
                 K 
                 K 
               
               
                 ABE3.3 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 F 
                 K 
                 K 
               
               
                 ABE3.4 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 F 
                 K 
                 K 
               
               
                 ABE3.5 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 F 
                 K 
                 K 
               
               
                 ABE3.6 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 F 
                 K 
                 K 
               
               
                 ABE3.7 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 F 
                 K 
                 K 
               
               
                 ABE3.8 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 F 
                 K 
                 K 
               
               
                 ABE4.1 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 L 
                 S 
                 V 
                 N 
                 H 
                 G 
                 N 
                 S 
                 Y 
                 R 
                 V 
                 I 
                 K 
                 K 
               
               
                 ABE4.2 
                 W 
                 G 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 L 
                 S 
                 V 
                 N 
                 H 
                 G 
                 N 
                 S 
                 Y 
                 R 
                 V 
                 I 
                 K 
                 K 
               
               
                 ABE4.3 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 N 
                 S 
                 Y 
                 R 
                 V 
                 F 
                 K 
                 K 
               
               
                 ABE5.1 
                 W 
                 R 
                 L 
                 N 
                 P 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE5.2 
                 W 
                 R 
                 H 
                 S 
                 P 
                   
                 R 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 F 
                 K 
                 T 
               
               
                 ABE5.3 
                 W 
                 R 
                 L 
                 N 
                 P 
                   
                 L 
                 N 
                 I 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE5.4 
                 W 
                 R 
                 H 
                 S 
                 P 
                   
                 R 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 F 
                 K 
                 T 
               
               
                 ABE5.5 
                 W 
                 R 
                 L 
                 N 
                 P 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE5.6 
                 W 
                 R 
                 L 
                 N 
                 P 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE5.7 
                 W 
                 R 
                 L 
                 N 
                 P 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE5.8 
                 W 
                 R 
                 L 
                 N 
                 P 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE5.9 
                 W 
                 R 
                 L 
                 N 
                 P 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE5.10 
                 W 
                 R 
                 L 
                 N 
                 P 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE5.11 
                 W 
                 R 
                 L 
                 N 
                 P 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE5.12 
                 W 
                 R 
                 L 
                 N 
                 P 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE5.13 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 L 
                 D 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 A 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 F 
                 K 
                 K 
               
               
                 ABE5.14 
                 W 
                 R 
                 H 
                 N 
                 S 
                   
                 L 
                 N 
                 F 
                 C 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 S 
                 Y 
                 R 
                 V 
                 F 
                 K 
                 K 
               
               
                 ABE6.1 
                 W 
                 R 
                 H 
                 N 
                 S 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 N 
                 S 
                 Y 
                 R 
                 V 
                 F 
                 K 
                 K 
               
               
                 ABE6.2 
                 W 
                 R 
                 H 
                 N 
                 T 
                 V 
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 N 
                 S 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE6.3 
                 W 
                 R 
                 L 
                 N 
                 S 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE6.4 
                 W 
                 R 
                 L 
                 N 
                 S 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 N 
                 C 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE6.5 
                 W 
                 R 
                 L 
                 N 
                 T 
                 V 
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE6.6 
                 W 
                 R 
                 L 
                 N 
                 T 
                 V 
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 N 
                 C 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE7.1 
                 W 
                 R 
                 L 
                 N 
                 A 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE7.2 
                 W 
                 R 
                 L 
                 N 
                 A 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 N 
                 C 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE7.3 
                 L 
                 R 
                 L 
                 N 
                 A 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE7.4 
                 R 
                 R 
                 L 
                 N 
                 A 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE7.5 
                 W 
                 R 
                 L 
                 N 
                 A 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 H 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE7.6 
                 W 
                 R 
                 L 
                 N 
                 A 
                   
                 L 
                 N 
                 I 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 P 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE7.7 
                 L 
                 R 
                 L 
                 N 
                 A 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 P 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE7.8 
                 L 
                 R 
                 L 
                 N 
                 A 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 N 
                 C 
                 Y 
                 R 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE7.9 
                 L 
                 R 
                 L 
                 N 
                 A 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 N 
                 C 
                 Y 
                 P 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE7.10 
                 R 
                 R 
                 L 
                 N 
                 A 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 P 
                 V 
                 F 
                 N 
                 K 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the base editor is an adenosine base eitor. In some embodiments, the adenosine base editor is an eighth generation ABE (ABE8). In some embodiments, the ABE8 contains a TadA*8 variant. In some embodiments, the ABE8 has a monomeric construct containing a TadA*8 variant (“ABE8.x-m”). In some embodiments, the ABE8 is ABE8.1-m, which has a monomeric construct containing TadA*7.10 with a Y147T mutation (TadA*8.1). In some embodiments, the ABE8 is ABE8.2-m, which has a monomeric construct containing TadA*7.10 with a Y147R mutation (TadA*8.2). In some embodiments, the ABE8 is ABE8.3-m, which has a monomeric construct containing TadA*7.10 with a Q154S mutation (TadA*8.3). In some embodiments, the ABE8 is ABE8.4-m, which has a monomeric construct containing TadA*7.10 with a Y123H mutation (TadA*8.4). In some embodiments, the ABE8 is ABE8.5-m, which has a monomeric construct containing TadA*7.10 with a V82S mutation (TadA*8.5). In some embodiments, the ABE8 is ABE8.6-m, which has a monomeric construct containing TadA*7.10 with a T166R mutation (TadA*8.6). In some embodiments, the ABE8 is ABE8.7-m, which has a monomeric construct containing TadA*7.10 with a Q154R mutation (TadA*8.7). In some embodiments, the ABE8 is ABE8.8-m, which has a monomeric construct containing TadA*7.10 with Y147R, Q154R, and Y123H mutations (TadA*8.8). In some embodiments, the ABE8 is ABE8.9-m, which has a monomeric construct containing TadA*7.10 with Y147R, Q154R and I76Y mutations (TadA*8.9). In some embodiments, the ABE8 is ABE8.10-m, which has a monomeric construct containing TadA*7.10 with Y147R, Q154R, and T166R mutations (TadA*8.10). In some embodiments, the ABE8 is ABE8.11-m, which has a monomeric construct containing TadA*7.10 with Y147T and Q154R mutations (TadA*8.11). In some embodiments, the ABE8 is ABE8.12-m, which has a monomeric construct containing TadA*7.10 with Y147T and Q154S mutations (TadA*8.12). In some embodiments, the ABE8 is ABE8.13-m, which has a monomeric construct containing TadA*7.10 with Y123H (Y123H reverted from H123Y), Y147R, Q154R and I76Y mutations (TadA*8.13). In some embodiments, the ABE8 is ABE8.14-m, which has a monomeric construct containing TadA*7.10 with I76Y and V82S mutations (TadA*8.14). In some embodiments, the ABE8 is ABE8.15-m, which has a monomeric construct containing TadA*7.10 with V82S and Y147R mutations (TadA*8.15). In some embodiments, the ABE8 is ABE8.16-m, which has a monomeric construct containing TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y) and Y147R mutations (TadA*8.16). In some embodiments, the ABE8 is ABE8.17-m, which has a monomeric construct containing TadA*7.10 with V82S and Q154R mutations (TadA*8.17). In some embodiments, the ABE8 is ABE8.18-m, which has a monomeric construct containing TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y) and Q154R mutations (TadA*8.18). In some embodiments, the ABE8 is ABE8.19-m, which has a monomeric construct containing TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y), Y147R and Q154R mutations (TadA*8.19). In some embodiments, the ABE8 is ABE8.20-m, which has a monomeric construct containing TadA*7.10 with I76Y, V82S, Y123H (Y123H reverted from H123Y), Y147R and Q154R mutations (TadA*8.20). In some embodiments, the ABE8 is ABE8.21-m, which has a monomeric construct containing TadA*7.10 with Y147R and Q154S mutations (TadA*8.21). In some embodiments, the ABE8 is ABE8.22-m, which has a monomeric construct containing TadA*7.10 with V82S and Q154S mutations (TadA*8.22). In some embodiments, the ABE8 is ABE8.23-m, which has a monomeric construct containing TadA*7.10 with V82S and Y123H (Y123H reverted from H123Y) mutations (TadA*8.23). In some embodiments, the ABE8 is ABE8.24-m, which has a monomeric construct containing TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y), and Y147T mutations (TadA*8.24). 
     In some embodiments, the ABE8 has a heterodimeric construct containing wild-type  E. coli  TadA fused to a TadA*8 variant (“ABE8.x-d”). In some embodiments, the ABE8 is ABE8.1-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with a Y147T mutation (TadA*8.1). In some embodiments, the ABE8 is ABE8.2-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with a Y147R mutation (TadA*8.2). In some embodiments, the ABE8 is ABE8.3-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with a Q154S mutation (TadA*8.3). In some embodiments, the ABE8 is ABE8.4-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with a Y123H mutation (TadA*8.4). In some embodiments, the ABE8 is ABE8.5-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with a V82S mutation (TadA*8.5). In some embodiments, the ABE8 is ABE8.6-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with a T166R mutation (TadA*8.6). In some embodiments, the ABE8 is ABE8.7-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with a Q154R mutation (TadA*8.7). In some embodiments, the ABE8 is ABE8.8-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with Y147R, Q154R, and Y123H mutations (TadA*8.8). In some embodiments, the ABE8 is ABE8.9-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with Y147R, Q154R and I76Y mutations (TadA*8.9). In some embodiments, the ABE8 is ABE8.10-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with Y147R, Q154R, and T166R mutations (TadA*8.10). In some embodiments, the ABE8 is ABE8.11-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with Y147T and Q154R mutations (TadA*8.11). In some embodiments, the ABE8 is ABE8.12-d, which has heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with Y147T and Q154S mutations (TadA*8.12). In some embodiments, the ABE8 is ABE8.13-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with Y123H (Y123H reverted from H123Y), Y147R, Q154R and I76Y mutations (TadA*8.13). In some embodiments, the ABE8 is ABE8.14-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with I76Y and V82S mutations (TadA*8.14). In some embodiments, the ABE8 is ABE8.15-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with V82S and Y147R mutations (TadA*8.15). In some embodiments, the ABE8 is ABE8.16-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y) and Y147R mutations (TadA*8.16). In some embodiments, the ABE8 is ABE8.17-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with V82S and Q154R mutations (TadA*8.17). In some embodiments, the ABE8 is ABE8.18-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y) and Q154R mutations (TadA*8.18). In some embodiments, the ABE8 is ABE8.19-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y), Y147R and Q154R mutations (TadA*8.19). In some embodiments, the ABE8 is ABE8.20-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with I76Y, V82S, Y123H (Y123H reverted from H123Y), Y147R and Q154R mutations (TadA*8.20). In some embodiments, the ABE8 is ABE8.21-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with Y147R and Q154S mutations (TadA*8.21). In some embodiments, the ABE8 is ABE8.22-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with V82S and Q154S mutations (TadA*8.22). In some embodiments, the ABE8 is ABE8.23-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with V82S and Y123H (Y123H reverted from H123Y) mutations (TadA*8.23). In some embodiments, the ABE8 is ABE8.24-d, which has a heterodimeric construct containing wild-type  E. coli  TadA fused to TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y), and Y147T mutations (TadA*8.24). 
     In some embodiments, the ABE8 has a heterodimeric construct containing TadA*7.10 fused to a TadA*8 variant (“ABE8.x-7”). In some embodiments, the ABE8 is ABE8.1-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with a Y147T mutation (TadA*8.1). In some embodiments, the ABE8 is ABE8.2-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with a Y147R mutation (TadA*8.2). In some embodiments, the ABE8 is ABE8.3-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with a Q154S mutation (TadA*8.3). In some embodiments, the ABE8 is ABE8.4-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with a Y123H mutation (TadA*8.4). In some embodiments, the ABE8 is ABE8.5-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with a V82S mutation (TadA*8.5). In some embodiments, the ABE8 is ABE8.6-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with a T166R mutation (TadA*8.6). In some embodiments, the ABE8 is ABE8.7-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with a Q154R mutation (TadA*8.7). In some embodiments, the ABE8 is ABE8.8-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y147R, Q154R, and Y123H mutations (TadA*8.8). In some embodiments, the ABE8 is ABE8.9-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y147R, Q154R and I76Y mutations (TadA*8.9). In some embodiments, the ABE8 is ABE8.10-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y147R, Q154R, and T166R mutations (TadA*8.10). In some embodiments, the ABE8 is ABE8.11-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y147T and Q154R mutations (TadA*8.11). In some embodiments, the ABE8 is ABE8.12-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y147T and Q154S mutations (TadA*8.12). In some embodiments, the ABE8 is ABE8.13-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y123H (Y123H reverted from H123Y), Y147R, Q154R and I76Y mutations (TadA*8.13). In some embodiments, the ABE8 is ABE8.14-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with I76Y and V82S mutations (TadA*8.14). In some embodiments, the ABE8 is ABE8.15-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S and Y147R mutations (TadA*8.15). In some embodiments, the ABE8 is ABE8.16-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y) and Y147R mutations (TadA*8.16). In some embodiments, the ABE8 is ABE8.17-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S and Q154R mutations (TadA*8.17). In some embodiments, the ABE8 is ABE8.18-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y) and Q154R mutations (TadA*8.18). In some embodiments, the ABE8 is ABE8.19-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y), Y147R and Q154R mutations (TadA*8.19). In some embodiments, the ABE8 is ABE8.20-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with I76Y, V82S, Y123H (Y123H reverted from H123Y), Y147R and Q154R mutations (TadA*8.20). In some embodiments, the ABE8 is ABE8.21-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with Y147R and Q154S mutations (TadA*8.21). In some embodiments, the ABE8 is ABE8.22-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S and Q154S mutations (TadA*8.22). In some embodiments, the ABE8 is ABE8.23-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S and Y123H (Y123H reverted from H123Y) mutations (TadA*8.23). In some embodiments, the ABE8 is ABE8.24-7, which has a heterodimeric construct containing TadA*7.10 fused to TadA*7.10 with V82S, Y123H (Y123H reverted from H123Y), and Y147T mutations (TadA*8.24). 
     In some embodiments, the ABE is ABE8.1-m, ABE8.2-m, ABE8.3-m, ABE8.4-m, ABE8.5-m, ABE8.6-m, ABE8.7-m, ABE8.8-m, ABE8.9-m, ABE8.10-m, ABE8.11-m, ABE8.12-m, ABE8.13-m, ABE8.14-m, ABE8.15-m, ABE8.16-m, ABE8.17-m, ABE8.18-m, ABE8.19-m, ABE8.20-m, ABE8.21-m, ABE8.22-m, ABE8.23-m, ABE8.24-m, ABE8.1-d, ABE8.2-d, ABE8.3-d, ABE8.4-d, ABE8.5-d, ABE8.6-d, ABE8.7-d, ABE8.8-d, ABE8.9-d, ABE8.10-d, ABE8.11-d, ABE8.12-d, ABE8.13-d, ABE8.14-d, ABE8.15-d, ABE8.16-d, ABE8.17-d, ABE8.18-d, ABE8.19-d, ABE8.20-d, ABE8.21-d, ABE8.22-d, ABE8.23-d, or ABE8.24-d as shown in Table 7 below. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 ABE8 Base Editors 
               
            
           
           
               
               
               
            
               
                 ABE8 Base 
                 Adenosine 
                   
               
               
                 Editor 
                 Deaminase 
                 Adenosine Deaminase Description 
               
               
                   
               
               
                 ABE8.1-m 
                 TadA*8.1 
                 Monomer_TadA*7.10 + Y147T 
               
               
                 ABE8.2-m 
                 TadA*8.2 
                 Monomer_TadA*7.10 + Y147R 
               
               
                 ABE8.3-m 
                 TadA*8.3 
                 Monomer_TadA*7.10 + Q154S 
               
               
                 ABE8.4-m 
                 TadA*8.4 
                 Monomer_TadA*7.10 + Y123H 
               
               
                 ABE8.5-m 
                 TadA*8.5 
                 Monomer_TadA*7.10 + V82S 
               
               
                 ABE8.6-m 
                 TadA*8.6 
                 Monomer_TadA*7.10 + T166R 
               
               
                 ABE8.7-m 
                 TadA*8.7 
                 Monomer_TadA*7.10 + Q154R 
               
               
                 ABE8.8-m 
                 TadA*8.8 
                 Monomer_TadA*7.10 + Y147R_Q154R_Y123H 
               
               
                 ABE8.9-m 
                 TadA*8.9 
                 Monomer_TadA*7.10 + Y147R_Q154R_I76Y 
               
               
                 ABE8.10-m 
                 TadA*8.10 
                 Monomer_TadA*7.10 + Y147R_Q154R_T166R 
               
               
                 ABE8.11-m 
                 TadA*8.11 
                 Monomer_TadA*7.10 + Y147T_Q154R 
               
               
                 ABE8.12-m 
                 TadA*8.12 
                 Monomer_TadA*7.10 + Y147T_Q154S 
               
               
                 ABE8.13-m 
                 TadA*8.13 
                 Monomer_TadA*7.10 + Y123H_Y147R_Q154R_I76Y 
               
               
                 ABE8.14-m 
                 TadA*8.14 
                 Monomer_TadA*7.10 + I76Y_V82S 
               
               
                 ABE8.15-m 
                 TadA*8.15 
                 Monomer_TadA*7.10 + V82S_Y147R 
               
               
                 ABE8.16-m 
                 TadA*8.16 
                 Monomer_TadA*7.10 + V82S_Y123H_Y147R 
               
               
                 ABE8.17-m 
                 TadA*8.17 
                 Monomer_TadA*7.10 + V82S_Q154R 
               
               
                 ABE8.18-m 
                 TadA*8.18 
                 Monomer_TadA*7.10 + V82S_Y123H_Q154R 
               
               
                 ABE8.19-m 
                 TadA*8.19 
                 Monomer_TadA*7.10 + V82S_Y123H_Y147R_Q154R 
               
               
                 ABE8.20-m 
                 TadA*8.20 
                 Monomer_TadA*7.10 + I76Y_V82S_Y123H_Y147R_Q154R 
               
               
                 ABE8.21-m 
                 TadA*8.21 
                 Monomer_TadA*7.10 + Y147R_Q154S 
               
               
                 ABE8.22-m 
                 TadA*8.22 
                 Monomer_TadA*7.10 + V82S_Q154S 
               
               
                 ABE8.23-m 
                 TadA*8.23 
                 Monomer_TadA*7.10 + V82S_Y123H 
               
               
                 ABE8.24-m 
                 TadA*8.24 
                 Monomer_TadA*7.10 + V82S_Y123H_Y147T 
               
               
                 ABE8.1-d 
                 TadA*8.1 
                 Heterodimer_(WT) + (TadA*7.10 + Y147T) 
               
               
                 ABE8.2-d 
                 TadA*8.2 
                 Heterodimer_(WT) + (TadA*7.10 + Y147R) 
               
               
                 ABE8.3-d 
                 TadA*8.3 
                 Heterodimer_(WT) + (TadA*7.10 + Q154S) 
               
               
                 ABE8.4-d 
                 TadA*8.4 
                 Heterodimer_(WT) + (TadA*7.10 + Y123H) 
               
               
                 ABE8.5-d 
                 TadA*8.5 
                 Heterodimer_(WT) + (TadA*7.10 + V82S) 
               
               
                 ABE8.6-d 
                 TadA*8.6 
                 Heterodimer_(WT) + (TadA*7.10 + T166R) 
               
               
                 ABE8.7-d 
                 TadA*8.7 
                 Heterodimer_(WT) + (TadA*7.10 + Q154R) 
               
               
                 ABE8.8-d 
                 TadA*8.8 
                 Heterodimer_(WT) + (TadA*7.10 + Y147R_Q154R_Y123H) 
               
               
                 ABE8.9-d 
                 TadA*8.9 
                 Heterodimer_(WT) + (TadA*7.10 + Y147R_Q154R_I76Y) 
               
               
                 ABE8.10-d 
                 TadA*8.10 
                 Heterodimer_(WT) + (TadA*7.10 + Y147R_Q154R_T166R) 
               
               
                 ABE8.11-d 
                 TadA*8.11 
                 Heterodimer_(WT) + (TadA*7.10 + Y147T_Q154R) 
               
               
                 ABE8.12-d 
                 TadA*8.12 
                 Heterodimer_(WT) + (TadA*7.10 + Y147T_Q154S) 
               
               
                 ABE8.13-d 
                 TadA*8.13 
                 Heterodimer_(WT) + (TadA*7.10 + Y123H_Y147T_Q154R_I76Y) 
               
               
                 ABE8.14-d 
                 TadA*8.14 
                 Heterodimer_(WT) + (TadA*7.10 + I76Y_V82S) 
               
               
                 ABE8.15-d 
                 TadA*8.15 
                 Heterodimer_(WT) + (TadA*7.10 + V82S_Y147R) 
               
               
                 ABE8.16-d 
                 TadA*8.16 
                 Heterodimer_(WT) + (TadA*7.10 + V82S_Y123H_Y147R) 
               
               
                 ABE8.17-d 
                 TadA*8.17 
                 Heterodimer_(WT) + (TadA*7.10 + V82S_Q154R) 
               
               
                 ABE8.18-d 
                 TadA*8.18 
                 Heterodimer_(WT) + (TadA*7.10 + V82S_Y123H_Q154R) 
               
               
                 ABE8.19-d 
                 TadA*8.19 
                 Heterodimer_(WT) + (TadA*7.10 + V82S_Y123H_Y147R_Q154R) 
               
               
                 ABE8.20-d 
                 TadA*8.20 
                 Heterodimer_(WT) + (TadA*7.10 + I76Y_V82S_Y123H_Y147R_Q154R) 
               
               
                 ABE8.21-d 
                 TadA*8.21 
                 Heterodimer_(WT) + (TadA*7.10 + Y147R_Q154S) 
               
               
                 ABE8.22-d 
                 TadA*8.22 
                 Heterodimer_(WT) + (TadA*7.10 + V82S_Q154S) 
               
               
                 ABE8.23-d 
                 TadA*8.23 
                 Heterodimer_(WT) + (TadA*7.10 + V82S_Y123H) 
               
               
                 ABE8.24-d 
                 TadA*8.24 
                 Heterodimer_(WT) + (TadA*7.10 + V82S_Y123H_Y147T) 
               
               
                   
               
            
           
         
       
     
     In some embodiments, base editors (e.g., ABE8) are generated by cloning an adenosine deaminase variant (e.g., TadA*8) into a scaffold that includes a circular permutant Cas9 (e.g., CP5 or CP6) and a bipartite nuclear localization sequence. In some embodiments, the base editor (e.g., ABE7.9, ABE7.10, or ABE8) is an NGC PAM CP5 variant ( S. pyrogenes  Cas9 or spVRQR Cas9). In some embodiments, the base editor (e.g., ABE7.9, ABE7.10, or ABE8) is an AGA PAM CP5 variant ( S. pyrogenes  Cas9 or spVRQR Cas9). In some embodiments, the base editor (e.g., ABE7.9, ABE7.10, or ABE8) is an NGC PAM CP6 variant ( S. pyrogenes  Cas9 or spVRQR Cas9). In some embodiments, the base editor (e.g. ABE7.9, ABE7.10, or ABE8) is an AGA PAM CP6 variant ( S. pyrogenes  Cas9 or spVRQR Cas9). 
     In some embodiments, the ABE has a genotype as shown in Table 8 below. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Genotypes of ABEs 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 23 
                 26 
                 36 
                 37 
                 48 
                 49 
                 51 
                 72 
                 84 
                 87 
                 105 
                 108 
                 123 
                 125 
                 142 
                 145 
                 147 
                 152 
                 155 
                 156 
                 157 
                 161 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 ABE7.9 
                 L 
                 R 
                 L 
                 N 
                 A 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 N 
                 C 
                 Y 
                 P 
                 V 
                 F 
                 N 
                 K 
               
               
                 ABE7.10 
                 R 
                 R 
                 L 
                 N 
                 A 
                   
                 L 
                 N 
                 F 
                 S 
                 V 
                 N 
                 Y 
                 G 
                 A 
                 C 
                 Y 
                 P 
                 V 
                 F 
                 N 
                 K 
               
               
                   
               
            
           
         
       
     
     As shown in Table 9 below, genotypes of 40 ABE8s are described. Residue positions in the evolved  E. coli  TadA portion of ABE are indicated. Mutational changes in ABE8 are shown when distinct from ABE7.10 mutations. In some embodiments, the ABE has a genotype of one of the ABEs as shown in Table 9 below. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Residue Identity in Evolved TadA 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 23 
                 36 
                 48 
                 51 
                 76 
                 82 
                 84 
                 106 
                 108 
                 123 
                 146 
                 147 
                 152 
                 154 
                 155 
                 156 
                 157 
                 166 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 ABE7.10 
                 R 
                 L 
                 A 
                 L 
                 I 
                 V 
                 F 
                 V 
                 N 
                 Y 
                 C 
                 Y 
                 P 
                 Q 
                 V 
                 F 
                 N 
                 T 
               
               
                 ABE8.1-m 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 T 
               
               
                 ABE8.2-m 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 R 
               
               
                 ABE8.3-m 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 S 
               
               
                 ABE8.4-m 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 H 
               
               
                 ABE8.5-m 
                   
                   
                   
                   
                   
                 S 
               
               
                 ABE8.6-m 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 R 
               
               
                 ABE8.7-m 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 R 
               
               
                 ABE8.8-m 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 H 
                   
                 R 
                   
                 R 
               
               
                 ABE8.9-m 
                   
                   
                   
                   
                 Y 
                   
                   
                   
                   
                   
                   
                 R 
                   
                 R 
               
               
                 ABE8.10-m 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 R 
                   
                 R 
                   
                   
                   
                 R 
               
               
                 ABE8.11-m 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 T 
                   
                 R 
               
               
                 ABE8.12-m 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 T 
                   
                 S 
               
               
                 ABE8.13-m 
                   
                   
                   
                   
                 Y 
                   
                   
                   
                   
                 H 
                   
                 R 
                   
                 R 
               
               
                 ABE8.14-m 
                   
                   
                   
                   
                 Y 
                 S 
               
               
                 ABE8.15-m 
                   
                   
                   
                   
                   
                 S 
                   
                   
                   
                   
                   
                 R 
               
               
                 ABE8.16-m 
                   
                   
                   
                   
                   
                 S 
                   
                   
                   
                 H 
                   
                 R 
               
               
                 ABE8.17-m 
                   
                   
                   
                   
                   
                 S 
                   
                   
                   
                   
                   
                   
                   
                 R 
               
               
                 ABE8.18-m 
                   
                   
                   
                   
                   
                 S 
                   
                   
                   
                 H 
                   
                   
                   
                 R 
               
               
                 ABE8.19-m 
                   
                   
                   
                   
                   
                 S 
                   
                   
                   
                 H 
                   
                 R 
                   
                 R 
               
               
                 ABE8.20-m 
                   
                   
                   
                   
                 Y 
                 S 
                   
                   
                   
                 H 
                   
                 R 
                   
                 R 
               
               
                 ABE8.21-m 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 R 
                   
                 S 
               
               
                 ABE8.22-m 
                   
                   
                   
                   
                   
                 S 
                   
                   
                   
                   
                   
                   
                   
                 S 
               
               
                 ABE8.23-m 
                   
                   
                   
                   
                   
                 S 
                   
                   
                   
                 H 
               
               
                 ABE8.24-m 
                   
                   
                   
                   
                   
                 S 
                   
                   
                   
                 H 
                   
                 T 
               
               
                 ABE8.1-d 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 T 
               
               
                 ABE8.2-d 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 R 
               
               
                 ABE8.3-d 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 S 
               
               
                 ABE8.4-d 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 H 
               
               
                 ABE8.5-d 
                   
                   
                   
                   
                   
                 S 
               
               
                 ABE8.6-d 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 R 
               
               
                 ABE8.7-d 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 R 
               
               
                 ABE8.8-d 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 H 
                   
                 R 
                   
                 R 
               
               
                 ABE8.9-d 
                   
                   
                   
                   
                 Y 
                   
                   
                   
                   
                   
                   
                 R 
                   
                 R 
               
               
                 ABE8.10-d 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 R 
                   
                 R 
                   
                   
                   
                 R 
               
               
                 ABE8.11-d 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 T 
                   
                 R 
               
               
                 ABE8.12-d 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 T 
                   
                 S 
               
               
                 ABE8.13-d 
                   
                   
                   
                   
                 Y 
                   
                   
                   
                   
                 H 
                   
                 R 
                   
                 R 
               
               
                 ABE8.14-d 
                   
                   
                   
                   
                 Y 
                 S 
               
               
                 ABE8.15-d 
                   
                   
                   
                   
                   
                 S 
                   
                   
                   
                   
                   
                 R 
               
               
                 ABE8.16-d 
                   
                   
                   
                   
                   
                 S 
                   
                   
                   
                 H 
                   
                 R 
               
               
                 ABE8.17-d 
                   
                   
                   
                   
                   
                 S 
                   
                   
                   
                   
                   
                   
                   
                 R 
               
               
                 ABE8.18-d 
                   
                   
                   
                   
                   
                 S 
                   
                   
                   
                 H 
                   
                   
                   
                 R 
               
               
                 ABE8.19-d 
                   
                   
                   
                   
                   
                 S 
                   
                   
                   
                 H 
                   
                 R 
                   
                 R 
               
               
                 ABE8.20-d 
                   
                   
                   
                   
                 Y 
                 S 
                   
                   
                   
                 H 
                   
                 R 
                   
                 R 
               
               
                 ABE8.21-d 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 R 
                   
                 S 
               
               
                 ABE8.22-d 
                   
                   
                   
                   
                   
                 S 
                   
                   
                   
                   
                   
                   
                   
                 S 
               
               
                 ABE8.23-d 
                   
                   
                   
                   
                   
                 S 
                   
                   
                   
                 H 
               
               
                 ABE8.24-d 
                   
                   
                   
                   
                   
                 S 
                   
                   
                   
                 H 
                   
                 T 
               
               
                   
               
            
           
         
       
     
     In some embodiments, the base editor is ABE8.1, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: 
     
       
         
           
               
               
            
               
                 ABE8.1_Y147T_CP5_NGC PAM_monomer 
                   
               
               
                 (SEQ ID NO: 254) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA 
                   
               
               
                   
               
               
                 LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP 
               
               
                   
               
               
                 GMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD      
               
               
                   
               
               
                 
                   
                   EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDK 
                 
               
               
                   
               
               
                 
                   GRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFMQPT 
                 
               
               
                   
               
               
                 
                   VAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
                 
               
               
                   
               
               
                 
                   YSLFELENGRKRMLASAKFLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH 
                 
               
               
                   
               
               
                 
                   KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAF 
                 
               
               
                   
               
               
                   KYFDTTIARKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD     
               
               
                   
               
               
                 
                   
                   DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE 
                 
               
               
                   
               
               
                 
                   ATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
                 
               
               
                   
               
               
                 
                   VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKL 
                 
               
               
                   
               
               
                 
                   FIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL 
                 
               
               
                   
               
               
                 
                   GLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRV 
                 
               
               
                   
               
               
                 
                   NTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQE 
                 
               
               
                   
               
               
                 
                   EFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF 
                 
               
               
                   
               
               
                 
                   LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIER 
                 
               
               
                   
               
               
                 
                   MTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN 
                 
               
               
                   
               
               
                 
                   RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED 
                 
               
               
                   
               
               
                 
                   IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL 
                 
               
               
                   
               
               
                 
                   DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVK 
                 
               
               
                   
               
               
                 
                   VVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT 
                 
               
               
                   
               
               
                 
                   QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGK 
                 
               
               
                   
               
               
                 
                   SDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQIT 
                 
               
               
                   
               
               
                 
                   KHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN 
                 
               
               
                   
               
               
                 
                   AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEGADKRTADGSEFESPKKKRKV* 
                 
               
            
           
         
       
     
     In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italics sequence denotes a linker sequence, and the underlined sequence denotes a bipartite nuclear localization sequence. 
     In some embodiments, the base editor is ABE8.1, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: 
     
       
         
           
               
               
            
               
                 pNMG-B335 ABE8.1Y147T_CP5_NGC PAM monomer 
                   
               
               
                 (SEQ ID NO: 254) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA 
                   
               
               
                   
               
               
                 LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP 
               
               
                   
               
               
                 GMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTDSGGSSG GSSGSETPGTSES   
               
               
                   
               
               
                 
                   ATPESSGGSSGGS 
                   EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDK 
                 
               
               
                   
               
               
                 
                   GRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFMQPT 
                 
               
               
                   
               
               
                 
                   VAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 
                 
               
               
                   
               
               
                 
                   YSLFELENGRKRMLASAKFLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQH 
                 
               
               
                   
               
               
                 
                   KHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAF 
                 
               
               
                   
               
               
                 
                   KYFDTTIARKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
                   GGSGGSGGSGGSGGSGGS 
                 
               
               
                   
               
               
                 
                   GGM 
                   DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE 
                 
               
               
                   
               
               
                 
                   ATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
                 
               
               
                   
               
               
                 
                   VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKL 
                 
               
               
                   
               
               
                 
                   FIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL 
                 
               
               
                   
               
               
                 
                   GLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRV 
                 
               
               
                   
               
               
                 
                   NTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQE 
                 
               
               
                   
               
               
                 
                   EFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF 
                 
               
               
                   
               
               
                 
                   LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIER 
                 
               
               
                   
               
               
                 
                   MTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN 
                 
               
               
                   
               
               
                 
                   RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILED 
                 
               
               
                   
               
               
                 
                   IVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL 
                 
               
               
                   
               
               
                 
                   DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVK 
                 
               
               
                   
               
               
                 
                   VVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT 
                 
               
               
                   
               
               
                 
                   QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGK 
                 
               
               
                   
               
               
                 
                   SDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQIT 
                 
               
               
                   
               
               
                 
                   KHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN 
                 
               
               
                   
               
               
                 
                   AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ 
                   EGADKRTADGSEFESPKKKRKV* 
                 
               
            
           
         
       
     
     In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italics sequence denotes a linker sequence, and the underlined sequence denotes a bipartite nuclear localization sequence. 
     In some embodiments, the base editor is ABE8.14, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: 
     
       
         
           
               
               
            
               
                 pNMG-357_ABE8.14 with NGC PAM CP5 
                   
               
               
                 (SEQ ID NO: 255) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMA 
                   
               
               
                   
               
               
                 LRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHP 
               
               
                   
               
               
                 GMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDGGSSGGSSGSETPGTSESA 
               
               
                   
               
               
                 TPESSGGSSGGSMSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
               
               
                   
               
               
                 LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTG 
               
               
                   
               
               
                 AAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD SGGSSGG   
               
               
                   
               
               
                 
                   SSGSETPGTSESATPESSGGSSGGS 
                   EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIE 
                 
               
               
                   
               
               
                 
                   TNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDW 
                 
               
               
                   
               
               
                 
                   DPKKYGGFMQPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYK 
                 
               
               
                   
               
               
                 
                   EVKKDLIIKLPKYSLFELENGRKRMLASAKFLQKGNELALPSKYVNFLYLASHYEKLKGSPE 
                 
               
               
                   
               
               
                 
                   DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHL 
                 
               
               
                   
               
               
                 
                   FTLTNLGAPRAFKYFDTTIARKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
                   GGSGGS 
                 
               
               
                   
               
               
                 
                   GGSGGSGGSGGSGGM 
                   DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI 
                 
               
               
                   
               
               
                 
                   GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEED 
                 
               
               
                   
               
               
                 
                   KKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG 
                 
               
               
                   
               
               
                 
                   DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKK 
                 
               
               
                   
               
               
                 
                   NGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNL 
                 
               
               
                   
               
               
                 
                   SDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNG 
                 
               
               
                   
               
               
                 
                   YAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELH 
                 
               
               
                   
               
               
                 
                   AILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
                 
               
               
                   
               
               
                 
                   DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ 
                 
               
               
                   
               
               
                 
                   KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDE 
                 
               
               
                   
               
               
                 
                   LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLIN 
                 
               
               
                   
               
               
                 
                   GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGS 
                 
               
               
                   
               
               
                 
                   PAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG 
                 
               
               
                   
               
               
                 
                   SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN 
                 
               
               
                   
               
               
                 
                   KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGE 
                 
               
               
                   
               
               
                 
                   IKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREI 
                 
               
               
                   
               
               
                 
                   NNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ 
                   EGADKRTADGSEF 
                 
               
               
                   
               
               
                 
                   ESPKKKRKV* 
                 
               
            
           
         
       
     
     In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italics sequence denotes a linker sequence, and the underlined sequence denotes a bipartite nuclear localization sequence. 
     In some embodiments, the base editor is ABE8.8-m, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: 
     
       
         
           
               
               
            
               
                   
                 ABE8.8-m 
               
               
                   
                 (SEQ ID NO: 256) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLV 
               
               
                   
               
               
                   
                 LNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVM 
               
               
                   
               
               
                   
                 QNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFG 
               
               
                   
               
               
                   
                 VRNAKTGAAGSLMDVLHHPGMNHRVEITEGILADE 
               
               
                   
               
               
                   
                 CAALLCRFFRMPRRVFNAQKKAQSSTD SGGSSGGS   
               
               
                   
               
               
                   
                   SGSETPGTSESATPESSGGSSGGS   DKKYSIGL     A     IG   
               
               
                   
               
               
                   
                 
                   TNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNL 
                 
               
               
                   
               
               
                   
                 
                   IGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
                 
               
               
                   
               
               
                   
                 
                   QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHP 
                 
               
               
                   
               
               
                   
                 
                   IFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLR 
                 
               
               
                   
               
               
                   
                 
                   LIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQ 
                 
               
               
                   
               
               
                   
                 
                   LVQTYNQLFEENPINASGVDAKAILSARLSKSRRL 
                 
               
               
                   
               
               
                   
                 
                   ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFD 
                 
               
               
                   
               
               
                   
                 
                   LAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLA 
                 
               
               
                   
               
               
                   
                 
                   AKNLSDAILLSDILRVNTEITKAPLSASMIKRYDE 
                 
               
               
                   
               
               
                   
                 
                   HHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY 
                 
               
               
                   
               
               
                   
                 
                   IDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED 
                 
               
               
                   
               
               
                   
                 
                   LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF 
                 
               
               
                   
               
               
                   
                 
                   LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTR 
                 
               
               
                   
               
               
                   
                 
                   KSEETITPWNFEEVVDKGASAQSFIERMTNFDKNL 
                 
               
               
                   
               
               
                   
                 
                   PNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKP 
                 
               
               
                   
               
               
                   
                 
                   AFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKI 
                 
               
               
                   
               
               
                   
                 
                   ECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFL 
                 
               
               
                   
               
               
                   
                 
                   DNEENEDILEDIVLTLTLFEDREMIEERLKTYAHL 
                 
               
               
                   
               
               
                   
                 
                   FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK 
                 
               
               
                   
               
               
                   
                 
                   TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQ 
                 
               
               
                   
               
               
                   
                 
                   VSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL 
                 
               
               
                   
               
               
                   
                 
                   VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK 
                 
               
               
                   
               
               
                   
                 
                   RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ 
                 
               
               
                   
               
               
                   
                 
                   NGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSI 
                 
               
               
                   
               
               
                   
                 
                   DNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLL 
                 
               
               
                   
               
               
                   
                 
                   NAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV 
                 
               
               
                   
               
               
                   
                 
                   ETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT 
                 
               
               
                   
               
               
                   
                 
                   LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAV 
                 
               
               
                   
               
               
                   
                 
                   VGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ 
                 
               
               
                   
               
               
                   
                 
                   EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPL 
                 
               
               
                   
               
               
                   
                 
                   IETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKK 
                 
               
               
                   
               
               
                   
                 
                   TEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYG 
                 
               
               
                   
               
               
                   
                 
                   GFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT 
                 
               
               
                   
               
               
                   
                 
                   IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS 
                 
               
               
                   
               
               
                   
                 
                   LFELENGRKRMLASAGELQKGNELALPSKYVNFLY 
                 
               
               
                   
               
               
                   
                 
                   LASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQ 
                 
               
               
                   
               
               
                   
                 
                   ISEFSKRVILADANLDKVLSAYNKHRDKPIREQAE 
                 
               
               
                   
               
               
                   
                 
                   NIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEV 
                 
               
               
                   
               
               
                   
                 
                   LDATLIHQSITGLYETRIDLSQLGGD 
                   EGADKRTAD 
                 
               
               
                   
               
               
                   
                   GSEFESPKKKRKV * 
               
            
           
         
       
     
     In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, underlined sequence denotes a bipartite nuclear localization sequence, and double underlined sequence indicates mutations. 
     In some embodiments, the base editor is ABE8.8-d, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: 
     
       
         
           
               
               
            
               
                 ABE.-d 
                   
               
               
                 (SEQ ID NO: 257) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMA 
                   
               
               
                   
               
               
                 LRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHP 
               
               
                   
               
               
                 GMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSES 
               
               
                   
               
               
                 ATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
               
               
                   
               
               
                 LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTG 
               
               
                   
               
               
                 AAGSLMDVLH H PGMNHRVEITEGILADECAALLC R FFRMPR R VFNAQKKAQSSTD SGGSSGG   
               
               
                   
               
               
                 
                   SSGSETPGTSESATPESSGGSSGGS 
                   DKKYSIGL 
                   
                     A 
                   
                   IGTNSVGWAVITDEYKVPSKKFKVLGNT 
                 
               
               
                   
               
               
                 
                   DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
                 
               
               
                   
               
               
                 
                   LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
                 
               
               
                   
               
               
                 
                   KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLEN 
                 
               
               
                   
               
               
                 
                   LIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQY 
                 
               
               
                   
               
               
                 
                   ADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYK 
                 
               
               
                   
               
               
                 
                   EIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSI 
                 
               
               
                   
               
               
                 
                   PHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEET 
                 
               
               
                   
               
               
                 
                   ITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGM 
                 
               
               
                   
               
               
                 
                   RKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHD 
                 
               
               
                   
               
               
                 
                   LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTG 
                 
               
               
                   
               
               
                 
                   WGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL 
                 
               
               
                   
               
               
                 
                   HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK 
                 
               
               
                   
               
               
                 
                   RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQ 
                 
               
               
                   
               
               
                 
                   SFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG 
                 
               
               
                   
               
               
                 
                   GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRK 
                 
               
               
                   
               
               
                 
                   DFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG 
                 
               
               
                   
               
               
                 
                   KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQV 
                 
               
               
                   
               
               
                 
                   NIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS 
                 
               
               
                   
               
               
                 
                   KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS 
                 
               
               
                   
               
               
                 
                   AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSK 
                 
               
               
                   
               
               
                 
                   RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTK 
                 
               
               
                   
               
               
                 EVLDATLIHQSITGLYETRIDLSQLGGD EGADKRTADGSEFESPKKKRKV * 
               
            
           
         
       
     
     In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, underlined sequence denotes a bipartite nuclear localization sequence, and double underlined sequence indicates mutations. 
     In some embodiments, the base editor is ABE8.13-m, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: 
     
       
         
           
               
               
            
               
                 ABE8.13-m 
                   
               
               
                 (SEQ ID NO: 258) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA 
                   
               
               
                   
               
               
                 LRQGGLVMQNYRL Y DATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLH H P 
               
               
                   
               
               
                 GMNHRVEITEGILADECAALLC R FFRMPR R VFNAQKKAQSSTD SGGSSGGSSGSETPGTSES   
               
               
                   
               
               
                 
                   ATPESSGGSSGGS 
                   DKKYSIGL 
                   
                     A 
                   
                   IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
                 
               
               
                   
               
               
                 
                   LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK 
                 
               
               
                   
               
               
                 
                   HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL 
                 
               
               
                   
               
               
                 
                   NPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG 
                 
               
               
                   
               
               
                 
                   LFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD 
                 
               
               
                   
               
               
                 
                   AILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA 
                 
               
               
                   
               
               
                 
                   GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAI 
                 
               
               
                   
               
               
                 
                   LRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDK 
                 
               
               
                   
               
               
                 
                   GASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKK 
                 
               
               
                   
               
               
                 
                   AIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLD 
                 
               
               
                   
               
               
                 
                   NEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGI 
                 
               
               
                   
               
               
                 
                   RDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA 
                 
               
               
                   
               
               
                 
                   IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQ 
                 
               
               
                   
               
               
                 
                   ILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV 
                 
               
               
                   
               
               
                 
                   LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK 
                 
               
               
                   
               
               
                 
                   RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINN 
                 
               
               
                   
               
               
                 
                   YHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI 
                 
               
               
                   
               
               
                 
                   MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG 
                 
               
               
                   
               
               
                 
                   FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI 
                 
               
               
                   
               
               
                 
                   TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELAL 
                 
               
               
                   
               
               
                 
                   PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKV 
                 
               
               
                   
               
               
                 
                   LSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI 
                 
               
               
                   
               
               
                   TGLYETRIDLSQLGGD   EGADKRTADGSEFESPKKKRKV * 
               
            
           
         
       
     
     In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, underlined sequence denotes a bipartite nuclear localization sequence, and double underlined sequence indicates mutations. 
     In some embodiments, the base editor is ABE8.13-d, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: 
     
       
         
           
               
               
            
               
                 ABE8.13-d 
                   
               
               
                 (SEQ ID NO: 259) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMA 
                   
               
               
                   
               
               
                 LRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHP 
               
               
                   
               
               
                 GMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD SGGSSGGSSGSETPGTSES   
               
               
                   
               
               
                   ATPESSGGSSGGSS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
               
               
                   
               
               
                 LHDPTAHAEIMALRQGGLVMQNYRL Y DATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTG 
               
               
                   
               
               
                 AAGSLMDVLH H PGMNHRVEITEGILADECAALLC R FFRMPR R VFNAQKKAQSSTD SGGSSGG   
               
               
                   
               
               
                 
                   SSGSETPGTSESATPESSGGSSGGS 
                   DKKYSIGL 
                   
                     A 
                   
                   IGTNSVGWAVITDEYKVPSKKFKVLGNT 
                 
               
               
                   
               
               
                 
                   DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
                 
               
               
                   
               
               
                 
                   LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
                 
               
               
                   
               
               
                 
                   KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLEN 
                 
               
               
                   
               
               
                 
                   LIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQY 
                 
               
               
                   
               
               
                 
                   ADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYK 
                 
               
               
                   
               
               
                 
                   EIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSI 
                 
               
               
                   
               
               
                 
                   PHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEET 
                 
               
               
                   
               
               
                 
                   ITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGM 
                 
               
               
                   
               
               
                 
                   RKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHD 
                 
               
               
                   
               
               
                 
                   LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTG 
                 
               
               
                   
               
               
                 
                   WGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL 
                 
               
               
                   
               
               
                 
                   HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK 
                 
               
               
                   
               
               
                 
                   RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQ 
                 
               
               
                   
               
               
                 
                   SFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG 
                 
               
               
                   
               
               
                 
                   GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRK 
                 
               
               
                   
               
               
                 
                   DFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG 
                 
               
               
                   
               
               
                 KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQV 
               
               
                   
               
               
                 
                   NIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS 
                 
               
               
                   
               
               
                 
                   KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS 
                 
               
               
                   
               
               
                 
                   AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSK 
                 
               
               
                   
               
               
                 
                   RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTK 
                 
               
               
                   
               
               
                   EVLDATLIHQSITGLYETRIDLSQLGGD   EGADKRTADGSEFESPKKKRKV * 
               
            
           
         
       
     
     In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, underlined sequence denotes a bipartite nuclear localization sequence, and double underlined sequence indicates mutations. 
     In some embodiments, the base editor is ABE8.17-m, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: 
     
       
         
           
               
               
            
               
                 ABE8.17-m 
                   
               
               
                 (SEQ ID NO: 260) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA 
                   
               
               
                   
               
               
                 LRQGGLVMQNYRLIDATLY S TFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP 
               
               
                   
               
               
                 GMNHRVEITEGILADECAALLCYFFRMPR R VFNAQKKAQSSTD SGGSSGGSSGSETPGTSES   
               
               
                   
               
               
                 
                   ATPESSGGSSGGS 
                   DKKYSIGL 
                   
                     A 
                   
                   IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
                 
               
               
                   
               
               
                 
                   LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK 
                 
               
               
                   
               
               
                 
                   HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL 
                 
               
               
                   
               
               
                 
                   NPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG 
                 
               
               
                   
               
               
                 
                   LFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD 
                 
               
               
                   
               
               
                 
                   AILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA 
                 
               
               
                   
               
               
                 
                   GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAI 
                 
               
               
                   
               
               
                 
                   LRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDK 
                 
               
               
                   
               
               
                 
                   GASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKK 
                 
               
               
                   
               
               
                 
                   AIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLD 
                 
               
               
                   
               
               
                 
                   NEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGI 
                 
               
               
                   
               
               
                 
                   RDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA 
                 
               
               
                   
               
               
                 
                   IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQ 
                 
               
               
                   
               
               
                 
                   ILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV 
                 
               
               
                   
               
               
                 
                   LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK 
                 
               
               
                   
               
               
                 
                   RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINN 
                 
               
               
                   
               
               
                 
                   YHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI 
                 
               
               
                   
               
               
                 
                   MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG 
                 
               
               
                   
               
               
                 
                   FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI 
                 
               
               
                   
               
               
                 
                   TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELAL 
                 
               
               
                   
               
               
                 
                   PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKV 
                 
               
               
                   
               
               
                 
                   LSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI 
                 
               
               
                   
               
               
                 TGLYETRIDLSQLGGD EGADKRTADGSEFESPKKKRKV * 
               
            
           
         
       
     
     In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, underlined sequence denotes a bipartite nuclear localization sequence, and double underlined sequence indicates mutations. 
     In some embodiments, the base editor is ABE8.17-d, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: 
     
       
         
           
               
               
            
               
                 ABE8.17-d 
                   
               
               
                 (SEQ ID NO: 261) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMA 
                   
               
               
                   
               
               
                 LRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHP 
               
               
                   
               
               
                 GMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD SGGSSGGSSGSETPGTSES   
               
               
                   
               
               
                   ATPESSGGSSGGSS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
               
               
                   
               
               
                 LHDPTAHAEIMALRQGGLVMQNYRLIDATLY S TFEPCVMCAGAMIHSRIGRVVFGVRNAKTG 
               
               
                   
               
               
                 AAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPR R VFNAQKKAQSSTD SGGSSGG   
               
               
                   
               
               
                 
                   SSGSETPGTSESATPESSGGSSGGS 
                   DKKYSIGL 
                   
                     A 
                   
                   IGTNSVGWAVITDEYKVPSKKFKVLGNT 
                 
               
               
                   
               
               
                 
                   DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
                 
               
               
                   
               
               
                 
                   LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
                 
               
               
                   
               
               
                 
                   KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLEN 
                 
               
               
                   
               
               
                 
                   LIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQY 
                 
               
               
                   
               
               
                 
                   ADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYK 
                 
               
               
                   
               
               
                 
                   EIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSI 
                 
               
               
                   
               
               
                 
                   PHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEET 
                 
               
               
                   
               
               
                 
                   ITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGM 
                 
               
               
                   
               
               
                 
                   RKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHD 
                 
               
               
                   
               
               
                 
                   LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTG 
                 
               
               
                   
               
               
                 
                   WGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL 
                 
               
               
                   
               
               
                 
                   HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK 
                 
               
               
                   
               
               
                 
                   RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQ 
                 
               
               
                   
               
               
                 
                   SFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG 
                 
               
               
                   
               
               
                 
                   GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRK 
                 
               
               
                   
               
               
                 
                   DFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG 
                 
               
               
                   
               
               
                 
                   KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQV 
                 
               
               
                   
               
               
                 
                   NIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS 
                 
               
               
                   
               
               
                 
                   KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDL11KLPKYSLFELENGRKRMLAS 
                 
               
               
                   
               
               
                 
                   AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSK 
                 
               
               
                   
               
               
                 
                   RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTK 
                 
               
               
                   
               
               
                   EVLDATLIHQSITGLYETRIDLSQLGGD   EGADKRTADGSEFESPKKKRKV * 
               
            
           
         
       
     
     In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, underlined sequence denotes a bipartite nuclear localization sequence, and double underlined sequence indicates mutations. 
     In some embodiments, the base editor is ABE8.20-m, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: 
     
       
         
           
               
               
            
               
                 ABE8.20-m 
                   
               
               
                 (SEQ ID NO: 262) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA 
                   
               
               
                   
               
               
                 LRQGGLVMQNYRL Y DATL Y STFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLH H P 
               
               
                   
               
               
                 GMNHRVEITEGILADECAALLC R FFRMPR R VFNAQKKAQSSTD SGGSSGGSSGSETPGTSES   
               
               
                   
               
               
                 
                   ATPESSGGSSGGS 
                   DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA 
                 
               
               
                   
               
               
                 
                   LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK 
                 
               
               
                   
               
               
                 
                   HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL 
                 
               
               
                   
               
               
                 
                   NPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG 
                 
               
               
                   
               
               
                 
                   LFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD 
                 
               
               
                   
               
               
                 
                   AILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA 
                 
               
               
                   
               
               
                 
                   GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAI 
                 
               
               
                   
               
               
                 
                   LRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDK 
                 
               
               
                   
               
               
                 
                   GASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKK 
                 
               
               
                   
               
               
                 
                   AIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLD 
                 
               
               
                   
               
               
                 
                   NEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGI 
                 
               
               
                   
               
               
                 
                   RDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA 
                 
               
               
                   
               
               
                 
                   IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQ 
                 
               
               
                   
               
               
                 
                   ILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV 
                 
               
               
                   
               
               
                 
                   LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK 
                 
               
               
                   
               
               
                 
                   RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINN 
                 
               
               
                   
               
               
                 
                   YHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI 
                 
               
               
                   
               
               
                 
                   MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG 
                 
               
               
                   
               
               
                 
                   FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI 
                 
               
               
                   
               
               
                 
                   TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELAL 
                 
               
               
                   
               
               
                 
                   PSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKV 
                 
               
               
                   
               
               
                 
                   LSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI 
                 
               
               
                   
               
               
                   TGLYETRIDLSQLGGD   EGADKRTADGSEFESPKKKRKV * 
               
            
           
         
       
     
     In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, underlined sequence denotes a bipartite nuclear localization sequence, and double underlined sequence indicates mutations. 
     In some embodiments, the base editor is ABE8.20-d, which comprises or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: 
     
       
         
           
               
               
            
               
                 ABE8.20-d 
                   
               
               
                 (SEQ ID NO: 263) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMA 
                   
               
               
                   
               
               
                 LRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHP 
               
               
                   
               
               
                 GMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSES 
               
               
                   
               
               
                 ATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
               
               
                   
               
               
                 LHDPTAHAEIMALRQGGLVMQNYRL Y DATLY S TFEPCVMCAGAMIHSRIGRVVFGVRNAKTG 
               
               
                   
               
               
                 AAGSLMDVLH H PGMNHRVEITEGILADECAALLCRFFRMPR R VFNAQKKAQSSTD SGGSSGG   
               
               
                   
               
               
                 
                   SSGSETPGTSESATPESSGGSSGGS 
                   DKKYSIGL 
                   
                     A 
                   
                   IGTNSVGWAVITDEYKVPSKKFKVLGNT 
                 
               
               
                   
               
               
                 
                   DRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
                 
               
               
                   
               
               
                 
                   LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
                 
               
               
                   
               
               
                 
                   KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLEN 
                 
               
               
                   
               
               
                 
                   LIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQY 
                 
               
               
                   
               
               
                 
                   ADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYK 
                 
               
               
                   
               
               
                 
                   EIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSI 
                 
               
               
                   
               
               
                 
                   PHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEET 
                 
               
               
                   
               
               
                 
                   ITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGM 
                 
               
               
                   
               
               
                 
                   RKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHD 
                 
               
               
                   
               
               
                 
                   LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTG 
                 
               
               
                   
               
               
                 
                   WGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL 
                 
               
               
                   
               
               
                 
                   HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK 
                 
               
               
                   
               
               
                 
                   RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQ 
                 
               
               
                   
               
               
                 
                   SFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG 
                 
               
               
                   
               
               
                 
                   GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRK 
                 
               
               
                   
               
               
                 
                   DFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG 
                 
               
               
                   
               
               
                 
                   KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQV 
                 
               
               
                   
               
               
                 
                   NIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS 
                 
               
               
                   
               
               
                 
                   KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDL11KLPKYSLFELENGRKRMLAS 
                 
               
               
                   
               
               
                 
                   AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSK 
                 
               
               
                   
               
               
                 
                   RVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTK 
                 
               
               
                   
               
               
                 
                   EVLDATLIHQSITGLYETRIDLSQLGGD 
                   
                     EGADKRTADGSEFESPKKKRRKV* 
                   
                 
               
            
           
         
       
     
     In the above sequence, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, underlined sequence denotes a bipartite nuclear localization sequence, and double underlined sequence indicates mutations. 
     In some embodiments, an ABE8 of the invention is selected from the following sequences: 
     
       
         
           
               
               
            
               
                   
                 01. monoABE8.1_bpNLS + Y147T 
               
               
                   
                 (SEQ ID NO: 264) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV 
               
               
                   
                   
               
               
                   
                 IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATL 
               
               
                   
                   
               
               
                   
                 YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV 
               
               
                   
                   
               
               
                   
                 LHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQK 
               
               
                   
                   
               
               
                   
                 KAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSD 
               
               
                   
                   
               
               
                   
                 KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS 
               
               
                   
                   
               
               
                   
                 IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
               
               
                   
                   
               
               
                   
                 QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
                   
               
               
                   
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
               
               
                   
                   
               
               
                   
                 KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN 
               
               
                   
                   
               
               
                   
                 ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI 
               
               
                   
                   
               
               
                   
                 ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
               
               
                   
                   
               
               
                   
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMI 
               
               
                   
                   
               
               
                   
                 KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY 
               
               
                   
                   
               
               
                   
                 IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ 
               
               
                   
                   
               
               
                   
                 RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
                   
               
               
                   
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
               
               
                   
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYN 
               
               
                   
                   
               
               
                   
                 ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK 
               
               
                   
                   
               
               
                   
                 QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK 
               
               
                   
                   
               
               
                   
                 DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHL 
               
               
                   
                   
               
               
                   
                 FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
               
               
                   
                   
               
               
                   
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHE 
               
               
                   
                   
               
               
                   
                 HIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
                   
               
               
                   
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE 
               
               
                   
                   
               
               
                   
                 NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIV 
               
               
                   
                   
               
               
                   
                 PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
               
               
                   
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV 
               
               
                   
                   
               
               
                   
                 ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL 
               
               
                   
                   
               
               
                   
                 VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP 
               
               
                   
                   
               
               
                   
                 KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI 
               
               
                   
                   
               
               
                   
                 MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
               
               
                   
                   
               
               
                   
                 KKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKE 
               
               
                   
                   
               
               
                   
                 LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS 
               
               
                   
                   
               
               
                   
                 LFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHY 
               
               
                   
                   
               
               
                   
                 EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
               
               
                   
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
               
               
                   
                   
               
               
                   
                 AFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDL 
               
               
                   
                   
               
               
                   
                 SQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
                   
               
               
                   
                 02. monoABE8.1_bpNLS + Y147R 
               
               
                   
                 (SEQ ID NO: 265) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV 
               
               
                   
                   
               
               
                   
                 IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATL 
               
               
                   
                   
               
               
                   
                 YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV 
               
               
                   
                   
               
               
                   
                 LHYPGMNHRVEITEGILADECAALLCRFFRMPRQVFNAQK 
               
               
                   
                   
               
               
                   
                 KAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSD 
               
               
                   
                   
               
               
                   
                 KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS 
               
               
                   
                   
               
               
                   
                 IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
               
               
                   
                   
               
               
                   
                 QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
                   
               
               
                   
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
               
               
                   
                   
               
               
                   
                 KFRGHFLIEGDLNPDNSDVDKLEIQLVQTYNQLFEENPIN 
               
               
                   
                   
               
               
                   
                 ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI 
               
               
                   
                   
               
               
                   
                 ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
               
               
                   
                   
               
               
                   
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMI 
               
               
                   
                   
               
               
                   
                 KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY 
               
               
                   
                   
               
               
                   
                 IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ 
               
               
                   
                   
               
               
                   
                 RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
                   
               
               
                   
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
               
               
                   
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYN 
               
               
                   
                   
               
               
                   
                 ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK 
               
               
                   
                   
               
               
                   
                 QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK 
               
               
                   
                   
               
               
                   
                 DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHL 
               
               
                   
                   
               
               
                   
                 FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
               
               
                   
                   
               
               
                   
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHE 
               
               
                   
                   
               
               
                   
                 HIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
                   
               
               
                   
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE 
               
               
                   
                   
               
               
                   
                 NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIV 
               
               
                   
                   
               
               
                   
                 PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
               
               
                   
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV 
               
               
                   
                   
               
               
                   
                 ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL 
               
               
                   
                   
               
               
                   
                 VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP 
               
               
                   
                   
               
               
                   
                 KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI 
               
               
                   
                   
               
               
                   
                 MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
               
               
                   
                   
               
               
                   
                 KKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKE 
               
               
                   
                   
               
               
                   
                 LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS 
               
               
                   
                   
               
               
                   
                 LFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHY 
               
               
                   
                   
               
               
                   
                 EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
               
               
                   
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
               
               
                   
                   
               
               
                   
                 AFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDL 
               
               
                   
                   
               
               
                   
                 SQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
                   
               
               
                   
                 03. monoABE8.1_bpNLS + Q154S 
               
               
                   
                 (SEQ ID NO: 266) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV 
               
               
                   
                   
               
               
                   
                 IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATL 
               
               
                   
                   
               
               
                   
                 YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV 
               
               
                   
                   
               
               
                   
                 LHYPGMNHRVEITEGILADECAALLCYFFRMPRSVFNAQK 
               
               
                   
                   
               
               
                   
                 KAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSD 
               
               
                   
                   
               
               
                   
                 KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS 
               
               
                   
                   
               
               
                   
                 IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
               
               
                   
                   
               
               
                   
                 QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
                   
               
               
                   
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
               
               
                   
                   
               
               
                   
                 KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN 
               
               
                   
                   
               
               
                   
                 ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI 
               
               
                   
                   
               
               
                   
                 ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
               
               
                   
                   
               
               
                   
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMI 
               
               
                   
                   
               
               
                   
                 KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY 
               
               
                   
                   
               
               
                   
                 IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ 
               
               
                   
                   
               
               
                   
                 RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
                   
               
               
                   
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
               
               
                   
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYN 
               
               
                   
                   
               
               
                   
                 ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK 
               
               
                   
                   
               
               
                   
                 QLKEDYFKKIECFDSVEISGVEDRENASLGTYHDLLKIIK 
               
               
                   
                   
               
               
                   
                 DKDELDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHL 
               
               
                   
                   
               
               
                   
                 FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
               
               
                   
                   
               
               
                   
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHE 
               
               
                   
                   
               
               
                   
                 HIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
                   
               
               
                   
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE 
               
               
                   
                   
               
               
                   
                 NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIV 
               
               
                   
                   
               
               
                   
                 PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
               
               
                   
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV 
               
               
                   
                   
               
               
                   
                 ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL 
               
               
                   
                   
               
               
                   
                 VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP 
               
               
                   
                   
               
               
                   
                 KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI 
               
               
                   
                   
               
               
                   
                 MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
               
               
                   
                   
               
               
                   
                 KKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKE 
               
               
                   
                   
               
               
                   
                 LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS 
               
               
                   
                   
               
               
                   
                 LFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHY 
               
               
                   
                   
               
               
                   
                 EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
               
               
                   
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
               
               
                   
                   
               
               
                   
                 AFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDL 
               
               
                   
                   
               
               
                   
                 SQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
                   
               
               
                   
                 04. monoABE8.1_bpNLS + Y123H 
               
               
                   
                 (SEQ ID NO: 267) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV 
               
               
                   
                   
               
               
                   
                 IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATL 
               
               
                   
                   
               
               
                   
                 YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV 
               
               
                   
                   
               
               
                   
                 LHHPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQK 
               
               
                   
                   
               
               
                   
                 KAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSD 
               
               
                   
                   
               
               
                   
                 KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS 
               
               
                   
                   
               
               
                   
                 IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
               
               
                   
                   
               
               
                   
                 QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
                   
               
               
                   
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
               
               
                   
                   
               
               
                   
                 KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN 
               
               
                   
                   
               
               
                   
                 ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI 
               
               
                   
                   
               
               
                   
                 ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
               
               
                   
                   
               
               
                   
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMI 
               
               
                   
                   
               
               
                   
                 KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY 
               
               
                   
                   
               
               
                   
                 IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ 
               
               
                   
                   
               
               
                   
                 RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
                   
               
               
                   
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
               
               
                   
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYN 
               
               
                   
                   
               
               
                   
                 ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK 
               
               
                   
                   
               
               
                   
                 QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK 
               
               
                   
                   
               
               
                   
                 DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHL 
               
               
                   
                   
               
               
                   
                 FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
               
               
                   
                   
               
               
                   
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHE 
               
               
                   
                   
               
               
                   
                 HIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
                   
               
               
                   
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE 
               
               
                   
                   
               
               
                   
                 NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIV 
               
               
                   
                   
               
               
                   
                 PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
               
               
                   
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV 
               
               
                   
                   
               
               
                   
                 ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL 
               
               
                   
                   
               
               
                   
                 VSDFRKDFQEYKVREINNYHHAHDAYLNAVVGTALIKKYP 
               
               
                   
                   
               
               
                   
                 KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI 
               
               
                   
                   
               
               
                   
                 MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
               
               
                   
                   
               
               
                   
                 KKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKE 
               
               
                   
                   
               
               
                   
                 LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS 
               
               
                   
                   
               
               
                   
                 LFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHY 
               
               
                   
                   
               
               
                   
                 EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
               
               
                   
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
               
               
                   
                   
               
               
                   
                 AFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDL 
               
               
                   
                   
               
               
                   
                 SQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
                   
               
               
                   
                 05. monoABE8.1_bpNLS + V82S 
               
               
                   
                 (SEQ ID NO: 268) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV 
               
               
                   
                   
               
               
                   
                 IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATL 
               
               
                   
                   
               
               
                   
                 YSTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV 
               
               
                   
                   
               
               
                   
                 LHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQK 
               
               
                   
                   
               
               
                   
                 KAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSD 
               
               
                   
                   
               
               
                   
                 KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS 
               
               
                   
                   
               
               
                   
                 IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
               
               
                   
                   
               
               
                   
                 QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
                   
               
               
                   
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
               
               
                   
                   
               
               
                   
                 KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN 
               
               
                   
                   
               
               
                   
                 ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI 
               
               
                   
                   
               
               
                   
                 ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
               
               
                   
                   
               
               
                   
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMI 
               
               
                   
                   
               
               
                   
                 KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY 
               
               
                   
                   
               
               
                   
                 IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ 
               
               
                   
                   
               
               
                   
                 RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
                   
               
               
                   
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
               
               
                   
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYN 
               
               
                   
                   
               
               
                   
                 ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK 
               
               
                   
                   
               
               
                   
                 QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK 
               
               
                   
                   
               
               
                   
                 DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHL 
               
               
                   
                   
               
               
                   
                 FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
               
               
                   
                   
               
               
                   
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHE 
               
               
                   
                   
               
               
                   
                 HIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
                   
               
               
                   
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE 
               
               
                   
                   
               
               
                   
                 NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIV 
               
               
                   
                   
               
               
                   
                 PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
               
               
                   
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV 
               
               
                   
                   
               
               
                   
                 ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL 
               
               
                   
                   
               
               
                   
                 VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP 
               
               
                   
                   
               
               
                   
                 KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI 
               
               
                   
                   
               
               
                   
                 MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
               
               
                   
                   
               
               
                   
                 KKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKE 
               
               
                   
                   
               
               
                   
                 LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS 
               
               
                   
                   
               
               
                   
                 LFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHY 
               
               
                   
                   
               
               
                   
                 EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
               
               
                   
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
               
               
                   
                   
               
               
                   
                 AFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDL 
               
               
                   
                   
               
               
                   
                 SQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
                   
               
               
                   
                 06. monoABE8.1_bpNLS + T166R 
               
               
                   
                 (SEQ ID NO: 269) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV 
               
               
                   
                   
               
               
                   
                 IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATL 
               
               
                   
                   
               
               
                   
                 YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV 
               
               
                   
                   
               
               
                   
                 LHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQK 
               
               
                   
                   
               
               
                   
                 KAQSSRDSGGSSGGSSGSETPGTSESATPESSGGSSGGSD 
               
               
                   
                   
               
               
                   
                 KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS 
               
               
                   
                   
               
               
                   
                 IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
               
               
                   
                   
               
               
                   
                 QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
                   
               
               
                   
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
               
               
                   
                   
               
               
                   
                 KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN 
               
               
                   
                   
               
               
                   
                 ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI 
               
               
                   
                   
               
               
                   
                 ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
               
               
                   
                   
               
               
                   
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMI 
               
               
                   
                   
               
               
                   
                 KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY 
               
               
                   
                   
               
               
                   
                 IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ 
               
               
                   
                   
               
               
                   
                 RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
                   
               
               
                   
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
               
               
                   
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYN 
               
               
                   
                   
               
               
                   
                 ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK 
               
               
                   
                   
               
               
                   
                 QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK 
               
               
                   
                   
               
               
                   
                 DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHL 
               
               
                   
                   
               
               
                   
                 FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
               
               
                   
                   
               
               
                   
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHE 
               
               
                   
                   
               
               
                   
                 HIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
                   
               
               
                   
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE 
               
               
                   
                   
               
               
                   
                 NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIV 
               
               
                   
                   
               
               
                   
                 PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
               
               
                   
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV 
               
               
                   
                   
               
               
                   
                 ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL 
               
               
                   
                   
               
               
                   
                 VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP 
               
               
                   
                   
               
               
                   
                 KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI 
               
               
                   
                   
               
               
                   
                 MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
               
               
                   
                   
               
               
                   
                 KKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKE 
               
               
                   
                   
               
               
                   
                 LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS 
               
               
                   
                   
               
               
                   
                 LFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHY 
               
               
                   
                   
               
               
                   
                 EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
               
               
                   
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
               
               
                   
                   
               
               
                   
                 AFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDL 
               
               
                   
                   
               
               
                   
                 SQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
                   
               
               
                   
                 07. monoABE8.1_bpNLS + Q154R 
               
               
                   
                 (SEQ ID NO: 270) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV 
               
               
                   
                   
               
               
                   
                 IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATL 
               
               
                   
                   
               
               
                   
                 YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV 
               
               
                   
                   
               
               
                   
                 LHYPGMNHRVEITEGILADECAALLCYEFRMPRRVFNAQK 
               
               
                   
                   
               
               
                   
                 KAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSD 
               
               
                   
                   
               
               
                   
                 KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS 
               
               
                   
                   
               
               
                   
                 IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
               
               
                   
                   
               
               
                   
                 QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
                   
               
               
                   
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
               
               
                   
                   
               
               
                   
                 KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN 
               
               
                   
                   
               
               
                   
                 ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI 
               
               
                   
                   
               
               
                   
                 ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
               
               
                   
                   
               
               
                   
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMI 
               
               
                   
                   
               
               
                   
                 KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY 
               
               
                   
                   
               
               
                   
                 IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ 
               
               
                   
                   
               
               
                   
                 RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
                   
               
               
                   
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
               
               
                   
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYN 
               
               
                   
                   
               
               
                   
                 ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK 
               
               
                   
                   
               
               
                   
                 QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK 
               
               
                   
                   
               
               
                   
                 DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHL 
               
               
                   
                   
               
               
                   
                 FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
               
               
                   
                   
               
               
                   
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHE 
               
               
                   
                   
               
               
                   
                 HIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
                   
               
               
                   
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE 
               
               
                   
                   
               
               
                   
                 NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIV 
               
               
                   
                   
               
               
                   
                 PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
               
               
                   
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV 
               
               
                   
                   
               
               
                   
                 ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL 
               
               
                   
                   
               
               
                   
                 VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP 
               
               
                   
                   
               
               
                   
                 KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI 
               
               
                   
                   
               
               
                   
                 MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
               
               
                   
                   
               
               
                   
                 KKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKE 
               
               
                   
                   
               
               
                   
                 LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS 
               
               
                   
                   
               
               
                   
                 LFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHY 
               
               
                   
                   
               
               
                   
                 EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
               
               
                   
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
               
               
                   
                   
               
               
                   
                 AFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDL 
               
               
                   
                   
               
               
                   
                 SQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
                   
               
               
                   
                 08. monoABE8.1_bpNLS + Y147R_Q154R_Y123H 
               
               
                   
                 (SEQ ID NO: 271) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV 
               
               
                   
                   
               
               
                   
                 IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATL 
               
               
                   
                   
               
               
                   
                 YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV 
               
               
                   
                   
               
               
                   
                 LHHPGMNHRVEITEGILADECAALLCRFFRMPRRVFNAQK 
               
               
                   
                   
               
               
                   
                 KAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSD 
               
               
                   
                   
               
               
                   
                 KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS 
               
               
                   
                   
               
               
                   
                 IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
               
               
                   
                   
               
               
                   
                 QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
                   
               
               
                   
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
               
               
                   
                   
               
               
                   
                 KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN 
               
               
                   
                   
               
               
                   
                 ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI 
               
               
                   
                   
               
               
                   
                 ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
               
               
                   
                   
               
               
                   
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMI 
               
               
                   
                   
               
               
                   
                 KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY 
               
               
                   
                   
               
               
                   
                 IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ 
               
               
                   
                   
               
               
                   
                 RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
                   
               
               
                   
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
               
               
                   
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYN 
               
               
                   
                   
               
               
                   
                 ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK 
               
               
                   
                   
               
               
                   
                 QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK 
               
               
                   
                   
               
               
                   
                 DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHL 
               
               
                   
                   
               
               
                   
                 FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
               
               
                   
                   
               
               
                   
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHE 
               
               
                   
                   
               
               
                   
                 HIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
                   
               
               
                   
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE 
               
               
                   
                   
               
               
                   
                 NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIV 
               
               
                   
                   
               
               
                   
                 PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
               
               
                   
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV 
               
               
                   
                   
               
               
                   
                 ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL 
               
               
                   
                   
               
               
                   
                 VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP 
               
               
                   
                   
               
               
                   
                 KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI 
               
               
                   
                   
               
               
                   
                 MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
               
               
                   
                   
               
               
                   
                 KKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKE 
               
               
                   
                   
               
               
                   
                 LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS 
               
               
                   
                   
               
               
                   
                 LFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHY 
               
               
                   
                   
               
               
                   
                 EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
               
               
                   
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
               
               
                   
                   
               
               
                   
                 AFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDL 
               
               
                   
                   
               
               
                   
                 SQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
                   
               
               
                   
                 09. monoABE8.1_bpNLS + Y147R_Q154R_I76Y 
               
               
                   
                 (SEQ ID NO: 272) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV 
               
               
                   
                   
               
               
                   
                 IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL 
               
               
                   
                   
               
               
                   
                 YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV 
               
               
                   
                   
               
               
                   
                 LHYPGMNHRVEITEGILADECAALLCRFFRMPRRVFNAQK 
               
               
                   
                   
               
               
                   
                 KAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSD 
               
               
                   
                   
               
               
                   
                 KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS 
               
               
                   
                   
               
               
                   
                 IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
               
               
                   
                   
               
               
                   
                 QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
                   
               
               
                   
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
               
               
                   
                   
               
               
                   
                 KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN 
               
               
                   
                   
               
               
                   
                 ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI 
               
               
                   
                   
               
               
                   
                 ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
               
               
                   
                   
               
               
                   
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMI 
               
               
                   
                   
               
               
                   
                 KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY 
               
               
                   
                   
               
               
                   
                 IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ 
               
               
                   
                   
               
               
                   
                 RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
                   
               
               
                   
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
               
               
                   
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYN 
               
               
                   
                   
               
               
                   
                 ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK 
               
               
                   
                   
               
               
                   
                 QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK 
               
               
                   
                   
               
               
                   
                 DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHL 
               
               
                   
                   
               
               
                   
                 FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDE 
               
               
                   
                   
               
               
                   
                 LKSDGFANRNEMQLIHDDSLTFKEDIQKAQVSGQGDSLHE 
               
               
                   
                   
               
               
                   
                 HIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
                   
               
               
                   
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE 
               
               
                   
                   
               
               
                   
                 NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIV 
               
               
                   
                   
               
               
                   
                 PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
               
               
                   
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV 
               
               
                   
                   
               
               
                   
                 ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL 
               
               
                   
                   
               
               
                   
                 VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP 
               
               
                   
                   
               
               
                   
                 KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI 
               
               
                   
                   
               
               
                   
                 MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
               
               
                   
                   
               
               
                   
                 KKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKE 
               
               
                   
                   
               
               
                   
                 LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS 
               
               
                   
                   
               
               
                   
                 LFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHY 
               
               
                   
                   
               
               
                   
                 EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
               
               
                   
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
               
               
                   
                   
               
               
                   
                 AFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDL 
               
               
                   
                   
               
               
                   
                 SQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
                   
               
               
                   
                 10. monoABE8.1_bpNLS + Y147R_Q154R_T166R 
               
               
                   
                 (SEQ ID NO: 273) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV 
               
               
                   
                   
               
               
                   
                 IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATL 
               
               
                   
                   
               
               
                   
                 YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV 
               
               
                   
                   
               
               
                   
                 LHYPGMNHRVEITEGILADECAALLCRFFRMPRRVFNAQK 
               
               
                   
                   
               
               
                   
                 KAQSSRDSGGSSGGSSGSETPGTSESATPESSGGSSGGSD 
               
               
                   
                   
               
               
                   
                 KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS 
               
               
                   
                   
               
               
                   
                 IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
               
               
                   
                   
               
               
                   
                 QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
                   
               
               
                   
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
               
               
                   
                   
               
               
                   
                 KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN 
               
               
                   
                   
               
               
                   
                 ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI 
               
               
                   
                   
               
               
                   
                 ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
               
               
                   
                   
               
               
                   
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMI 
               
               
                   
                   
               
               
                   
                 KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY 
               
               
                   
                   
               
               
                   
                 IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ 
               
               
                   
                   
               
               
                   
                 RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
                   
               
               
                   
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
               
               
                   
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYN 
               
               
                   
                   
               
               
                   
                 ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK 
               
               
                   
                   
               
               
                   
                 QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK 
               
               
                   
                   
               
               
                   
                 DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHL 
               
               
                   
                   
               
               
                   
                 FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
               
               
                   
                   
               
               
                   
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHE 
               
               
                   
                   
               
               
                   
                 HIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
                   
               
               
                   
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE 
               
               
                   
                   
               
               
                   
                 NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIV 
               
               
                   
                   
               
               
                   
                 PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
               
               
                   
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV 
               
               
                   
                   
               
               
                   
                 ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL 
               
               
                   
                   
               
               
                   
                 VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP 
               
               
                   
                   
               
               
                   
                 KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI 
               
               
                   
                   
               
               
                   
                 MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
               
               
                   
                   
               
               
                   
                 KKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKE 
               
               
                   
                   
               
               
                   
                 LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS 
               
               
                   
                   
               
               
                   
                 LFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHY 
               
               
                   
                   
               
               
                   
                 EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
               
               
                   
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
               
               
                   
                   
               
               
                   
                 AFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDL 
               
               
                   
                   
               
               
                   
                 SQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
                   
               
               
                   
                 11. monoABE8.1_bpNLS + Y147T_Q154R 
               
               
                   
                 (SEQ ID NO: 274) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV 
               
               
                   
                   
               
               
                   
                 IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATL 
               
               
                   
                   
               
               
                   
                 YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV 
               
               
                   
                   
               
               
                   
                 LHYPGMNHRVEITEGILADECAALLCTFFRMPRRVFNAQK 
               
               
                   
                   
               
               
                   
                 KAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSD 
               
               
                   
                   
               
               
                   
                 KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS 
               
               
                   
                   
               
               
                   
                 IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
               
               
                   
                   
               
               
                   
                 QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
                   
               
               
                   
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
               
               
                   
                   
               
               
                   
                 KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN 
               
               
                   
                   
               
               
                   
                 ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI 
               
               
                   
                   
               
               
                   
                 ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
               
               
                   
                   
               
               
                   
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMI 
               
               
                   
                   
               
               
                   
                 KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY 
               
               
                   
                   
               
               
                   
                 IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ 
               
               
                   
                   
               
               
                   
                 RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
                   
               
               
                   
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
               
               
                   
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYN 
               
               
                   
                   
               
               
                   
                 ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK 
               
               
                   
                   
               
               
                   
                 QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK 
               
               
                   
                   
               
               
                   
                 DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHL 
               
               
                   
                   
               
               
                   
                 FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
               
               
                   
                   
               
               
                   
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHE 
               
               
                   
                   
               
               
                   
                 HIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
                   
               
               
                   
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE 
               
               
                   
                   
               
               
                   
                 NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIV 
               
               
                   
                   
               
               
                   
                 PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
               
               
                   
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV 
               
               
                   
                   
               
               
                   
                 ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL 
               
               
                   
                   
               
               
                   
                 VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP 
               
               
                   
                   
               
               
                   
                 KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI 
               
               
                   
                   
               
               
                   
                 MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
               
               
                   
                   
               
               
                   
                 KKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKE 
               
               
                   
                   
               
               
                   
                 LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS 
               
               
                   
                   
               
               
                   
                 LFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHY 
               
               
                   
                   
               
               
                   
                 EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
               
               
                   
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
               
               
                   
                   
               
               
                   
                 AFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDL 
               
               
                   
                   
               
               
                   
                 SQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
                   
               
               
                   
                 12. monoABE8.1_bpNLS + Y147T_Q154S 
               
               
                   
                 (SEQ ID NO: 275) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV 
               
               
                   
                   
               
               
                   
                 IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATL 
               
               
                   
                   
               
               
                   
                 YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV 
               
               
                   
                   
               
               
                   
                 LHYPGMNHRVEITEGILADECAALLCTFFRMPRSVFNAQK 
               
               
                   
                   
               
               
                   
                 KAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSD 
               
               
                   
                   
               
               
                   
                 KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS 
               
               
                   
                   
               
               
                   
                 IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
               
               
                   
                   
               
               
                   
                 QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
                   
               
               
                   
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
               
               
                   
                   
               
               
                   
                 KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN 
               
               
                   
                   
               
               
                   
                 ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI 
               
               
                   
                   
               
               
                   
                 ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
               
               
                   
                   
               
               
                   
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMI 
               
               
                   
                   
               
               
                   
                 KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY 
               
               
                   
                   
               
               
                   
                 IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ 
               
               
                   
                   
               
               
                   
                 RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
                   
               
               
                   
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
               
               
                   
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYN 
               
               
                   
                   
               
               
                   
                 ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK 
               
               
                   
                   
               
               
                   
                 QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK 
               
               
                   
                   
               
               
                   
                 DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHL 
               
               
                   
                   
               
               
                   
                 FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
               
               
                   
                   
               
               
                   
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHE 
               
               
                   
                   
               
               
                   
                 HIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
                   
               
               
                   
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE 
               
               
                   
                   
               
               
                   
                 NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIV 
               
               
                   
                   
               
               
                   
                 PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
               
               
                   
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV 
               
               
                   
                   
               
               
                   
                 ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL 
               
               
                   
                   
               
               
                   
                 VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP 
               
               
                   
                   
               
               
                   
                 KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI 
               
               
                   
                   
               
               
                   
                 MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
               
               
                   
                   
               
               
                   
                 KKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKE 
               
               
                   
                   
               
               
                   
                 LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS 
               
               
                   
                   
               
               
                   
                 LFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHY 
               
               
                   
                   
               
               
                   
                 EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
               
               
                   
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
               
               
                   
                   
               
               
                   
                 AFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDL 
               
               
                   
                   
               
               
                   
                 SQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
                   
               
               
                   
                 13. monoABE8.1_bpNLS + H123_Y123_HY147R_ 
               
               
                   
                 Q154R_I76Y 
               
               
                   
                 (SEQ ID NO: 276) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV 
               
               
                   
                   
               
               
                   
                 IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLYDATL 
               
               
                   
                   
               
               
                   
                 YVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV 
               
               
                   
                   
               
               
                   
                 LHHPGMNHRVEITEGILADECAALLCRFFRMPRRVFNAQK 
               
               
                   
                   
               
               
                   
                 KAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSD 
               
               
                   
                   
               
               
                   
                 KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS 
               
               
                   
                   
               
               
                   
                 IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
               
               
                   
                   
               
               
                   
                 QEIFSNEMAKVDDSEFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
                   
               
               
                   
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
               
               
                   
                   
               
               
                   
                 KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN 
               
               
                   
                   
               
               
                   
                 ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI 
               
               
                   
                   
               
               
                   
                 ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
               
               
                   
                   
               
               
                   
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMI 
               
               
                   
                   
               
               
                   
                 KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY 
               
               
                   
                   
               
               
                   
                 IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ 
               
               
                   
                   
               
               
                   
                 RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
                   
               
               
                   
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
               
               
                   
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYN 
               
               
                   
                   
               
               
                   
                 ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK 
               
               
                   
                   
               
               
                   
                 QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK 
               
               
                   
                   
               
               
                   
                 DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHL 
               
               
                   
                   
               
               
                   
                 FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
               
               
                   
                   
               
               
                   
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHE 
               
               
                   
                   
               
               
                   
                 HIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
                   
               
               
                   
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE 
               
               
                   
                   
               
               
                   
                 NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIV 
               
               
                   
                   
               
               
                   
                 PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
               
               
                   
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV 
               
               
                   
                   
               
               
                   
                 ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL 
               
               
                   
                   
               
               
                   
                 VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP 
               
               
                   
                   
               
               
                   
                 KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI 
               
               
                   
                   
               
               
                   
                 MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
               
               
                   
                   
               
               
                   
                 KKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKE 
               
               
                   
                   
               
               
                   
                 LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS 
               
               
                   
                   
               
               
                   
                 LFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHY 
               
               
                   
                   
               
               
                   
                 EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
               
               
                   
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
               
               
                   
                   
               
               
                   
                 AFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDL 
               
               
                   
                   
               
               
                   
                 SQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
                   
               
               
                   
                 14. monoABE8.1_bpNLS + V82S + Q154R 
               
               
                   
                 (SEQ ID NO: 277) 
               
               
                   
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRV 
               
               
                   
                   
               
               
                   
                 IGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATL 
               
               
                   
                   
               
               
                   
                 YSTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDV 
               
               
                   
                   
               
               
                   
                 LHYPGMNHRVEITEGILADECAALLCYFFRMPRRVFNAQK 
               
               
                   
                   
               
               
                   
                 KAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSD 
               
               
                   
                   
               
               
                   
                 KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHS 
               
               
                   
                   
               
               
                   
                 IKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL 
               
               
                   
                   
               
               
                   
                 QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
                   
               
               
                   
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
               
               
                   
                   
               
               
                   
                 KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPIN 
               
               
                   
                   
               
               
                   
                 ASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI 
               
               
                   
                   
               
               
                   
                 ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI 
               
               
                   
                   
               
               
                   
                 GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMI 
               
               
                   
                   
               
               
                   
                 KRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGY 
               
               
                   
                   
               
               
                   
                 IDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ 
               
               
                   
                   
               
               
                   
                 RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
                   
               
               
                   
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV 
               
               
                   
                   
               
               
                   
                 DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYN 
               
               
                   
                   
               
               
                   
                 ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK 
               
               
                   
                   
               
               
                   
                 QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIK 
               
               
                   
                   
               
               
                   
                 DKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHL 
               
               
                   
                   
               
               
                   
                 FDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF 
               
               
                   
                   
               
               
                   
                 LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHE 
               
               
                   
                   
               
               
                   
                 HIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
                   
               
               
                   
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE 
               
               
                   
                   
               
               
                   
                 NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIV 
               
               
                   
                   
               
               
                   
                 PQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNY 
               
               
                   
                   
               
               
                   
                 WRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLV 
               
               
                   
                   
               
               
                   
                 ETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL 
               
               
                   
                   
               
               
                   
                 VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYP 
               
               
                   
                   
               
               
                   
                 KLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNI 
               
               
                   
                   
               
               
                   
                 MNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
                   
               
               
                   
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
               
               
                   
                   
               
               
                   
                 KKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKE 
               
               
                   
                   
               
               
                   
                 LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS 
               
               
                   
                   
               
               
                   
                 LFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHY 
               
               
                   
                   
               
               
                   
                 EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
               
               
                   
                   
               
               
                   
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA 
               
               
                   
                   
               
               
                   
                 AFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDL 
               
               
                   
                   
               
               
                   
                 SQLGGDEGADKRTADGSEFESPKKKRKV 
               
            
           
         
       
     
     In some embodiments, the base editor is a fusion protein comprising a polynucleotide programmable nucleotide binding domain (e.g., Cas9-derived domain) fused to a nucleobase editing domain (e.g., all or a portion of a deaminase domain). In certain embodiments, the fusion proteins provided herein comprise one or more features that improve the base editing activity of the fusion proteins. For example, any of the fusion proteins provided herein may comprise a Cas9 domain that has reduced nuclease activity. In some embodiments, any of the fusion proteins provided herein may have a Cas9 domain that does not have nuclease activity (dCas9), or a Cas9 domain that cuts one strand of a duplexed DNA molecule, referred to as a Cas9 nickase (nCas9). 
     In some embodiments, the base editor further comprises a domain comprising all or a portion of a uracil glycosylase inhibitor (UGI). In some embodiments, the base editor comprises a domain comprising all or a portion of a uracil binding protein (UBP), such as a uracil DNA glycosylase (UDG). In some embodiments, the base editor comprises a domain comprising all or a portion of a nucleic acid polymerase. In some embodiments, a nucleic acid polymerase or portion thereof incorporated into a base editor is a translesion DNA polymerase. 
     In some embodiments, a domain of the base editor can comprise multiple domains. For example, the base editor comprising a polynucleotide programmable nucleotide binding domain derived from Cas9 can comprise an REC lobe and an NUC lobe corresponding to the REC lobe and NUC lobe of a wild-type or natural Cas9. In another example, the base editor can comprise one or more of a RuvCI domain, BH domain, REC1 domain, REC2 domain, RuvCII domain, L1 domain, HNH domain, L2 domain, RuvCIII domain, WED domain, TOPO domain or CTD domain. In some embodiments, one or more domains of the base editor comprise a mutation (e.g., substitution, insertion, deletion) relative to a wild-type version of a polypeptide comprising the domain. For example, an HNH domain of a polynucleotide programmable DNA binding domain can comprise an H840A substitution. In another example, a RuvCI domain of a polynucleotide programmable DNA binding domain can comprise a D10A substitution. 
     Different domains (e.g., adjacent domains) of the base editor disclosed herein can be connected to each other with or without the use of one or more linker domains (e.g., an XTEN linker domain). In some embodiments, a linker domain can be a bond (e.g., covalent bond), chemical group, or a molecule linking two molecules or moieties, e.g., two domains of a fusion protein, such as, for example, a first domain (e.g., Cas9-derived domain) and a second domain (e.g., an adenosine deaminase domain). In some embodiments, a linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-hetero atom bond, etc.). In certain embodiments, a linker is a carbon nitrogen bond of an amide linkage. In certain embodiments, a linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, a linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, a linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In some embodiments, a linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In some embodiments, a linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, a linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, a linker comprises a polyethylene glycol moiety (PEG). In certain embodiments, a linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. A linker can include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile can be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates. In some embodiments, a linker j oins a gRNA binding domain of an RNA-programmable nuclease, including a Cas9 nuclease domain, and the catalytic domain of a nucleic acid editing protein. In some embodiments, a linker joins a dCas9 and a second domain (e.g., UGI, etc.). 
     Typically, a linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, a linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, a linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, a linker is 2-100 amino acids in length, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. In some embodiments, the linker is about 3 to about 104 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100) amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, a linker domain comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 67), which can also be referred to as the XTEN linker. Any method for linking the fusion protein domains can be employed (e.g., ranging from very flexible linkers of the form (SGGS)n (SEQ ID NO: 278), (GGGS)n (SEQ ID NO: 279), (GGGGS)n (SEQ ID NO: 280), and (G)n, to more rigid linkers of the form (EAAAK)n (SEQ ID NO: 281), (GGS)n, SGSETPGTSESATPES (SEQ ID NO: 67) (see, e.g., Guilinger J P, Thompson D B, Liu D R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82; the entire contents are incorporated herein by reference), or (XP) n  motif, in order to achieve the optimal length for activity for the nucleobase editor. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS) n  motif, wherein n is 1, 3, or 7 (SEQ ID NO: 282). In some embodiments, the Cas9 domain of the fusion proteins provided herein are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 67). In some embodiments, a linker comprises a plurality of proline residues and is 5-21, 5-14, 5-9, 5-7 amino acids in length, e.g., PAPAP (SEQ ID NO: 283), PAPAPA (SEQ ID NO: 284), PAPAPAP (SEQ ID NO: 285), PAPAPAPA (SEQ ID NO: 286), P(AP) 4  (SEQ ID NO: 287), P(AP) 7  (SEQ ID NO: 288), P(AP) 10  (SEQ ID NO: 289) (see, e.g., Tan J, Zhang F, Karcher D, Bock R. Engineering of high-precision base editors for site-specific single nucleotide replacement. Nat Commun. 2019 Jan. 25; 10(1):439; the entire contents are incorporated herein by reference). Such proline-rich linkers are also termed “rigid” linkers. 
     A fusion protein of the invention comprises a nucleic acid editing domain. In some embodiments, the deaminase is an adenosine deaminase. In some embodiments, the deaminase is a vertebrate deaminase. In some embodiments, the deaminase is an invertebrate deaminase. In some embodiments, the deaminase is a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse deaminase. In some embodiments, the deaminase is a human deaminase. In some embodiments, the deaminase is a rat deaminase. 
     As used herein, “heterodimer” can refer to a fusion protein comprising a wild type TadA domain and a variant of TadA*7.10 domain or to two variant TadA domains (e.g., TadA7.10 and TadA7.10 with Y147T and Q154S alterations. 
     In some embodiments, the base editor comprises a fusion protein comprising a heterologous polypeptide fused internally or inserted in a napDNAbp. The heterologous polypeptide (e.g., deaminase) can be inserted in the napDNAbp (e.g., Cas9) at a suitable location, for example, such that the napDNAbp retains its ability to bind the target polynucleotide and a guide nucleic acid. A deaminase can be inserted into a napDNAbp without compromising function of the deaminase (e.g., base editing activity) or the napDNAbp (e.g., ability to bind to target nucleic acid and guide nucleic acid). A deaminase can be inserted in the napDNAbp at, for example, a disordered region or a region comprising a high temperature factor or B-factor as shown by crystallographic studies. Regions of a protein that are less ordered, disordered, or unstructured, for example solvent exposed regions and loops, can be used for insertion without compromising structure or function. A deaminase can be inserted in the napDNAbp in a flexible loop region or a solvent-exposed region. In some embodiments, the deaminase is inserted in a flexible loop of the Cas9 polypeptide. 
     In some embodiments, the insertion location of a deaminase is determined by B-factor analysis of the crystal structure of Cas9 polypeptide. In some embodiments, the deaminase is inserted in regions of the Cas9 polypeptide comprising higher than average B-factors (e.g., higher B factors compared to the total protein or the protein domain comprising the disordered region). B-factor or temperature factor can indicate the fluctuation of atoms from their average position (for example, as a result of temperature-dependent atomic vibrations or static disorder in a crystal lattice). A high B-factor (e.g., higher than average B-factor) for backbone atoms can be indicative of a region with relatively high local mobility. Such a region can be used for inserting a deaminase without compromising structure or function. A deaminase can be inserted at a location with a residue having a Cα atom with a B-factor that is 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or greater than 200% more than the average B-factor for the total protein. A deaminase can be inserted at a location with a residue having a Cα atom with a B-factor that is 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or greater than 200% more than the average B-factor for a Cas9 protein domain comprising the residue. Cas9 polypeptide positions comprising a higher than average B-factor can include, for example, residues 768, 792, 1052, 1015, 1022, 1026, 1029, 1067, 1040, 1054, 1068, 1246, 1247, and 1248 as numbered in SEQ ID No:1. Cas9 polypeptide regions comprising a higher than average B-factor can include, for example, residues 792-872, 792-906, and 2-791 as numbered in SEQ ID No:1. 
     A heterologous polypeptide (e.g., deaminase) can be inserted in the napDNAbp at an amino acid residue selected from the group consisting of: 768, 791, 792, 1015, 1016, 1022, 1023, 1026, 1029, 1040, 1052, 1054, 1067, 1068, 1069, 1246, 1247, and 1248 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the heterologous polypeptide is inserted between amino acid positions 768-769, 791-792, 792-793, 1015-1016, 1022-1023, 1026-1027, 1029-1030, 1040-1041, 1052-1053, 1054-1055, 1067-1068, 1068-1069, 1247-1248, or 1248-1249 as numbered in SEQ ID NO: 1 or corresponding amino acid positions thereof. In some embodiments, the heterologous polypeptide is inserted between amino acid positions 769-770, 792-793, 793-794, 1016-1017, 1023-1024, 1027-1028, 1030-1031, 1041-1042, 1053-1054, 1055-1056, 1068-1069, 1069-1070, 1248-1249, or 1249-1250 as numbered in SEQ ID NO: 1 or corresponding amino acid positions thereof. In some embodiments, the heterologous polypeptide replaces an amino acid residue selected from the group consisting of: 768, 791, 792, 1015, 1016, 1022, 1023, 1026, 1029, 1040, 1052, 1054, 1067, 1068, 1069, 1246, 1247, and 1248 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. It should be understood that the reference to SEQ ID NO:1 with respect to insertion positions is for illustrative purpose. The insertions as discussed herein are not limited to the Cas9 polypeptide sequence of SEQ ID NO: 1, but include insertion at corresponding locations in variant Cas9 polypeptides, for example a Cas9 nickase (nCas9), nuclease dead Cas9 (dCas9), a Cas9 variant lacking a nuclease domain, a truncated Cas9, or a Cas9 domain lacking partial or complete HNH domain. 
     A heterologous polypeptide (e.g., deaminase) can be inserted in the napDNAbp at an amino acid residue selected from the group consisting of: 768, 792, 1022, 1026, 1040, 1068, and 1247 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the heterologous polypeptide is inserted between amino acid positions 768-769, 792-793, 1022-1023, 1026-1027, 1029-1030, 1040-1041, 1068-1069, or 1247-1248 as numbered in SEQ ID NO: 1 or corresponding amino acid positions thereof. In some embodiments, the heterologous polypeptide is inserted between amino acid positions 769-770, 793-794, 1023-1024, 1027-1028, 1030-1031, 1041-1042, 1069-1070, or 1248-1249 as numbered in SEQ ID NO: 1 or corresponding amino acid positions thereof. In some embodiments, the heterologous polypeptide replaces an amino acid residue selected from the group consisting of: 768, 792, 1022, 1026, 1040, 1068, and 1247 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, an ABE (e.g., TadA) is inserted at an amino acid residue selected from the group consisting of: 1015, 1022, 1029, 1040, 1068, 1247, 1054, 1026, 768, 1067, 1248, 1052, and 1246 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, an ABE (e.g., TadA) is inserted in place of residues 792-872, 792-906, or 2-791 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the N-terminus of an amino acid selected from the group consisting of: 1015, 1022, 1029, 1040, 1068, 1247, 1054, 1026, 768, 1067, 1248, 1052, and 1246 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the C-terminus of an amino acid selected from the group consisting of: 1015, 1022, 1029, 1040, 1068, 1247, 1054, 1026, 768, 1067, 1248, 1052, and 1246 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted to replace an amino acid selected from the group consisting of: 1015, 1022, 1029, 1040, 1068, 1247, 1054, 1026, 768, 1067, 1248, 1052, and 1246 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, a CBE (e.g., APOBEC1) is inserted at an amino acid residue selected from the group consisting of: 1016, 1023, 1029, 1040, 1069, and 1247 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the N-terminus of an amino acid selected from the group consisting of: 1016, 1023, 1029, 1040, 1069, and 1247 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the C-terminus of an amino acid selected from the group consisting of: 1016, 1023, 1029, 1040, 1069, and 1247 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted to replace an amino acid selected from the group consisting of: 1016, 1023, 1029, 1040, 1069, and 1247 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, the deaminase is inserted at amino acid residue 768 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the N-terminus of amino acid 768 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the C-terminus of amino acid 768 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted to replace amino acid 768 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, the deaminase is inserted at amino acid residue 791 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the N-terminus of amino acid 791 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the C-terminus of amino acid 791 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted to replace amino acid 791 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, the deaminase is inserted at amino acid residue 792 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the N-terminus of amino acid 792 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the C-terminus of amino acid 792 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted to replace amino acid 792 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, the deaminase is inserted at amino acid residue 1016 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the N-terminus of amino acid 1016 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the C-terminus of amino acid 1016 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted to replace amino acid 1016 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, the deaminase is inserted at amino acid residue 1022 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the N-terminus of amino acid 1022 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the C-terminus of amino acid 1022 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted to replace amino acid 1022 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, the deaminase is inserted at amino acid residue 1023 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the N-terminus of amino acid 1023 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the C-terminus of amino acid 1023 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted to replace amino acid 1023 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, the deaminase is inserted at amino acid residue 1026 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the N-terminus of amino acid 1026 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the C-terminus of amino acid 1026 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted to replace amino acid 1026 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, the deaminase is inserted at amino acid residue 1029 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the N-terminus of amino acid 1029 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the C-terminus of amino acid 1029 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted to replace amino acid 1029 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, the deaminase is inserted at amino acid residue 1040 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the N-terminus of amino acid 140 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the C-terminus of amino acid 1040 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted to replace amino acid 1040 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, the deaminase is inserted at amino acid residue 1052 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the N-terminus of amino acid 1052 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the C-terminus of amino acid 1052 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted to replace amino acid 1052 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, the deaminase is inserted at amino acid residue 1054 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the N-terminus of amino acid 1054 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the C-terminus of amino acid 1054 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted to replace amino acid 1054 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, the deaminase is inserted at amino acid residue 1067 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the N-terminus of amino acid 1067 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the C-terminus of amino acid 1067 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted to replace amino acid 1067 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, the deaminase is inserted at amino acid residue 1068 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the N-terminus of amino acid 1068 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the C-terminus of amino acid 1068 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted to replace amino acid 1068 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, the deaminase is inserted at amino acid residue 1069 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the N-terminus of amino acid 1069 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the C-terminus of amino acid 1069 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted to replace amino acid 1069 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, the deaminase is inserted at amino acid residue 1246 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the N-terminus of amino acid 1246 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the C-terminus of amino acid 1246 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted to replace amino acid 1246 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, the deaminase is inserted at amino acid residue 1247 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the N-terminus of amino acid 1247 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the C-terminus of amino acid 1247 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted to replace amino acid 1247 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, the deaminase is inserted at amino acid residue 1248 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the N-terminus of amino acid 1248 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted at the C-terminus of amino acid 1248 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the ABE is inserted to replace amino acid 1248 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     In some embodiments, a heterologous polypeptide (e.g., deaminase) is inserted in a flexible loop of a Cas9 polypeptide. The flexible loop portions can be selected from the group consisting of 530-537, 569-570, 686-691, 943-947, 1002-1025, 1052-1077, 1232-1247, or 1298-1300 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. The flexible loop portions can be selected from the group consisting of: 1-529, 538-568, 580-685, 692-942, 948-1001, 1026-1051, 1078-1231, or 1248-1297 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     A heterologous polypeptide (e.g., deaminase) can be inserted into a Cas9 polypeptide region corresponding to amino acid residues: 1017-1069, 1242-1247, 1052-1056, 1060-1077, 1002-1003, 943-947, 530-537, 568-579, 686-691,1242-1247, 1298-1300, 1066-1077, 1052-1056, or 1060-1077 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     A heterologous polypeptide (e.g., deaminase) can be inserted in place of a deleted region of a Cas9 polypeptide. The deleted region can correspond to an N-terminal or C-terminal portion of the Cas9 polypeptide. In some embodiments, the deleted region corresponds to residues 792-872 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deleted region corresponds to residues 792-906 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deleted region corresponds to residues 2-791 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     A heterologous polypeptide (e.g., deaminase) can be inserted within a structural or functional domain of a Cas9 polypeptide. A heterologous polypeptide (e.g., deaminase) can be inserted between two structural or functional domains of a Cas9 polypeptide. A heterologous polypeptide (e.g., deaminase) can be inserted in place of a structural or functional domain of a Cas9 polypeptide, for example, after deleting the domain from the Cas9 polypeptide. The structural or functional domains of a Cas9 polypeptide can include, for example, RuvC I, RuvC II, RuvC III, Rec1, Rec2, PI, or HNH. 
     In some embodiments, the Cas9 polypeptide lacks one or more domains selected from the group consisting of: RuvC I, RuvC II, RuvC III, Rec1, Rec2, PI, or HNH domain. In some embodiments, the Cas9 polypeptide lacks a nuclease domain. In some embodiments, the Cas9 polypeptide lacks a HNH domain. In some embodiments, the Cas9 polypeptide lacks a portion of the HNH domain such that the Cas9 polypeptide has reduced or abolished HNH activity. 
     In some embodiments, the Cas9 polypeptide comprises a deletion of the nuclease domain and the deaminase is inserted to replace the nuclease domain. In some embodiments, the HNH domain is deleted and the deaminase is inserted in its place. In some embodiments, one or more of the RuvC domains is deleted and the deaminase is inserted in its place. 
     A fusion protein comprising a heterologous polypeptide can be flanked by a N-terminal and a C-terminal fragment of a napDNAbp. In some embodiments, the fusion protein comprises a deaminase flanked by a N-terminal fragment and a C-terminal fragment of a Cas9 polypeptide. The N terminal fragment or the C terminal fragment can bind the target polynucleotide sequence. The C-terminus of the N terminal fragment or the N-terminus of the C terminal fragment can comprise a part of a flexible loop of a Cas9 polypeptide. The C-terminus of the N terminal fragment or the N-terminus of the C terminal fragment can comprise a part of an alpha-helix structure of the Cas9 polypeptide. The N-terminal fragment or the C-terminal fragment can comprise a DNA binding domain. The N-terminal fragment or the C-terminal fragment can comprise a RuvC domain. The N-terminal fragment or the C-terminal fragment can comprise a HNH domain. In some embodiments, neither of the N-terminal fragment and the C-terminal fragment comprises a HNH domain. 
     In some embodiments, the C-terminus of the N terminal Cas9 fragment comprises an amino acid that is in proximity to a target nucleobase when the fusion protein deaminates the target nucleobase. In some embodiments, the N-terminus of the C terminal Cas9 fragment comprises an amino acid that is in proximity to a target nucleobase when the fusion protein deaminates the target nucleobase. The insertion location of different deaminases can be different in order to have proximity between the target nucleobase and an amino acid in the C-terminus of the N terminal Cas9 fragment or the N-terminus of the C terminal Cas9 fragment. For example, the insertion position of an ABE can be at an amino acid residue selected from the group consisting of: 1015, 1022, 1029, 1040, 1068, 1247, 1054, 1026, 768, 1067, 1248, 1052, and 1246 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. A suitable insertion position of a CBE can be an amino acid residue selected from the group consisting of: 1016, 1023, 1029, 1040, 1069, and 1247 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. In certain embodiments, the insertion of the ABE can be inserted to the N terminus or the C terminus of any one of the above listed amino acid residues. In some embodiemnts, the insertion of the ABE can be inserted to replace any one of the above listed amino acid residues. 
     The N-terminal Cas9 fragment of a fusion protein (i.e. the N-terminal Cas9 fragment flanking the deaminase in a fusion protein) can comprise the N-terminus of a Cas9 polypeptide. The N-terminal Cas9 fragment of a fusion protein can comprise a length of at least about: 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids. The N-terminal Cas9 fragment of a fusion protein can comprise a sequence corresponding to amino acid residues: 1-56, 1-95, 1-200, 1-300, 1-400, 1-500, 1-600, 1-700, 1-718, 1-765, 1-780, 1-906, 1-918, or 1-1100 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. The N-terminal Cas9 fragment can comprise a sequence comprising at least: 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to amino acid residues: 1-56, 1-95, 1-200, 1-300, 1-400, 1-500, 1-600, 1-700, 1-718, 1-765, 1-780, 1-906, 1-918, or 1-1100 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     The C-terminal Cas9 fragment of a fusion protein (i.e. the C-terminal Cas9 fragment flanking the deaminase in a fusion protein) can comprise the C-terminus of a Cas9 polypeptide. The C-terminal Cas9 fragment of a fusion protein can comprise a length of at least about: 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids. The C-terminal Cas9 fragment of a fusion protein can comprise a sequence corresponding to amino acid residues: 1099-1368, 918-1368, 906-1368, 780-1368, 765-1368, 718-1368, 94-1368, or 56-1368 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. The N-terminal Cas9 fragment can comprise a sequence comprising at least: 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to amino acid residues: 1099-1368, 918-1368, 906-1368, 780-1368, 765-1368, 718-1368, 94-1368, or 56-1368 as numbered in SEQ ID NO: 1, or a corresponding amino acid residue in another Cas9 polypeptide. 
     The N-terminal Cas9 fragment and C-terminal Cas9 fragment of a fusion protein taken together may not correspond to a full-length naturally occurring Cas9 polypeptide sequence, for example, as set forth in SEQ ID NO: 1. 
     The fusion protein described herein can effect targeted deamination with reduced deamination at non-target sites (e.g., off-target sites), such as reduced genome wide spurious deamination. The fusion protein described herein can effect targeted deamination with reduced bystander deamination at non-target sites. The undesired deamination or off-target deamination can be reduced by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% compared with, for example, an end terminus fusion protein comprising the deaminase fused to a N terminus or a C terminus of a Cas9 polypeptide. The undesired deamination or off-target deamination can be reduced by at least one-fold, at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least tenfold, at least fifteen fold, at least twenty fold, at least thirty fold, at least forty fold, at least fifty fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, or at least hundred fold, compared with, for example, an end terminus fusion protein comprising the deaminase fused to a N terminus or a C terminus of a Cas9 polypeptide. 
     In some embodiments, the deaminase of the fusion protein deaminates no more than two nucleobases within the range of a R-loop. In some embodiments, the deaminase of the fusion protein deaminates no more than three nucleobases within the range of the R-loop. In some embodiments, the deaminase of the fusion protein deaminates no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobases within the range of the R-loop. A R-loop is a three-stranded nucleic acid structure including a DNA:RNA hybrid, a DNA:DNA or a RNA:RNA complementary structure and the associated with single-stranded DNA. As used herein, a R-loop may be formed when a target polynucleotide is contacted with a CRISPR complex or a base editing complex, wherein a portion of a guide polynucleotide, e.g. a guide RNA, hybridizes with and displaces with a portion of a target polynucleotide, e.g. a target DNA. In some embodiments, a R-loop comprises a hybridized region of a spacer sequence and a target DNA complementary sequence. A R-loop region may be of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nuclebase pairs in length. In some embodiments, the R-loop region is about 20 nucleobase pairs in length. It should be understood that, as used herein, a R-loop region is not limited to the target DNA strand that hybridizes with the gudie polynucleotide. For example, editing of a target nucleobase within a R-loop region may be to a DNA strand that comprises the complementary strand to a guide RNA or may be to a DNA strand that is the opposing strand of the strand complementary to the guide RNA. In some embodiments, editing in the region of the R-loop comprises editing a nucleobase on non-complementary strand (protospacer strand) to a guide RNA in a target DNA sequence. 
     The fusion protein described herein can effect target deamination in an editing window different from canonical base editing. In some embodiments, a target nucleobase is from about 1 to about 20 bases upstream of a PAM sequence in the target polynucleotide sequence. In some embodiments, a target nucleobase is from about 2 to about 12 bases upstream of a PAM sequence in the target polynucleotide sequence. In some embodiments, a target nucleobase is from about 1 to 9 base pairs, about 2 to 10 base pairs, about 3 to 11 base pairs, about 4 to 12 base pairs, about 5 to 13 base pairs, about 6 to 14 base paris, about 7 to 15 base pairs, about 8 to 16 base pairs, about 9 to 17 base pairs, about 10 to 18 base pairs, about 11 to 19 base pairs, about 12 to 20 base pairs, about 1 to 7 base pairs, about 2 to 8 base pairs, about 3 to 9 base pairs, about 4 to 10 base pairs, about 5 to 11 base pairs, about 6 to 12 base pairs, about 7 to 13 base pairs, about 8 to 14 base pairs, about 9 to 15 base pairs, about 10 to 16 base pairs, about 11 to 17 base pairs, about 12 to 18 base pairs, about 13 to 19 base pairs, about 14 to 20 base pairs, about 1 to 5 base pairs, about 2 to 6 base pairs, about 3 to 7 base pairs, about 4 to 8 base pairs, about 5 to 9 base pairs, about 6 to 10 base pairs, about 7 to 11 base pairs, about 8 to 12 base pairs, about 9 to 13 base pairs, about 10 to 14 base pairs, about 11 to 15 base pairs, about 12 to 16 base paris, about 13 to 17 base paris, about 14 to 18 base pairs, about 15 to 19 base pairs, about 16 to 20 base pairs, about 1 to 3 base pairs, about 2 to 4 base pairs, about 3 to 5 base pairs, about 4 to 6 base pairs, about 5 to 7 base pairs, about 6 to 8 base pairs, about 7 to 9 base pairs, about 8 to 10 base pairs, about 9 to 11 base pairs, about 10 to 12 base pairs, about 11 to 13 base pairs, about 12 to 14 base pairs, about 13 to 15 base pairs, about 14 to 16 base pairs, about 15 to 17 base pairs, about 16 to 18 base pairs, about 17 to 19 base pairs, about 18 to 20 base pairs away or upstream of the PAM sequence. In some embodiments, a target nucleobase is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more base pairs away or upstream of the PAM sequence. In some embodiemtns, a target nucleobase is about 1, 2, 3, 4, 5, 6, 7, 8, or 9 base pairs upstream of the PAM sequence. In some embodiments, a target nucleobase is about 2, 3, 4, or 6 base pairs upstream of the PAM sequence. 
     Accordingly, also provided herein are fusion protein libraries and method for using same to optimize base editing that allow for alternative preferred base editing windows compared to canonical base editors, e.g. BE4. In some embodiments, the disclosure provides a protein library for optimized base editing comprising a plurality of fusion proteins, wherein each one of the plurality of fusion proteins comprises a deaminase flanked by a N-terminal fragment and a C-terminal fragment of a Cas9 polypeptide, wherein the N-terminal fragment of each one of the fusion proteins differs from the N-terminal fragments of the rest of the plurality of fusion proteins or wherein the C-terminal fragment of each one of the fusion proteins differs from the C-terminal fragments of the rest of the plurality of fusion proteins, wherein the deaminase of each one of the fusion proteins deaminates a target nucleobase in proximity to a Protospacer Adjacent Motif (PAM) sequence in a target polynucleotide sequence, and wherein the N terminal fragment or the C terminal fragment binds the target polynucleotide sequence. In some embodiments, for each nucleobase within a CRISPR R-loop, at least one of the plurality of fusion proteins deaminates the nucleobase. In some embodiments, for each nucleobase within of a target polynucleotide from 1 to 20 base pairs away of a PAM sequence, at least one of the plurality of fusion proteins deaminates the nucleobase. In some embodiments, provided herein is a kit comprising the fusion protein library that allows for optimized base editing. 
     The fusion protein can comprise more than one heterologous polypeptide. For example, the fusion protein can additionally comprise one or more UGI domains and/or one or more nuclear localization signals. The two or more heterologous domains can be inserted in tandem. The two or more heterologous domains can be inserted at locations such that they are not in tandem in the NapDNAbp. 
     In some embodiments, the base editor comprises a fusion protein comprising a napDNAbp domain (e.g., Cas12-derived domain) with an internally fused nucleobase editing domain (e.g., all or a portion of a deaminase domain). In some embodiments, the napDNAbp is a Cas12b. In some embodiments, the base editor comprises a BhCas12b domain with an internally fused TadA*8 domain inserted at the loci provided in the Table A below. 
     
       
         
           
               
             
               
                 TABLE A 
               
             
            
               
                   
               
               
                 Insertion loci in Cas12b proteins 
               
            
           
           
               
               
               
            
               
                   
                   
                 Inserted 
               
               
                   
                 Insertion site 
                 between aa 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 BhCas12b 
                   
                   
               
               
                   
                 position 1 
                 153 
                 PS 
               
               
                   
                 position 2 
                 255 
                 KE 
               
               
                   
                 position 3 
                 306 
                 DE 
               
               
                   
                 position 4 
                 980 
                 DG 
               
               
                   
                 position 5 
                 1019 
                 KL 
               
               
                   
                 position 6 
                 534 
                 FP 
               
               
                   
                 position 7 
                 604 
                 KG 
               
               
                   
                 position 8 
                 344 
                 HF 
               
               
                   
                 BvCas12b 
               
               
                   
                 position 1 
                 147 
                 PD 
               
               
                   
                 position 2 
                 248 
                 GG 
               
               
                   
                 position 3 
                 299 
                 PE 
               
               
                   
                 position 4 
                 991 
                 GE 
               
               
                   
                 position 5 
                 1031 
                 KM 
               
               
                   
                 AaCas12b 
               
               
                   
                 position 1 
                 157 
                 PG 
               
               
                   
                 position 2 
                 258 
                 VG 
               
               
                   
                 position 3 
                 310 
                 DP 
               
               
                   
                 position 4 
                 1008 
                 GE 
               
               
                   
                 position 5 
                 1044 
                 GK 
               
               
                   
                   
               
            
           
         
       
     
     In some embodiments, a base editor can comprise multiple domains. For example, the base editor comprising a napDNAbp domain derived from a Cas12 protein can comprise an REC lobe and an NUC lobe corresponding to the REC lobe and NUC lobe of a wild-type or natural Cas12. In another example, the base editor can comprise one or more of a RuvC domainWED domain. In some embodiments, one or more domains of the base editor comprise a mutation (e.g., substitution, insertion, deletion) relative to a wild type version of a polypeptide comprising the domain. 
     A fusion protein of the invention comprises a nucleic acid editing domain. In some embodiments, the nucleic acid editing domain can catalyze a C to U base change. In some embodiments, the nucleic acid editing domain is a deaminase domain. In some embodiments, the deaminase is a cytidine deaminase or an adenosine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC1 deaminase. In some embodiments, the deaminase is an APOBEC2 deaminase. In some embodiments, the deaminase is an APOBEC3 deaminase. In some embodiments, the deaminase is an APOBEC3 A deaminase. In some embodiments, the deaminase is an APOBEC3B deaminase. In some embodiments, the deaminase is an APOBEC3C deaminase. In some embodiments, the deaminase is an APOBEC3D deaminase. In some embodiments, the deaminase is an APOBEC3E deaminase. In some embodiments, the deaminase is an APOBEC3F deaminase. In some embodiments, the deaminase is an APOBEC3G deaminase. In some embodiments, the deaminase is an APOBEC3H deaminase. In some embodiments, the deaminase is an APOBEC4 deaminase. 
     In some embodiments, the deaminase is an activation-induced deaminase (AID). 
     In some embodiments, the deaminase is a vertebrate deaminase. In some embodiments, the deaminase is an invertebrate deaminase. In some embodiments, the deaminase is a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse deaminase. In some embodiments, the deaminase is a human deaminase. In some embodiments, the deaminase is a rat deaminase, e.g., rAPOBEC1. In some embodiments, the deaminase is a  Petromyzon marinus  cytidine deaminase 1 (pmCDA1). In some embodiments, the deaminase is a human APOBEC3G. In some embodiments, the deaminase is a fragment of the human APOBEC3G. In some embodiments, the deaminase is a human APOBEC3G variant comprising a D316R and a D317R mutation. In some embodiments, the deaminase is a fragment of the human APOBEC3G and comprising mutations corresponding to the D316R and D317R mutations. In some embodiments, the nucleic acid editing domain is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or at least 99.5% identical to the deaminase domain of any deaminase described herein. 
     Linkers 
     In certain embodiments, linkers may be used to link any of the peptides or peptide domains of the invention. The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In certain embodiments, the linker is a polypeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises amino acids. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates. 
     In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is a bond (e.g., a covalent bond), an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is about 3 to about 104 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100) amino acids in length. 
     In some embodiments, the adenosine deaminase and the napDNAbp are fused via a linker that is 4, 16, 32, or 104 amino acids in length. In some embodiments, the linker is about 3 to about 104 amino acids in length. In some embodiments, any of the fusion proteins provided herein, comprise an adenosine deaminase and a Cas9 domain that are fused to each other via a linker. Various linker lengths and flexibilities between the deaminase domain (e.g., an engineered ecTadA) and the Cas9 domain can be employed (e.g., ranging from very flexible linkers of the form (GGGS) n  (SEQ ID NO: 279), (GGGGS) n  (SEQ ID NO: 280), and (G) n  to more rigid linkers of the form (EAAAK) n  (SEQ ID NO: 281), (SGGS) n  (SEQ ID NO: 278), SGSETPGTSESATPES (SEQ ID NO: 67) (see, e.g., Guilinger J P, Thompson D B, Liu D R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82; the entire contents are incorporated herein by reference) and (XP)n) in order to achieve the optimal length for activity for the nucleobase editor. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS), motif, wherein n is 1, 3, or 7 (SEQ ID NO: 282). In some embodiments, the adenosine deaminase and the Cas9 domain of any of the fusion proteins provided herein are fused via a linker (e.g., an XTEN linker) comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 67). 
     Cas9 Complexes with Guide RNAs 
     Some aspects of this disclosure provide complexes comprising any of the fusion proteins provided herein, and a guide RNA (e.g., a guide that targets A \ mutation) bound to a CAS9 domain (e.g., a dCas9, a nuclease active Cas9, or a Cas9 nickase) of fusion protein. These complexes are also termed ribonucleoproteins (RNPs). Any method for linking the fusion protein domains can be employed (e.g., ranging from very flexible linkers of the form (GGGS) n  (SEQ ID NO: 279), (GGGGS) n  (SEQ ID NO: 280), and (G) n  to more rigid linkers of the form (EAAAK) n  (SEQ ID NO: 281), (SGGS) n  (SEQ ID NO: 278), SGSETPGTSESATPES (SEQ ID NO: 67) (see, e.g., Guilinger J P, Thompson D B, Liu D R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82; the entire contents are incorporated herein by reference) and (XP)n) in order to achieve the optimal length for activity for the nucleobase editor. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS), motif, wherein n is 1, 3, or 7 (SEQ ID NO: 282). In some embodiments, the Cas9 domain of the fusion proteins provided herein are fused via a linker comprising the amino acid sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 67) 
               
               
                   
                 SGSETPGTSESATPES. 
               
            
           
         
       
     
     In some embodiments, the guide nucleic acid (e.g., guide RNA) is from 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. In some embodiments, the guide RNA comprises a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the target sequence is a DNA sequence. In some embodiments, the target sequence is a sequence in the genome of a bacteria, yeast, fungi, insect, plant, or animal. In some embodiments, the target sequence is a sequence in the genome of a human. In some embodiments, the 3′ end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3′ end of the target sequence is immediately adjacent to a non-canonical PAM sequence (e.g., a sequence listed in Table 1 or 5′-NAA-3′). In some embodiments, the guide nucleic acid (e.g., guide RNA) is complementary to a sequence in a gene of interest (e.g., a gene associated with a disease or disorder). 
     Some aspects of this disclosure provide methods of using the fusion proteins, or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule with any of the fusion proteins provided herein, and with at least one guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the 3′ end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3′ end of the target sequence is not immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3′ end of the target sequence is immediately adjacent to an AGC, GAG, TTT, GTG, or CAA sequence. In some embodiments, the 3′ end of the target sequence is immediately adjacent to an NGA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, NGTN, NGTN, or 5′ (TTTV) sequence. 
     It will be understood that the numbering of the specific positions or residues in the respective sequences depends on the particular protein and numbering scheme used. Numbering might be different, e.g., in precursors of a mature protein and the mature protein itself, and differences in sequences from species to species may affect numbering. One of skill in the art will be able to identify the respective residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues. 
     It will be apparent to those of skill in the art that in order to target any of the fusion proteins disclosed herein, to a target site, e.g., a site comprising a mutation to be edited, it is typically necessary to co-express the fusion protein together with a guide RNA. As explained in more detail elsewhere herein, a guide RNA typically comprises a tracrRNA framework allowing for Cas9 binding, and a guide sequence, which confers sequence specificity to the Cas9:nucleic acid editing enzyme/domain fusion protein. Alternatively, the guide RNA and tracrRNA may be provided separately, as two nucleic acid molecules. In some embodiments, the guide RNA comprises a structure, wherein the guide sequence comprises a sequence that is complementary to the target sequence. The guide sequence is typically 20 nucleotides long. The sequences of suitable guide RNAs for targeting Cas9:nucleic acid editing enzyme/domain fusion proteins to specific genomic target sites will be apparent to those of skill in the art based on the instant disclosure. Such suitable guide RNA sequences typically comprise guide sequences that are complementary to a nucleic sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting any of the provided fusion proteins to specific target sequences are provided herein. 
     Methods of Using Fusion Proteins Comprising Adenosine Deaminase Variant and a Cas9 Domain 
     Some aspects of this disclosure provide methods of using the fusion proteins, or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule encoding a mutant form of a protein with any of the fusion proteins provided herein, and with at least one guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the 3′ end of the target sequence is immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3′ end of the target sequence is not immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3′ end of the target sequence is immediately adjacent to an AGC, GAG, TTT, GTG, or CAA sequence. In some embodiments, the 3′ end of the target sequence is immediately adjacent to an NGA, NGCG, NGN, NNGRRT, NNNRRT, NGCG, NGCN, NGTN, NGTN, NGTN, or 5′ (TTTV) sequence. 
     It will be understood that the numbering of the specific positions or residues in the respective sequences depends on the particular protein and numbering scheme used. Numbering might be different, e.g., in precursors of a mature protein and the mature protein itself, and differences in sequences from species to species may affect numbering. One of skill in the art will be able to identify the respective residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues. 
     It will be apparent to those of skill in the art that in order to target any of the fusion proteins comprising a Cas9 domain and an adenosine deaminase variant (e.g., ABE8), as disclosed herein, to a target site, e.g., a site comprising a mutation to be edited, it is typically necessary to co-express the fusion protein together with a guide RNA, e.g., an sgRNA. As explained in more detail elsewhere herein, a guide RNA typically comprises a tracrRNA framework allowing for Cas9 binding, and a guide sequence, which confers sequence specificity to the Cas9:nucleic acid editing enzyme/domain fusion protein. Alternatively, the guide RNA and tracrRNA may be provided separately, as two nucleic acid molecules. In some embodiments, the guide RNA comprises a structure, wherein the guide sequence comprises a sequence that is complementary to the target sequence. The guide sequence is typically 20 nucleotides long. The sequences of suitable guide RNAs for targeting Cas9:nucleic acid editing enzyme/domain fusion proteins to specific genomic target sites will be apparent to those of skill in the art based on the instant disclosure. Such suitable guide RNA sequences typically comprise guide sequences that are complementary to a nucleic sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting any of the provided fusion proteins to specific target sequences are provided herein. 
     Cas12 Complexes with Guide RNAs 
     Some aspects of this disclosure provide complexes comprising any of the fusion proteins provided herein, and a guide RNA (e.g., a guide that targets a target polynucleotide for editing). 
     In some embodiments, the guide nucleic acid (e.g., guide RNA) is from 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. In some embodiments, the guide RNA comprises a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the target sequence is a DNA sequence. In some embodiments, the target sequence is a sequence in the genome of a bacteria, yeast, fungi, insect, plant, or animal. In some embodiments, the target sequence is a sequence in the genome of a human. In some embodiments, the 3′ end of the target sequence is immediately adjacent to a canonical PAM sequence. In some embodiments, the 3′ end of the target sequence is immediately adjacent to a non-canonical PAM sequence. 
     Some aspects of this disclosure provide methods of using the fusion proteins, or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule with any of the fusion proteins provided herein, and with at least one guide RNA, wherein the guide RNA is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the 3′ end of the target sequence is immediately adjacent to an e.g., TTN, DTTN, GTTN, ATTN, ATTC, DTTNT, WTTN, HATY, TTTN, TTTV, TTTC, TG, RTR, or YTN PAM site. 
     It will be understood that the numbering of the specific positions or residues in the respective sequences depends on the particular protein and numbering scheme used. Numbering might be different, e.g., in precursors of a mature protein and the mature protein itself, and differences in sequences from species to species may affect numbering. One of skill in the art will be able to identify the respective residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g., by sequence alignment and determination of homologous residues. 
     It will be apparent to those of skill in the art that in order to target any of the fusion proteins disclosed herein, to a target site, e.g., a site comprising a mutation to be edited, it is typically necessary to co-express the fusion protein together with a guide RNA. As explained in more detail elsewhere herein, a guide RNA typically comprises a tracrRNA framework allowing for Cas12 binding, and a guide sequence, which confers sequence specificity to the Cas12:nucleic acid editing enzyme/domain fusion protein. Alternatively, the guide RNA and tracrRNA may be provided separately, as two nucleic acid molecules. In some embodiments, the guide RNA comprises a structure, wherein the guide sequence comprises a sequence that is complementary to the target sequence. The guide sequence is typically 20 nucleotides long. The sequences of suitable guide RNAs for targeting Cas12:nucleic acid editing enzyme/domain fusion proteins to specific genomic target sites will be apparent to those of skill in the art based on the instant disclosure. Such suitable guide RNA sequences typically comprise guide sequences that are complementary to a nucleic sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting any of the provided fusion proteins to specific target sequences are provided herein. 
     The domains of the base editor disclosed herein can be arranged in any order as long as the deaminase domain is internalized in the Cas12 protein. Non-limiting examples of a base editor comprising a fusion protein comprising e.g., a Cas12 domain and a deaminase domain can be arranged as following: 
     NH2-[Cas12 domain]-Linker1-[ABE8]-Linker2-[Cas12 domain]-COOH;
 
NH2-[Cas12 domain]-Linker1-[ABE8]-[Cas12 domain]-COOH;
 
NH2-[Cas12 domain]-[ABE8]-Linker2-[Cas12 domain]-COOH;
 
NH2-[Cas12 domain]-[ABE8]-[Cas12 domain]-COOH;
 
NH2-[Cas12 domain]-Linker1-[ABE8]-Linker2-[Cas12 domain]-[inosine BER inhibitor]-COOH;
 
NH2-[Cas12 domain]-Linker1-[ABE8]-[Cas12 domain]-[inosine BER inhibitor]-COOH;
 
NH2-[Cas12 domain]-[ABE8]-Linker2-[Cas12 domain]-[inosine BER inhibitor]-COOH;
 
NH2-[Cas12 domain]-[ABE8]-[Cas12 domain]-[inosine BER inhibitor]-COOH;
 
NH2-[inosine BER inhibitor]-[Cas12 domain]-Linker1-[ABE8]-Linker2-[Cas12 domain]-COOH;
 
NH2-[inosine BER inhibitor]-[Cas12 domain]-Linker1-[ABE8]-[Cas12 domain]-COOH;
 
NH2-[inosine BER inhibitor]-[Cas12 domain]-[ABE8]-Linker2-[Cas12 domain]-COOH;
 
NH2-[inosine BER inhibitor]NH2-[Cas12 domain]-[ABE8]-[Cas12 domain]-COOH;
 
     Additionally, in some cases, a Gam protein can be fused to an N terminus of a base editor. In some cases, a Gam protein can be fused to a C terminus of a base editor. The Gam protein of bacteriophage Mu can bind to the ends of double strand breaks (DSBs) and protect them from degradation. In some embodiments, using Gam to bind the free ends of DSB can reduce indel formation during the process of base editing. In some embodiments, 174-residue Gam protein is fused to the N terminus of the base editors. See. Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017). In some cases, a mutation or mutations can change the length of a base editor domain relative to a wild type domain. For example, a deletion of at least one amino acid in at least one domain can reduce the length of the base editor. In another case, a mutation or mutations do not change the length of a domain relative to a wild type domain. For example, substitution(s) in any domain does/do not change the length of the base editor. Non-limiting examples of such base editors, where the length of all the domains is the same as the wild type domains, can include: 
     NH2-[Cas12 domain]-Linker1-[APOBEC1]-Linker2-[Cas12 domain]-COOH;
 
NH2-[Cas12 domain]-Linker1-[APOBEC1]-[Cas12 domain]-COOH;
 
NH2-[Cas12 domain]-[APOBEC1]-Linker2-[Cas12 domain]-COOH;
 
NH2-[Cas12 domain]-[APOBEC1]-[Cas12 domain]-COOH;
 
NH2-[Cas12 domain]-Linker1-[APOBEC1]-Linker2-[Cas12 domain][UGI]-COOH;
 
NH2-[Cas12 domain]-Linker1-[APOBEC1]-[Cas12 domain]-[UGI]-COOH;
 
NH2-[Cas12 domain]-[APOBEC1]-Linker2-[Cas12 domain][UGI]-COOH;
 
NH2-[Cas12 domain]-[APOBEC1]-[Cas12 domain][UGI]-COOH;
 
NH2-[UGI]-[Cas12 domain]-Linker1-[APOBEC1]-Linker2-[Cas12 domain]-COOH;
 
NH2-[UGI]-[Cas12 domain]-Linker1-[APOBEC1]-[Cas12 domain]-COOH;
 
NH2-[UGI]-[Cas12 domain]-[APOBEC1]-Linker2-[Cas12 domain]-COOH;
 
NH2-[UGI]-[Cas12 domain]-[APOBEC1]-[Cas12 domain]-COOH; In some embodiments, the base editing fusion proteins provided herein need to be positioned at a precise location, for example, where a target base is placed within a defined region (e.g., a “deamination window”). In some cases, a target can be within a 4-base region. In some cases, such a defined target region can be approximately 15 bases upstream of the PAM. See Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), the entire contents of which are hereby incorporated by reference.
 
     A defined target region can be a deamination window. A deamination window can be the defined region in which a base editor acts upon and deaminates a target nucleotide. In some embodiments, the deamination window is within a 2, 3, 4, 5, 6, 7, 8, 9, or 10 base regions. In some embodiments, the deamination window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases upstream of the PAM. 
     The base editors of the present disclosure can comprise any domain, feature or amino acid sequence which facilitates the editing of a target polynucleotide sequence. For example, in some embodiments, the base editor comprises a nuclear localization sequence (NLS). In some embodiments, an NLS of the base editor is localized between a deaminase domain and a napDNAbp domain. In some embodiments, an NLS of the base editor is localized C-terminal to a napDNAbp domain. 
     Protein domains included in the fusion protein can be a heterologous functional domain. Non-limiting examples of protein domains which can be included in the fusion protein include a deaminase domain (e.g., cytidine deaminase and/or adenosine deaminase), a uracil glycosylase inhibitor (UGI) domain, epitope tags, and reporter gene sequences. Protein domains can be a heterologous functional domain, for example, having one or more of the following activities: transcriptional activation activity, transcriptional repression activity, transcription release factor activity, gene silencing activity, chromatin modifying activity, epigenetic modifying activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Such heterologous functional domains can confer a function activity, such as modification of a target polypeptide associated with target DNA (e.g., a histone, a DNA binding protein, etc.), leading to, for example, histone methylation, histone acetylation, histone ubiquitination, and the like. Other functions and/or activities conferred can include transposase activity, integrase activity, recombinase activity, ligase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, or any combination of the above. 
     A domain may be detected or labeled with an epitope tag, a reporter protein, other binding domains. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). Additional protein sequences can include amino acid sequences that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. 
     In some embodiments, BhCas12b guide polynucleotide has the following sequence: 
     
       
         
           
               
            
               
                 BhCas12b sgRNA scaffold (underlined) + 20 nt to  
               
               
                 23 nt guide sequence (denoted by Nn) 
               
               
                 (SEQ ID NO: 142) 
               
               
                 5′ GTTCTGTCTTTTGGTCAGGACAACCGTCTAGCTATAAGTGCTGCAGG   
               
               
                 
                   GTGTGAGAAACTCCTATTGCTGGACGATGTCTCTTACGAGGCATTAGCA 
                 
               
               
                   C NNNNNNNNNNNNNNNNNNNN-3′ 
               
            
           
         
       
     
     In some embodiments, BvCas12b and AaCas12b guide polynucleotides have the following sequences: 
     
       
         
           
               
            
               
                 BvCas12b sgRNA scaffold (underlined) + 20 nt to 
               
               
                 23 nt guide sequence (denoted by Nn) 
               
               
                 (SEQ ID NO: 143) 
               
               
                 5′ GACCTATAGGGTCAATGAATCTGTGCGTGTGCCATAAGTAATTAAAA   
               
               
                   ATTACCCACCACAGGAGCACCTGAAAACAGGTGCTTGGCAC NNNNNNNN 
               
               
                 NNNNNNNNNNNN-3′  
               
            
           
         
       
     
     
       
         
           
               
            
               
                 AaCas12b sgRNA scaffold (underlined) + 20 nt to 
               
               
                 23 nt guide sequence (denoted by Nn) 
               
               
                 (SEQ ID NO: 144) 
               
               
                 5′ GTCTAAAGGACAGAATTTTTCAACGGGTGTGCCAATGGCCACTTTCC   
               
               
                 
                   AGGTGGCAAAGCCCGTTGAACTTCTCAAAAAGAACGATCTGAGAAGTGG 
                 
               
               
                   CAC NNNNNNNNNNNNNNNNNNNN-3′ 
               
            
           
         
       
     
     Base Editor Efficiency 
     CRISPR-Cas9 nucleases have been widely used to mediate targeted genome editing. In most genome editing applications, Cas9 forms a complex with a guide polynucleotide (e.g., single guide RNA (sgRNA)) and induces a double-stranded DNA break (DSB) at the target site specified by the sgRNA sequence. Cells primarily respond to this DSB through the non-homologous end-joining (NHEJ) repair pathway, which results in stochastic insertions or deletions (indels) that can cause frameshift mutations that disrupt the gene. In the presence of a donor DNA template with a high degree of homology to the sequences flanking the DSB, gene correction can be achieved through an alternative pathway known as homology directed repair (HDR). Unfortunately, under most non-perturbative conditions, HDR is inefficient, dependent on cell state and cell type, and dominated by a larger frequency of indels. As most of the known genetic variations associated with human disease are point mutations, methods that can more efficiently and cleanly make precise point mutations are needed. Base editing systems as provided herein provide a new way to provide genome editing without generating double-strand DNA breaks, without requiring a donor DNA template, and without inducing an excess of stochastic insertions and deletions. 
     The fusion proteins of the invention advantageously modify a specific nucleotide base encoding a protein comprising a mutation without generating a significant proportion of indels. An “indel,” as used herein, refers to the insertion or deletion of a nucleotide base within a nucleic acid. Such insertions or deletions can lead to frame shift mutations within a coding region of a gene. In some embodiments, it is desirable to generate base editors that efficiently modify (e.g., mutate or deaminate) a specific nucleotide within a nucleic acid, without generating a large number of insertions or deletions (i.e., indels) in the nucleic acid. In certain embodiments, any of the base editors provided herein are capable of generating a greater proportion of intended modifications (e.g., mutations or deaminations) versus indels. 
     In some embodiments, any of base editor systems provided herein result in less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% indel formation in the target polynucleotide sequence. 
     In some embodiments, any of base editor systems comprising one of the ABE8 base editor variants described herein result in less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% indel formation in the target polynucleotide sequence. In some embodiments, any of base editor systems comprising one of the ABE8 base editor variants described herein result in less than 0.8% indel formation in the target polynucleotide sequence. In some embodiments, any of base editor systems comprising one of the ABE8 base editor variants described herein result in at most 0.8% indel formation in the target polynucleotide sequence. In some embodiments, any of base editor systems comprising one of the ABE8 base editor variants described herein result in less than 0.3% indel formation in the target polynucleotide sequence. In some embodiments, any of base editor systems comprising one of the ABE8 base editor variants described results in lower indel formation in the target polynucleotide sequence compared to a base editor system comprising one of ABE7 base editors. In some embodiments, any of base editor systems comprising one of the ABE8 base editor variants described herein results in lower indel formation in the target polynucleotide sequence compared to a base editor system comprising an ABE7.10. 
     In some embodiments, any of base editor systems comprising one of the ABE8 base editor variants described herein has reduction in indel frequency compared to a base editor system comprising one of the ABE7 base editors. In some embodiments, any of base editor systems comprising one of the ABE8 base editor variants described herein has at least 0.01%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% reduction in indel frequency compared to a base editor system comprising one of the ABE7 base editors. In some embodiments, a base editor system comprising one of the ABE8 base editor variants described herein has at least 0.01%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% reduction in indel frequency compared to a base editor system comprising an ABE7.10. 
     The invention provides adenosine deaminase variants (e.g., ABE8 variants) that have increased efficiency and specificity. In particular, the adenosine deaminase variants described herein are more likely to edit a desired base within a polynucleotide, and are less likely to edit bases that are not intended to be altered in the base editing window (e.g., “bystanders”). 
     In some embodiments, any of the base editing system comprising one of the ABE8 base editor variants described herein has reduced bystander editing or mutations. In some embodiments, an unintended editing or mutation is a bystander mutation or bystander editing, for example, base editing of a target base (e.g., A or C) in an unintended or non-target position in a target window of a target nucleotide sequence. In some embodiments, any of the base editing system comprising one of the ABE8 base editor variants described herein has reduced bystander editing or mutations compared to a base editor system comprising an ABE7 base editor, e.g., ABE7.10. In some embodiments, any of the base editing system comprising one of the ABE8 base editor variants described herein has reduced bystander editing or mutations by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% compared to a base editor system comprising an ABE7 base editor, e.g., ABE7.10. In some embodiments, any of the base editing system comprising one of the ABE8 base editor variants described herein has reduced bystander editing or mutations by at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, or at least 3.0 fold compared to a base editor system comprising an ABE7 base editor, e.g., ABE7.10. 
     In some embodiments, any of the base editing system comprising one of the ABE8 base editor variants described herein has reduced spurious editing. In some embodiments, an unintended editing or mutation is a spurious mutation or spurious editing, for example, non-specific editing or guide independent editing of a target base (e.g., A or C) in an unintended or non-target region of the genome. In some embodiments, any of the base editing system comprising one of the ABE8 base editor variants described herein has reduced spurious editing compared to a base editor system comprising an ABE7 base editor, e.g., ABE7.10. In some embodiments, any of the base editing system comprising one of the ABE8 base editor variants described herein has reduced spurious editing by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% compared to a base editor system comprising an ABE7 base editor, e.g., ABE7.10. In some embodiments, any of the base editing system comprising one of the ABE8 base editor variants described herein has reduced spurious editing by at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, or at least 3.0 fold compared to a base editor system comprising an ABE7 base editor, e.g., ABE7.10. 
     Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of efficiently generating an intended mutation, such as a point mutation, in a nucleic acid (e.g., a nucleic acid within a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations (i.e., mutation of bystanders). In some embodiments, any of the base editors provided herein are capable of generating at least 0.01% of intended mutations (i.e., at least 0.01% base editing efficiency). In some embodiments, any of the base editors provided herein are capable of generating at least 0.01%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of intended mutations. 
     In some embodiments, any of the ABE8 base editor variants described herein have at least 0.01%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% base editing efficiency. In some embodiments, the base editing efficiency may be measured by calculating the percentage of edited nucleobases in a population of cells. In some embodiments, any of the ABE8 base editor variants described herein have base editing efficiency of at least 0.01%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% as measured by edited nucleobases in a population of cells. 
     In some embodiments, any of the ABE8 base editor variants described herein has higher base editing efficiency compared to the ABE7 base editors. In some embodiments, any of the ABE8 base editor variants described herein have at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 155%, at least 160%, at least 165%, at least 170%, at least 175%, at least 180%, at least 185%, at least 190%, at least 195%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 450%, or at least 500% higher base editing efficiency compared to an ABE7 base editor, e.g., ABE7.10. 
     In some embodiments, any of the ABE8 base editor variants described herein has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3.0 fold, at least 3.1 fold, at least 3.2, at least 3.3 fold, at least 3.4 fold, at least 3.5 fold, at least 3.6 fold, at least 3.7 fold, at least 3.8 fold, at least 3.9 fold, at least 4.0 fold, at least 4.1 fold, at least 4.2 fold, at least 4.3 fold, at least 4.4 fold, at least 4.5 fold, at least 4.6 fold, at least 4.7 fold, at least 4.8 fold, at least 4.9 fold, or at least 5.0 fold higher base editing efficiency compared to an ABE7 base editor, e.g., ABE7.10. 
     In some embodiments, any of the ABE8 base editor variants described herein have at least 0.01%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% on-target base editing efficiency. In some embodiments, any of the ABE8 base editor variants described herein have on-target base editing efficiency of at least 0.01%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% as measured by edited target nucleobases in a population of cells. 
     In some embodiments, any of the ABE8 base editor variants described herein has higher on-target base editing efficiency compared to the ABE7 base editors. In some embodiments, any of the ABE8 base editor variants described herein have at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 155%, at least 160%, at least 165%, at least 170%, at least 175%, at least 180%, at least 185%, at least 190%, at least 195%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 450%, or at least 500% higher on-target base editing efficiency compared to an ABE7 base editor, e.g., ABE7.10. 
     In some embodiments, any of the ABE8 base editor variants described herein has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3.0 fold, at least 3.1 fold, at least 3.2 fold, at least 3.3 fold, at least 3.4 fold, at least 3.5 fold, at least 3.6 fold, at least 3.7 fold, at least 3.8 fold, at least 3.9 fold, at least 4.0 fold, at least 4.1 fold, at least 4.2 fold, at least 4.3 fold, at least 4.4 fold, at least 4.5 fold, at least 4.6 fold, at least 4.7 fold, at least 4.8 fold, at least 4.9 fold, or at least 5.0 fold higher on-target base editing efficiency compared to an ABE7 base editor, e.g., ABE7.10. 
     The ABE8 base editor variants described herein may be delivered to a host cell via a plasmid, a vector, a LNP complex, or an mRNA. In some embodiments, any of the ABE8 base editor variants described herein is delivered to a host cell as an mRNA. In some embodiments, an ABE8 base editor delivered via a nucleic acid based delivery system, e.g., an mRNA, has on-target editing efficiency of at least at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% as measured by edited nucleobases. In some embodiments, an ABE8 base editor delivered by an mRNA system has higher base editing efficiency compared to an ABE8 base editor delivered by a plasmid or vector system. In some embodiments, any of the ABE8 base editor variants described herein has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 155%, at least 160%, at least 165%, at least 170%, at least 175%, at least 180%, at least 185%, at least 190%, at least 195%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300% higher, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 450%, or at least 500% on-target editing efficiency when delivered by an mRNA system compared to when delivered by a plasmid or vector system. In some embodiments, any of the ABE8 base editor variants described herein has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3.0 fold, at least 3.1 fold, at least 3.2 fold, at least 3.3 fold, at least 3.4 fold, at least 3.5 fold, at least 3.6 fold, at least 3.7 fold, at least 3.8 fold, at least 3.9 fold, at least 4.0 fold, at least 4.1 fold, at least 4.2 fold, at least 4.3 fold, at least 4.4 fold, at least 4.5 fold, at least 4.6 fold, at least 4.7 fold, at least 4.8 fold, at least 4.9 fold, or at least 5.0 fold higher on-target editing efficiency when delivered by an mRNA system compared to when delivered by a plasmid or vector system. 
     In some embodiments, any of base editor systems comprising one of the ABE8 base editor variants described herein result in less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% off-target editing in the target polynucleotide sequence. 
     In some embodiments, any of the ABE8 base editor variants described herein has lower guided off-target editing efficiency when delivered by an mRNA system compared to when delivered by a plasmid or vector system. In some embodiments, any of the ABE8 base editor variants described herein has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% lower guided off-target editing efficiency when delivered by an mRNA system compared to when delivered by a plasmid or vector system. In some embodiments, any of the ABE8 base editor variants described herein has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, or at least 3.0 fold lower guided off-target editing efficiency when delivered by an mRNA system compared to when delivered by a plasmid or vector system. In some embodiments, any of the ABE8 base editor variants described herein has at least about 2.2 fold decrease in guided off-target editing efficiency when delivered by an mRNA system compared to when delivered by a plasmid or vector system. 
     In some embodiments, any of the ABE8 base editor variants described herein has lower guide-independent off-target editing efficiency when delivered by an mRNA system compared to when delivered by a plasmid or vector system. In some embodiments, any of the ABE8 base editor variants described herein has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% lower guide-independent off-target editing efficiency when delivered by an mRNA system compared to when delivered by a plasmid or vector system. In some embodiments, any of the ABE8 base editor variants described herein has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3.0 fold, at least 5.0 fold, at least 10.0 fold, at least 20.0 fold, at least 50.0 fold, at least 70.0 fold, at least 100.0 fold, at least 120.0 fold, at least 130.0 fold, or at least 150.0 fold lower guide-independent off-target editing efficiency when delivered by an mRNA system compared to when delivered by a plasmid or vector system. In some embodiments, ABE8 base editor variants described herein has 134.0 fold decrease in guide-independent off-target editing efficiency (e.g., spurious RNA deamination) when delivered by an mRNA system compared to when delivered by a plasmid or vector system. In some embodiments, ABE8 base editor variants described herein does not increase guide-independent mutation rates across the genome. 
     In some embodiments, the base editors provided herein are capable of generating a ratio of intended mutations to indels that is greater than 1:1. In some embodiments, the base editors provided herein are capable of generating a ratio of intended mutations to indels that is at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 200:1, at least 300:1, at least 400:1, at least 500:1, at least 600:1, at least 700:1, at least 800:1, at least 900:1, or at least 1000:1, or more. 
     The number of intended mutations and indels can be determined using any suitable method, for example, as described in International PCT Application Nos. PCT/2017/045381 (WO2018/027078) and PCT/US2016/058344 (WO2017/070632); Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017); the entire contents of which are hereby incorporated by reference. 
     In some embodiments, to calculate indel frequencies, sequencing reads are scanned for exact matches to two 10-bp sequences that flank both sides of a window in which indels can occur. If no exact matches are located, the read is excluded from analysis. If the length of this indel window exactly matches the reference sequence the read is classified as not containing an indel. If the indel window is two or more bases longer or shorter than the reference sequence, then the sequencing read is classified as an insertion or deletion, respectively. In some embodiments, the base editors provided herein can limit formation of indels in a region of a nucleic acid. In some embodiments, the region is at a nucleotide targeted by a base editor or a region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide targeted by a base editor. 
     The number of indels formed at a target nucleotide region can depend on the amount of time a nucleic acid (e.g., a nucleic acid within the genome of a cell) is exposed to a base editor. In some embodiments, the number or proportion of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing the target nucleotide sequence (e.g., a nucleic acid within the genome of a cell) to a base editor. It should be appreciated that the characteristics of the base editors as described herein can be applied to any of the fusion proteins, or methods of using the fusion proteins provided herein. 
     In some embodiments, the base editors provided herein are capable of limiting formation of indels in a region of a nucleic acid. In some embodiments, the region is at a nucleotide targeted by a base editor or a region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide targeted by a base editor. In some embodiments, any of the base editors provided herein are capable of limiting the formation of indels at a region of a nucleic acid to less than 1%, less than 1.5%, less than 2%, less than 2.5%, less than 3%, less than 3.5%, less than 4%, less than 4.5%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 15%, or less than 20%. The number of indels formed at a nucleic acid region may depend on the amount of time a nucleic acid (e.g., a nucleic acid within the genome of a cell) is exposed to a base editor. In some embodiments, any number or proportion of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing a nucleic acid (e.g., a nucleic acid within the genome of a cell) to a base editor. 
     Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of efficiently generating an intended mutation in a nucleic acid (e.g., a nucleic acid within a genome of a subject) without generating a significant number of unintended mutations (e.g., spurious off-target editing or bystander editing). In some embodiments, an intended mutation is a mutation that is generated by a specific base editor bound to a gRNA, specifically designed to alter or correct a mutation in a target gene. In some embodiments, an intended mutation is a mutation that is generated by a specific base editor bound to a gRNA, specifically designed to alter or correct an HBG mutation. In some embodiments, any of the base editors provided herein are capable of generating a ratio of intended mutations to unintended mutations (e.g., intended mutations:unintended mutations) that is greater than 1:1. In some embodiments, any of the base editors provided herein are capable of generating a ratio of intended mutations to unintended mutations that is at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 150:1, at least 200:1, at least 250:1, at least 500:1, or at least 1000:1, or more. It should be appreciated that the characteristics of the base editors described herein may be applied to any of the fusion proteins, or methods of using the fusion proteins provided herein. 
     Multiplex Editing 
     In some embodiments, the base editor system provided herein is capable of multiplex editing of a plurality of nucleobase pairs in one or more genes. In some embodiments, the plurality of nucleobase pairs is located in the same gene. In some embodiments, the plurality of nucleobase pairs is located in one or more gene, wherein at least one gene is located in a different locus. In some embodiments, the multiplex editing can comprise one or more guide polynucleotides. In some embodiments, the multiplex editing can comprise one or more base editor system. In some embodiments, the multiplex editing can comprise one or more base editor systems with a single guide polynucleotide. In some embodiments, the multiplex editing can comprise one or more base editor system with a plurality of guide polynucleotides. In some embodiments, the multiplex editing can comprise one or more guide polynucleotide with a single base editor system. In some embodiments, the multiplex editing can comprise at least one guide polynucleotide that does not require a PAM sequence to target binding to a target polynucleotide sequence. In some embodiments, the multiplex editing can comprise at least one guide polynucleotide that requires a PAM sequence to target binding to a target polynucleotide sequence. In some embodiments, the multiplex editing can comprise a mix of at least one guide polynucleotide that does not require a PAM sequence to target binding to a target polynucleotide sequence and at least one guide polynucleotide that require a PAM sequence to target binding to a target polynucleotide sequence. It should be appreciated that the characteristics of the multiplex editing using any of the base editors as described herein can be applied to any of combination of the methods of using any of the base editor provided herein. It should also be appreciated that the multiplex editing using any of the base editors as described herein can comprise a sequential editing of a plurality of nucleobase pairs. 
     In some embodiments, the plurality of nucleobase pairs are in one more genes. In some embodiments, the plurality of nucleobase pairs is in the same gene. In some embodiments, at least one gene in the one more genes is located in a different locus. 
     In some embodiments, the editing is editing of the plurality of nucleobase pairs in at least one protein coding region. In some embodiments, the editing is editing of the plurality of nucleobase pairs in at least one protein non-coding region. In some embodiments, the editing is editing of the plurality of nucleobase pairs in at least one protein coding region and at least one protein non-coding region. 
     In some embodiments, the editing is in conjunction with one or more guide polynucleotides. In some embodiments, the base editor system can comprise one or more base editor system. In some embodiments, the base editor system can comprise one or more base editor systems in conjunction with a single guide polynucleotide. In some embodiments, the base editor system can comprise one or more base editor system in conjunction with a plurality of guide polynucleotides. In some embodiments, the editing is in conjunction with one or more guide polynucleotide with a single base editor system. In some embodiments, the editing is in conjunction with at least one guide polynucleotide that does not require a PAM sequence to target binding to a target polynucleotide sequence. In some embodiments, the editing is in conjunction with at least one guide polynucleotide that require a PAM sequence to target binding to a target polynucleotide sequence. In some embodiments, the editing is in conjunction with a mix of at least one guide polynucleotide that does not require a PAM sequence to target binding to a target polynucleotide sequence and at least one guide polynucleotide that require a PAM sequence to target binding to a target polynucleotide sequence. It should be appreciated that the characteristics of the multiplex editing using any of the base editors as described herein can be applied to any of combination of the methods of using any of the base editors provided herein. It should also be appreciated that the editing can comprise a sequential editing of a plurality of nucleobase pairs. 
     In some embodiments, the base editor system capable of multiplex editing of a plurality of nucleobase pairs in one or more genes comprises one of the ABE8 base editor variants described herein. In some embodiments, the base editor system capable of multiplex editing of a plurality of nucleobase pairs in one or more genes comprises one of ABE7 base editors. In some embodiments, the base editor system capable of multiplex editing comprising one of the ABE8 base editor variants described herein has higher multiplex editing efficiency compared the base editor system capable of multiplex editing comprising one of ABE7 base editors. In some embodiments, the base editor system capable of multiplex editing comprising one of the ABE8 base editor variants described herein has at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 155%, at least 160%, at least 165%, at least 170%, at least 175%, at least 180%, at least 185%, at least 190%, at least 195%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300% higher, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 450%, or at least 500% higher multiplex editing efficiency compared the base editor system capable of multiplex editing comprising one of ABE7 base editors. In some embodiments, the base editor system capable of multiplex editing comprising one of the ABE8 base editor variants described herein has at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2.0 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3.0 fold, at least 3.1 fold, at least 3.2 fold, at least 3.3 fold, at least 3.4 fold, at least 3.5 fold, at least 4.0 fold, at least 4.5 fold, at least 5.0 fold, at least 5.5 fold, or at least 6.0 fold higher multiplex editing efficiency compared the base editor system capable of multiplex editing comprising one of ABE7 base editors. 
     Methods for Editing Nucleic Acids 
     Some aspects of the disclosure provide methods for editing a nucleic acid. In some embodiments, the method is a method for editing a nucleobase of a nucleic acid molecule encoding a protein (e.g., a base pair of a double-stranded DNA sequence). In some embodiments, the method comprises the steps of: a) contacting a target region of a nucleic acid (e.g., a double-stranded DNA sequence) with a complex comprising a base editor (e.g., a Cas9 domain fused to an adenosine deaminase) and a guide nucleic acid (e.g., gRNA), b) inducing strand separation of said target region, c) converting a first nucleobase of said target nucleobase pair in a single strand of the target region to a second nucleobase, and d) cutting no more than one strand of said target region using the nCas9, where a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase. In some embodiments, the method results in less than 20% indel formation in the nucleic acid. It should be appreciated that in some embodiments, step b is omitted. In some embodiments, the method results in less than 19%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, 0.2%, or less than 0.1% indel formation. In some embodiments, the method further comprises replacing the second nucleobase with a fifth nucleobase that is complementary to the fourth nucleobase, thereby generating an intended edited base pair (e.g., G•C to A•T). In some embodiments, at least 5% of the intended base pairs are edited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended base pairs are edited. 
     In some embodiments, the ratio of intended products to unintended products in the target nucleotide is at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 200:1, or more. In some embodiments, the ratio of intended mutation to indel formation is greater than 1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1, or more. In some embodiments, the cut single strand (nicked strand) is hybridized to the guide nucleic acid. In some embodiments, the cut single strand is opposite to the strand comprising the first nucleobase. In some embodiments, the base editor comprises a dCas9 domain. In some embodiments, the base editor protects or binds the non-edited strand. In some embodiments, the intended edited base pair is upstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended edited base pair is downstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream stream of the PAM site. In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the nucleobase editor comprises a linker. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length. In some embodiments, linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In one embodiment, the linker is 32 amino acids in length. In another embodiment, a “long linker” is at least about 60 amino acids in length. In other embodiments, the linker is between about 3-100 amino acids in length. In some embodiments, the target region comprises a target window, wherein the target window comprises the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotides in length. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended edited base pair is within the target window. In some embodiments, the target window comprises the intended edited base pair. In some embodiments, the method is performed using any of the base editors provided herein. In some embodiments, a target window is a methylation window. 
     In some embodiments, the disclosure provides methods for editing a nucleotide (e.g., SNP in a gene encoding a protein). In some embodiments, the disclosure provides a method for editing a nucleobase pair of a double-stranded DNA sequence. In some embodiments, the method comprises a) contacting a target region of the double-stranded DNA sequence with a complex comprising a base editor and a guide nucleic acid (e.g., gRNA), where the target region comprises a target nucleobase pair, b) inducing strand separation of said target region, c) converting a first nucleobase of said target nucleobase pair in a single strand of the target region to a second nucleobase, d) cutting no more than one strand of said target region, wherein a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase, and the second nucleobase is replaced with a fifth nucleobase that is complementary to the fourth nucleobase, thereby generating an intended edited base pair, wherein the efficiency of generating the intended edited base pair is at least 5%. It should be appreciated that in some embodiments, step b is omitted. In some embodiments, at least 5% of the intended base pairs are edited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended base pairs are edited. In some embodiments, the method causes less than 19%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, 0.2%, or less than 0.1% indel formation. In some embodiments, the ratio of intended product to unintended products at the target nucleotide is at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 200:1, or more. In some embodiments, the ratio of intended mutation to indel formation is greater than 1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1, or more. In some embodiments, the cut single strand is hybridized to the guide nucleic acid. In some embodiments, the cut single strand is opposite to the strand comprising the first nucleobase. In some embodiments, the intended edited base pair is upstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended edited base pair is downstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream stream of the PAM site. In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length. In some embodiments, the linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the target region comprises a target window, wherein the target window comprises the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotides in length. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended edited base pair occurs within the target window. In some embodiments, the target window comprises the intended edited base pair. In some embodiments, the nucleobase editor is any one of the base editors provided herein. 
     Expression of Fusion Proteins in a Host Cell 
     Fusion proteins of the invention comprising an adenosine deaminase variant may be expressed in virtually any host cell of interest, including but not limited to bacteria, yeast, fungi, insects, plants, and animal cells using routine methods known to the skilled artisan. For example, a DNA encoding an adenosine deaminase of the invention can be cloned by designing suitable primers for the upstream and downstream of CDS based on the cDNA sequence. The cloned DNA may be directly, or after digestion with a restriction enzyme when desired, or after addition of a suitable linker and/or a nuclear localization signal ligated with a DNA encoding one or more additional components of a base editing system. The base editing system is translated in a host cell to form a complex. 
     Fusion proteins are generated by operably linking one or more polynucleotides encoding one or more domains having nucleobase modifying activity (e.g., an adenosine deaminase) to a polynucleotide encoding a napDNAbp to prepare a polynucleotide that encodes a fusion protein of the invention. In some embodiments, a polynucleotide encoding a napDNAbp, and a DNA encoding a domain having nucleobase modifying activity may each be fused with a DNA encoding a binding domain or a binding partner thereof, or both DNAs may be fused with a DNA encoding a separation intein, whereby the nucleic acid sequence-recognizing conversion module and the nucleic acid base converting enzyme are translated in a host cell to form a complex. In these cases, a linker and/or a nuclear localization signal can be linked to a suitable position of one of or both DNAs when desired. 
     A DNA encoding a protein domain described herein can be obtained by chemically synthesizing the DNA, or by connecting synthesized partly overlapping oligoDNA short chains by utilizing the PCR method and the Gibson Assembly method to construct a DNA encoding the full length thereof. The advantage of constructing a full-length DNA by chemical synthesis or a combination of PCR method or Gibson Assembly method is that the codon to be used can be designed in CDS full-length according to the host into which the DNA is introduced. In the expression of a heterologous DNA, the protein expression level is expected to increase by converting the DNA sequence thereof to a codon highly frequently used in the host organism. As the data of codon use frequency in host to be used, for example, the genetic code use frequency database (/www.kazusa.or.jp/codon/index.html) disclosed in the home page of Kazusa DNA Research Institute can be used, or documents showing the codon use frequency in each host may be referred to. By reference to the obtained data and the DNA sequence to be introduced, codons showing low use frequency in the host from among those used for the DNA sequence may be converted to a codon coding the same amino acid and showing high use frequency. 
     An expression vector containing a DNA encoding a nucleic acid sequence-recognizing module and/or a nucleic acid base converting enzyme can be produced, for example, by linking the DNA to the downstream of a promoter in a suitable expression vector. 
     As the expression vector,  Escherichia coli -derived plasmids (e.g., pBR322, pBR325, pUC12, pUC13);  Bacillus subtilis -derived plasmids (e.g., pUB110, pTP5, pC194); yeast-derived plasmids (e.g., pSH19, pSH15); insect cell expression plasmids (e.g., pFast-Bac); animal cell expression plasmids (e.g., pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo); bacteriophages such as .lambda.phage and the like; insect virus vectors such as baculovirus and the like (e.g., BmNPV, AcNPV); animal virus vectors such as retrovirus, vaccinia virus, adenovirus and the like, and the like are used. 
     As the promoter, any promoter appropriate for a host to be used for gene expression can be used. In a conventional method using DSB, since the survival rate of the host cell sometimes decreases markedly due to the toxicity, it is desirable to increase the number of cells by the start of the induction by using an inductive promoter. However, since sufficient cell proliferation can also be afforded by expressing the nucleic acid-modifying enzyme complex of the present invention, a constitution promoter can also be used without limitation. 
     For example, when the host is an animal cell, SR.alpha. promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney mouse leukemia virus) LTR, HSV-TK (simple herpes virus thymidine kinase) promoter and the like are used. Of these, CMV promoter, SR.alpha. promoter and the like are preferable. When the host is  Escherichia coli , trp promoter, lac promoter, recA promoter, lambda.P.sub.L promoter, lpp promoter, T7 promoter and the like are preferable. When the host is genus  Bacillus , SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable. When the host is a yeast, Gal1/10 promoter, PHOS promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, polyhedrin promoter, P10 promoter and the like are preferable. When the host is a plant cell, CaMV35S promoter, CaMV19S promoter, NOS promoter and the like are preferable. 
     In some embodiments, the expression vector may contain an enhancer, splicing signal, terminator, polyA addition signal, a selection marker such as drug resistance gene, auxotrophic complementary gene and the like, replication origin and the like on demand. 
     An RNA encoding a protein domain described herein can be prepared by, for example, transcription to mRNA in a vitro transcription system known per se by using a vector encoding DNA encoding the above-mentioned nucleic acid sequence-recognizing module and/or a nucleic acid base converting enzyme as a template. 
     A fusion protein of the invention can be intracellularly expressed by introducing an expression vector containing a DNA encoding a nucleic acid sequence-recognizing module and/or a nucleic acid base converting enzyme into a host cell, and culturing the host cell. 
     Host cells useful in the invention include bacterial cells, yeast, insect cells, animal cells and the like. 
     The genus  Escherichia  includes  Escherichia coli  K12.cndot.DH1 (Proc. Natl. Acad. Sci. USA, 60, 160 (1968)),  Escherichia coli  JM103 (Nucleic Acids Research, 9, 309 (1981)),  Escherichia coli  JA221 (Journal of Molecular Biology, 120, 517 (1978)),  Escherichia coli  HB101 (Journal of Molecular Biology, 41, 459 (1969)),  Escherichia coli  C600 (Genetics, 39, 440 (1954)) and the like. 
     The genus  Bacillus  includes  Bacillus subtilis  M1114 (Gene, 24, 255 (1983)),  Bacillus subtilis  207-21 (Journal of Biochemistry, 95, 87 (1984)) and the like. 
     Yeast useful for expression the fusion protein of the present invention include,  Saccharomyces cerevisiae  AH22, AH22R − , NA87-11A, DKD-5D, 20B-12 , Schizosaccharomyces pombe  NCYC1913, NCYC2036 , Pichia pastoris  KM71 and the like. 
     Fusion proteins are expressed in insect cells using, for example, viral vectors, such as AcNPV. Insect host cells include any of the following cell lines: cabbage armyworm larva-derived established line ( Spodoptera frugiperda  cell; Sf cell), MG1 cells derived from the mid-intestine of  Trichoplusia ni , High Five™. cells derived from an egg of  Trichoplusia ni, Mamestra brassicae -derived cells,  Estigmena acrea -derived cells and the like are used. When the virus is BmNPV, cells of  Bombyx mori -derived established line ( Bombyx mori  N cell; BmN cell) and the like are used as insect cells. As the Sf cell, for example, Sf9 cell (ATCC CRL1711), Sf21 cell (all above, In Vivo, 13, 213-217 (1977)) and the like. 
     As the insect, for example, larva of  Bombyx mori, Drosophila , cricket and the like are used to express fusion proteins of the invention (Nature, 315, 592 (1985)). 
     Mammalian cell lines may be used to express fusion proteins. Such cell lines include monkey COS-7 cell, monkey Vero cell, Chinese hamster ovary (CHO) cell, dhfr gene-deficient CHO cell, mouse L cell, mouse AtT-20 cell, mouse myeloma cell, rat GH3 cell, human FL cell and the like, pluripotent stem cells such as iPS cell, ES cell and the like of human and other mammals, and primary cultured cells prepared from various tissues. Furthermore, zebrafish embryo,  Xenopus  oocyte and the like can also be used. 
     Plant cells may be maintained in culture using methods well known to the skilled artisan. Plant cell culture involves suspending cultured cells, callus, protoplast, leaf segment, root segment and the like prepared from various plants (e.g., grain such as rice, wheat, corn and the like, product crops such as tomato, cucumber, eggplant, carnations,  Eustoma russellianum , tobacco,  Arabidopsis thaliana ). 
     All the above-mentioned host cells may be haploid (monoploid), or polyploid (e.g., diploid, triploid, tetraploid and the like). In the conventional mutation introduction methods, mutation is, in principle, introduced into only one homologous chromosome to produce a hetero gene type. Therefore, desired phenotype is not expressed unless dominant mutation occurs, and homozygousness inconveniently requires labor and time. In contrast, according to the present invention, since mutation can be introduced into any allele on the homologous chromosome in the genome, desired phenotype can be expressed in a single generation even in the case of recessive mutation, which is extremely useful since the problem of the conventional method can be solved. 
     Expression vectors encoding a fusion protein of the invention are introduced into host cells using any transfection method (e.g., lysozyme method, competent method, PEG method, CaCl 2  coprecipitation method, electroporation method, the microinjection method, the particle gun method, lipofection method,  Agrobacterium  method). The transfection method is selected based on the host cell to be transfected. 
       Escherichia coli  can be transformed according to the methods described in, for example, Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982) and the like. 
     The genus  Bacillus  can be introduced into a vector according to the methods described in, for example, Molecular &amp; General Genetics, 168, 111 (1979) and the like. 
     Yeast cells can be introduced into a vector according to the methods described in, for example, Methods in Enzymology, 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and the like. 
     Insect cells can be introduced into a vector according to the methods described in, for example, Bio/Technology, 6, 47-55 (1988) and the like. 
     Mammalian cells can be introduced into a vector according to the methods described in, for example, Cell Engineering additional volume 8, New Cell Engineering Experiment Protocol, 263-267 (1995) (published by Shujunsha), and Virology, 52, 456 (1973). Cells comprising expression vectors of the invention are cultured according to known methods, which vary depending on the host. 
     For example, when  Escherichia coli  or genus  Bacillus  are cultured, a liquid medium is preferable as a medium to be used for the culture. The medium preferably contains a carbon source, nitrogen source, inorganic substance and the like necessary for the growth of the transformant. Examples of the carbon source include glucose, dextrin, soluble starch, sucrose and the like; examples of the nitrogen source include inorganic or organic substances such as ammonium salts, nitrate salts, corn steep liquor, peptone, casein, meat extract, soybean cake, potato extract and the like; and examples of the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. The medium may contain yeast extract, vitamins, growth promoting factor and the like. The pH of the medium is preferably about 5-about 8. 
     As a medium for culturing  Escherichia coli , for example, M9 medium containing glucose, casamino acid (Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972) is preferable. Where necessary, for example, agents such as 3.beta.-indolylacrylic acid may be added to the medium to ensure an efficient function of a promoter.  Escherichia coli  is cultured at generally about 15-about 43° C. Where necessary, aeration and stirring may be performed. 
     The genus  Bacillus  is cultured at generally about 30-about 40° C. Where necessary, aeration and stirring may be performed. 
     Examples of the medium for culturing yeast include Burkholder minimum medium (Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)), SD medium containing 0.5% casamino acid (Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)) and the like. The pH of the medium is preferably about 5-about 8. The culture is performed at generally about 20° C.-about 35° C. Where necessary, aeration and stirring may be performed. 
     As a medium for culturing an insect cell or insect, for example, Grace&#39;s Insect Medium (Nature, 195, 788 (1962)) containing an additive such as inactivated 10% bovine serum and the like as appropriate and the like are used. The pH of the medium is preferably about 6.2 to about 6.4. The culture is performed at generally about 27° C. Where necessary, aeration and stirring may be performed. 
     As a medium for culturing an animal cell, for example, minimum essential medium (MEM) containing about 5-about 20% of fetal bovine serum (Science, 122, 501 (1952)), Dulbecco&#39;s modified Eagle medium (DMEM) (Virology, 8, 396 (1959)), RPMI 1640 medium (The Journal of the American Medical Association, 199, 519 (1967)), 199 medium (Proceeding of the Society for the Biological Medicine, 73, 1 (1950)) and the like are used. The pH of the medium is preferably about 6-about 8. The culture is performed at generally about 30° C.-about 40° C. Where necessary, aeration and stirring may be performed. 
     As a medium for culturing a plant cell, for example, MS medium, LS medium, B5 medium and the like are used. The pH of the medium is preferably about 5-about 8. The culture is performed at generally about 20° C.-about 30° C. Where necessary, aeration and stirring may be performed. 
     When a higher eukaryotic cell, such as animal cell, insect cell, plant cell and the like is used as a host cell, a DNA encoding a base editing system of the present invention (e.g., comprising an adenosine deaminase variant) is introduced into a host cell under the regulation of an inducible promoter (e.g., metallothionein promoter (induced by heavy metal ion), heat shock protein promoter (induced by heat shock), Tet-ON/Tet-OFF system promoter (induced by addition or removal of tetracycline or a derivative thereof), steroid-responsive promoter (induced by steroid hormone or a derivative thereof) etc.), the induction substance is added to the medium (or removed from the medium) at an appropriate stage to induce expression of the nucleic acid-modifying enzyme complex, culture is performed for a given period to carry out a base editing and, introduction of a mutation into a target gene, transient expression of the base editing system can be realized. 
     Prokaryotic cells such as  Escherichia coli  and the like can utilize an inducible promoter. Examples of the inducible promoter include, but are not limited to, lac promoter (induced by IPTG), cspA promoter (induced by cold shock), araBAD promoter (induced by arabinose) and the like. 
     Alternatively, the above-mentioned inductive promoter can also be utilized as a vector removal mechanism when higher eukaryotic cells, such as animal cell, insect cell, plant cell and the like are used as a host cell. That is, a vector is mounted with a replication origin that functions in a host cell, and a nucleic acid encoding a protein necessary for replication (e.g., SV40 on and large T antigen, oriP and EBNA-1 etc. for animal cells), of the expression of the nucleic acid encoding the protein is regulated by the above-mentioned inducible promoter. As a result, while the vector is autonomously replicatable in the presence of an induction substance, when the induction substance is removed, autonomous replication is not available, and the vector naturally falls off along with cell division (autonomous replication is not possible by the addition of tetracycline and doxycycline in Tet-OFF system vector). 
     Methods of Using Base Editors 
     The correction of point mutations in disease-associated genes and alleles provides new strategies for gene correction with applications in therapeutics and basic research. 
     The present disclosure provides methods for the treatment of a subject diagnosed with a disease associated with or caused by a point mutation that can be corrected by a base editor system provided herein. For example, in some embodiments, a method is provided that comprises administering to a subject having such a disease, e.g., a disease caused by a genetic mutation, an effective amount of a nucleobase editor (e.g., an adenosine deaminase base editor or a cytidine deaminase base editor) that corrects the point mutation in the disease associated gene. The present disclosure provides methods for the treatment of diseases that are associated with or caused by a point mutation that can be corrected by deaminase-mediated gene editing. Suitable diseases that can be treated with the strategies and fusion proteins provided herein will be apparent to those of skill in the art based on the instant disclosure. Provided herein are methods of using a base editor or base editor system for editing a nucleobase in a target nucleotide sequence associated with a disease or disorder. In some embodiments, the activity of the base editor (e.g., comprising an adenosine deaminase and a Cas12 domain) results in a correction of the point mutation. In some embodiments, the target DNA sequence comprises a G→A point mutation associated with a disease or disorder, and deamination of the mutant A base results in a sequence that is not associated with a disease or disorder. In some embodiments, the target DNA sequence comprises a T→C point mutation associated with a disease or disorder, and deamination of the mutant C base results in a sequence that is not associated with a disease or disorder. 
     In some embodiments, the target DNA sequence encodes a protein, and the point mutation is in a codon and results in a change in the amino acid encoded by the mutant codon as compared to the wild-type codon. In some embodiments, the deamination of the mutant A results in a change of the amino acid encoded by the mutant codon. In some embodiments, the deamination of the mutant A results in the codon encoding the wild-type amino acid. In some embodiments, the deamination of the mutant C results in a change of the amino acid encoded by the mutant codon. In some embodiments, the deamination of the mutant C results in the codon encoding the wild-type amino acid. In some embodiments, the subject has or has been diagnosed with a disease or disorder. 
     In some embodiments, the adenosine deaminases provided herein are capable of deaminating a deoxyadenosine residue of DNA. Other aspects of the disclosure provide fusion proteins that comprise an adenosine deaminase (e.g., an adenosine deaminase that deaminates deoxyadenosine in DNA as described herein) and a domain (e.g., a Cas12) capable of binding to a specific nucleotide sequence. For example, the adenosine can be converted to an inosine residue, which typically base pairs with a cytosine residue. Such fusion proteins are useful, inter alia, for targeted editing of nucleic acid sequences. Such fusion proteins can be used for targeted editing of DNA in vitro, e.g., for the generation of mutant cells or animals; for the introduction of targeted mutations, e.g., for the correction of genetic defects in cells ex vivo, e.g., in cells obtained from a subject that are subsequently re-introduced into the same or another subject; and for the introduction of targeted mutations in vivo, e.g., the correction of genetic defects or the introduction of deactivating mutations in disease-associated genes in a G to A, or a T to C to mutation can be treated using the nucleobase editors provided herein. The present disclosure provides deaminases, fusion proteins, nucleic acids, vectors, cells, compositions, methods, kits, systems, etc. that utilize the deaminases and nucleobase editors. 
     Generating an Intended Mutation 
     In some embodiments, the purpose of the methods provided herein is to restore the function of a dysfunctional gene via gene editing. In some embodiments, the function of a dysfunctional gene is restored by introducing an intended mutation. In some embodiments, the methods provided herein can be used to disrupt the normal function of a gene product. The nucleobase editing proteins provided herein can be validated for gene editing-based human therapeutics in vitro, e.g., by correcting a disease-associated mutation in human cell culture. It will be understood by the skilled artisan that the nucleobase editing proteins provided herein, e.g., the fusion proteins comprising a napDNAbp domain (e.g., Cas12) and a nucleobase editing domain (e.g., an adenosine deaminase domain or a cytidine deaminase domain) can be used to correct any single point A to G or C to T mutation. In the first case, deamination of the mutant A to I corrects the mutation, and in the latter case, deamination of the A that is base-paired with the mutant T, followed by a round of replication, corrects the mutation. 
     In some embodiments, the present disclosure provides base editors that efficiently generate an intended mutation, such as a point mutation, in a nucleic acid (e.g., a nucleic acid within a genome of a subject) without generating a significant number of unintended mutations, such as unintended point mutations. In some embodiments, an intended mutation is a mutation that is generated by a specific base editor (e.g., cytidine base editor or adenosine base editor) bound to a guide polynucleotide (e.g., gRNA), specifically designed to generate the intended mutation. In some embodiments, the intended mutation is a mutation associated with a disease or disorder. In some embodiments, the intended mutation is an adenine (A) to guanine (G) point mutation associated with a disease or disorder. In some embodiments, the intended mutation is a cytosine (C) to thymine (T) point mutation associated with a disease or disorder. In some embodiments, the intended mutation is an adenine (A) to guanine (G) point mutation within the coding region or non-coding region of a gene. In some embodiments, the intended mutation is a cytosine (C) to thymine (T) point mutation within the coding region or non-coding region of a gene. In some embodiments, the intended mutation is a point mutation that generates a stop codon, for example, a premature stop codon within the coding region of a gene. In some embodiments, the intended mutation is a mutation that eliminates a stop codon. 
     In some embodiments, any of the base editors provided herein are capable of generating a ratio of intended mutations to unintended mutations (e.g., intended point mutations:unintended point mutations) that is greater than 1:1. In some embodiments, any of the base editors provided herein are capable of generating a ratio of intended mutations to unintended mutations (e.g., intended point mutations:unintended point mutations) that is at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 150:1, at least 200:1, at least 250:1, at least 500:1, or at least 1000:1, or more 
     Details of base editor efficiency are described in International PCT Application Nos. PCT/2017/045381 (WO 2018/027078) and PCT/US2016/058344 (WO 2017/070632), each of which is incorporated herein by reference for its entirety. Also see Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), the entire contents of which are hereby incorporated by reference. 
     In some embodiments, editing of a plurality of nucleobase pairs in one or more genes using the methods provided herein results in formation of at least one intended mutation. In some embodiments, said formation of said at least one intended mutation results in a precise correction of a disease-causing mutation. It should be appreciated that multiplex editing can be accomplished using any method or combination of methods provided herein. 
     Delivery System 
     Nucleic Acid-Based Delivery of a Nucleobase Editors and gRNAs 
     Nucleic acids encoding base editing systems according to the present disclosure can be administered to subjects or delivered into cells in vitro or in vivo by art-known methods or as described herein. In one embodiment, nucleobase editors can be delivered by, e.g., vectors (e.g., viral or non-viral vectors), non-vector based methods (e.g., using naked DNA, DNA complexes, mRNA, lipid nanoparticles), or a combination thereof. 
     Nucleic acids encoding nucleobase editors can be delivered directly to cells (e.g., hematopoietic cells or their progenitors, hematopoietic stem cells, and/or induced pluripotent stem cells) as naked DNA or RNA, e.g., mRNA, for instance by means of transfection or electroporation, or can be conjugated to molecules (e.g., N-acetylgalactosamine) promoting uptake by the target cells. Nucleic acid vectors, such as the vectors described herein can also be used. 
     Nucleic acid vectors can comprise one or more sequences encoding a domain of a fusion protein described herein. A vector can also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization), associated with (e.g., inserted into or fused to) a sequence coding for a protein. As one example, a nucleic acid vectors can include a Cas9 coding sequence that includes one or more nuclear localization sequences (e.g., a nuclear localization sequence from SV40), and an adenosine deaminase variant (e.g., ABE8). 
     The nucleic acid vector can also include any suitable number of regulatory/control elements, e.g., promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences, or internal ribosome entry sites (IRES). These elements are well known in the art. For hematopoietic cells suitable promoters can include IFNbeta or CD45. 
     Nucleic acid vectors according to this disclosure include recombinant viral vectors. Exemplary viral vectors are set forth herein. Other viral vectors known in the art can also be used. In addition, viral particles can be used to deliver base editing system components in nucleic acid and/or peptide form. For example, “empty” viral particles can be assembled to contain any suitable cargo. Viral vectors and viral particles can also be engineered to incorporate targeting ligands to alter target tissue specificity. 
     In addition to viral vectors, non-viral vectors can be used to deliver nucleic acids encoding genome editing systems according to the present disclosure. One important category of non-viral nucleic acid vectors are nanoparticles, which can be organic or inorganic. Nanoparticles are well known in the art. Any suitable nanoparticle design can be used to deliver genome editing system components or nucleic acids encoding such components. For instance, organic (e.g. lipid and/or polymer) nanoparticles can be suitable for use as delivery vehicles in certain embodiments of this disclosure. Exemplary lipids for use in nanoparticle formulations, and/or gene transfer are shown in Table 10 (below). 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Lipids Used for Gene Transfer 
               
            
           
           
               
               
               
            
               
                 Lipid 
                 Abbreviation 
                 Feature 
               
               
                   
               
               
                 1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine 
                 DOPC 
                 Helper 
               
               
                 1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine 
                 DOPE 
                 Helper 
               
               
                 Cholesterol 
                   
                 Helper 
               
               
                 N-[1-(2,3-Dioleyloxy)prophyl]N,N,N-trimethylammonium 
                 DOTMA 
                 Cationic 
               
               
                 chloride 
               
               
                 1,2-Dioleoyloxy-3-trimethylammonium-propane 
                 DOTAP 
                 Cationic 
               
               
                 Dioctadecylamidoglycylspermine 
                 DOGS 
                 Cationic 
               
               
                 N-(3-Aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1- 
                 GAP-DLRIE 
                 Cationic 
               
               
                 propanaminium bromide 
               
               
                 Cetyltrimethylammonium bromide 
                 CTAB 
                 Cationic 
               
               
                 6-Lauroxyhexyl ornithinate 
                 LHON 
                 Cationic 
               
               
                 1-(2,3-Dioleoyloxypropyl)-2,4,6-trimethylpyridinium 
                 2Oc 
                 Cationic 
               
               
                 2,3-Dioleyloxy-N-[2(sperminecarboxamido-ethyl]-N,N- 
                 DOSPA 
                 Cationic 
               
               
                 dimethyl-1-propanaminium trifluoroacetate 
               
               
                 1,2-Dioleyl-3-trimethylammonium-propane 
                 DOPA 
                 Cationic 
               
               
                 N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1- 
                 MDRIE 
                 Cationic 
               
               
                 propanaminium bromide 
               
               
                 Dimyristooxypropyl dimethyl hydroxyethyl ammonium bromide 
                 DMRI 
                 Cationic 
               
               
                 3β-[N-(N′,N′-Dimethylaminoethane)-carbamoyl]cholesterol 
                 DC-Chol 
                 Cationic 
               
               
                 Bis-guanidium-tren-cholesterol 
                 BGTC 
                 Cationic 
               
               
                 1,3-Diodeoxy-2-(6-carboxy-spermyl)-propylamide 
                 DOSPER 
                 Cationic 
               
               
                 Dimethyloctadecylammonium bromide 
                 DDAB 
                 Cationic 
               
               
                 Dioctadecylamidoglicylspermidin 
                 DSL 
                 Cationic 
               
               
                 rac-[(2,3-Dioctadecyloxypropyl)(2-hydroxyethyl)]- 
                 CLIP-1 
                 Cationic 
               
               
                 dimethylammonium chloride 
               
               
                 rac-[2(2,3-Dihexadecyloxypropyl- 
                 CLIP-6 
                 Cationic 
               
               
                 oxymethyloxy)ethyl]trimethylammoniun bromide 
               
               
                 Ethyldimyristoylphosphatidylcholine 
                 EDMPC 
                 Cationic 
               
               
                 1,2-Distearyloxy-N,N-dimethyl-3-aminopropane 
                 DSDMA 
                 Cationic 
               
               
                 1,2-Dimyristoyl-trimethylammonium propane 
                 DMTAP 
                 Cationic 
               
               
                 O,O′-Dimyristyl-N-lysyl aspartate 
                 DMKE 
                 Cationic 
               
               
                 1,2-Distearoyl-sn-glycero-3-ethylpho sphocholine 
                 DSEPC 
                 Cationic 
               
               
                 N-Palmitoyl D-erythro-sphingosyl carbamoyl-spermine 
                 CCS 
                 Cationic 
               
               
                 N-t-Butyl-N0-tetradecyl-3-tetradecylaminopropionamidine 
                 diC14-amidine 
                 Cationic 
               
               
                 Octadecenolyoxy[ethyl-2-heptadecenyl-3 hydroxyethyl] 
                 DOTIM 
                 Cationic 
               
               
                 imidazolinium chloride 
               
               
                 N1-Cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine 
                 CDAN 
                 Cationic 
               
               
                 2-(3-[Bis(3-amino-propyl)-amino]propylamino)-N- 
                 RPR209120 
                 Cationic 
               
               
                 ditetradecylcarbamoylme-ethyl-acetamide 
               
               
                 1,2-dilinoleyloxy-3-dimethylaminopropane 
                 DLinDMA 
                 Cationic 
               
               
                 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane 
                 DLin-KC2-DMA 
                 Cationic 
               
               
                 dilinoleyl-methyl-4-dimethylaminobutyrate 
                 DLin-MC3-DMA 
                 Cationic 
               
               
                   
               
            
           
         
       
     
     Table 11 lists exemplary polymers for use in gene transfer and/or nanoparticle formulations. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Polymers Used for Gene Transfer 
               
            
           
           
               
               
               
            
               
                   
                 Polymer 
                 Abbreviation 
               
               
                   
                   
               
               
                   
                 Poly(ethylene)glycol 
                 PEG 
               
               
                   
                 Polyethylenimine 
                 PEI 
               
               
                   
                 Dithiobis (succinimidylpropionate) 
                 DSP 
               
               
                   
                 Dimethyl-3,3′-dithiobispropionimidate 
                 DTBP 
               
               
                   
                 Poly(ethylene imine)biscarbamate 
                 PEIC 
               
               
                   
                 Poly(L-lysine) 
                 PLL 
               
               
                   
                 Histidine modified PLL 
               
               
                   
                 Poly(N-vinylpyrrolidone) 
                 PVP 
               
               
                   
                 Poly(propylenimine) 
                 PPI 
               
               
                   
                 Poly(amidoamine) 
                 PAMAM 
               
               
                   
                 Poly(amidoethylenimine) 
                 SS-PAEI 
               
               
                   
                 Triethylenetetramine 
                 TETA 
               
               
                   
                 Poly(β-aminoester) 
               
               
                   
                 Poly(4-hydroxy-L-proline ester) 
                 PHP 
               
               
                   
                 Poly(allylamine) 
               
               
                   
                 Poly(α-[4-aminobutyl]-L-glycolic acid) 
                 PAGA 
               
               
                   
                 Poly(D,L-lactic-co-glycolic acid) 
                 PLGA 
               
               
                   
                 Poly(N-ethyl-4-vinylpyridinium bromide) 
               
               
                   
                 Poly(phosphazene)s 
                 PPZ 
               
               
                   
                 Poly(phosphoester)s 
                 PPE 
               
               
                   
                 Poly(phosphoramidate)s 
                 PPA 
               
               
                   
                 Poly(N-2-hydroxypropylmethacrylamide) 
                 pHPMA 
               
               
                   
                 Poly (2-(dimethylamino)ethyl methacrylate) 
                 pDMAEMA 
               
               
                   
                 Poly(2-aminoethyl propylene phosphate) 
                 PPE-EA 
               
               
                   
                 Chitosan 
               
               
                   
                 Galactosylated chitosan 
               
               
                   
                 N-Dodacylated chitosan 
               
               
                   
                 Histone 
               
               
                   
                 Collagen 
               
               
                   
                 Dextran-spermine 
                 D-SPM 
               
               
                   
                   
               
            
           
         
       
     
     Table 12 summarizes delivery methods for a polynucleotide encoding a fusion protein described herein. 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 12 
               
               
                   
               
               
                   
                   
                 Delivery into 
                   
                   
                 Type of 
               
               
                   
                   
                 Non-Dividing 
                 Duration of 
                 Genome 
                 Molecule 
               
               
                 Delivery 
                 Vector/Mode 
                 Cells 
                 Expression 
                 Integration 
                 Delivered 
               
               
                   
               
             
            
               
                 Physical 
                 (e.g., 
                 YES 
                 Transient 
                 NO 
                 Nucleic Acids 
               
               
                   
                 electroporation, 
                   
                   
                   
                 and Proteins 
               
               
                   
                 particle gun, 
               
               
                   
                 Calcium 
               
               
                   
                 Phosphate 
               
               
                   
                 transfection 
               
               
                 Viral 
                 Retrovirus 
                 NO 
                 Stable 
                 YES 
                 RNA 
               
               
                   
                 Lentivirus 
                 YES 
                 Stable 
                 YES/NO with 
                 RNA 
               
               
                   
                   
                   
                   
                 modification 
               
               
                   
                 Adenovirus 
                 YES 
                 Transient 
                 NO 
                 DNA 
               
               
                   
                 Adeno- 
                 YES 
                 Stable 
                 NO 
                 DNA 
               
               
                   
                 Associated 
               
               
                   
                 Virus (AAV) 
               
               
                   
                 Vaccinia Virus 
                 YES 
                 Very 
                 NO 
                 DNA 
               
               
                   
                   
                   
                 Transient 
               
               
                   
                 Herpes Simplex 
                 YES 
                 Stable 
                 NO 
                 DNA 
               
               
                   
                 Virus 
               
               
                 Non-Viral 
                 Cationic 
                 YES 
                 Transient 
                 Depends on 
                 Nucleic Acids 
               
               
                   
                 Liposomes 
                   
                   
                 what is 
                 and Proteins 
               
               
                   
                   
                   
                   
                 delivered 
               
               
                   
                 Polymeric 
                 YES 
                 Transient 
                 Depends on 
                 Nucleic Acids 
               
               
                   
                 Nanoparticles 
                   
                   
                 what is 
                 and Proteins 
               
               
                   
                   
                   
                   
                 delivered 
               
               
                 Biological 
                 Attenuated 
                 YES 
                 Transient 
                 NO 
                 Nucleic Acids 
               
               
                 Non-Viral 
                 Bacteria 
               
               
                 Delivery 
                 Engineered 
                 YES 
                 Transient 
                 NO 
                 Nucleic Acids 
               
               
                 Vehicles 
                 Bacteriophages 
                 YES 
                 Transient 
                 NO 
                 Nucleic Acids 
               
               
                   
                 Mammalian 
               
               
                   
                 Virus-like 
               
               
                   
                 Particles 
               
               
                   
                 Biological 
                 YES 
                 Transient 
                 NO 
                 Nucleic Acids 
               
               
                   
                 liposomes: 
               
               
                   
                 Erythrocyte 
               
               
                   
                 Ghosts and 
               
               
                   
                 Exosomes 
               
               
                   
               
            
           
         
       
     
     In another aspect, the delivery of genome editing system components or nucleic acids encoding such components, for example, a nucleic acid binding protein such as, for example, Cas9 or variants thereof, and a gRNA targeting a genomic nucleic acid sequence of interest, may be accomplished by delivering a ribonucleoprotein (RNP) to cells. The RNP comprises the nucleic acid binding protein, e.g., Cas9, in complex with the targeting gRNA. RNPs may be delivered to cells using known methods, such as electroporation, nucleofection, or cationic lipid-mediated methods, for example, as reported by Zuris, J. A. et al., 2015,  Nat. Biotechnology,  33(1):73-80. RNPs are advantageous for use in CRISPR base editing systems, particularly for cells that are difficult to transfect, such as primary cells. In addition, RNPs can also alleviate difficulties that may occur with protein expression in cells, especially when eukaryotic promoters, e.g., CMV or EF1A, which may be used in CRISPR plasmids, are not well-expressed. Advantageously, the use of RNPs does not require the delivery of foreign DNA into cells. Moreover, because an RNP comprising a nucleic acid binding protein and gRNA complex is degraded over time, the use of RNPs has the potential to limit off-target effects. In a manner similar to that for plasmid based techniques, RNPs can be used to deliver binding protein (e.g., Cas9 variants) and to direct homology directed repair (HDR). 
     A promoter used to drive base editor coding nucleic acid molecule expression can include AAV ITR. This can be advantageous for eliminating the need for an additional promoter element, which can take up space in the vector. The additional space freed up can be used to drive the expression of additional elements, such as a guide nucleic acid or a selectable marker. ITR activity is relatively weak, so it can be used to reduce potential toxicity due to over expression of the chosen nuclease. 
     Any suitable promoter can be used to drive expression of the base editor and, where appropriate, the guide nucleic acid. For ubiquitous expression, promoters that can be used include CMV, CAG, CBh, PGK, SV40, Ferritin heavy or light chains, etc. For brain or other CNS cell expression, suitable promoters can include: SynapsinI for all neurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. For liver cell expression, suitable promoters include the Albumin promoter. For lung cell expression, suitable promoters can include SP-B. For endothelial cells, suitable promoters can include ICAM. For hematopoietic cells suitable promoters can include IFNbeta or CD45. For Osteoblasts suitable promoters can include OG-2. 
     In some embodiments, a base editor of the present disclosure is of small enough size to allow separate promoters to drive expression of the base editor and a compatible guide nucleic acid within the same nucleic acid molecule. For instance, a vector or viral vector can comprise a first promoter operably linked to a nucleic acid encoding the base editor and a second promoter operably linked to the guide nucleic acid. 
     The promoter used to drive expression of a guide nucleic acid can include: Pol III promoters such as U6 or H1 Use of Pol II promoter and intronic cassettes to express gRNA Adeno Associated Virus (AAV). 
     Viral Vectors 
     A base editor described herein can therefore be delivered with viral vectors. In some embodiments, a base editor disclosed herein can be encoded on a nucleic acid that is contained in a viral vector. In some embodiments, one or more components of the base editor system can be encoded on one or more viral vectors. For example, a base editor and guide nucleic acid can be encoded on a single viral vector. In other embodiments, the base editor and guide nucleic acid are encoded on different viral vectors. In either case, the base editor and guide nucleic acid can each be operably linked to a promoter and terminator. The combination of components encoded on a viral vector can be determined by the cargo size constraints of the chosen viral vector. 
     The use of RNA or DNA viral based systems for the delivery of a base editor takes advantage of highly evolved processes for targeting a virus to specific cells in culture or in the host and trafficking the viral payload to the nucleus or host cell genome. Viral vectors can be administered directly to cells in culture, patients (in vivo), or they can be used to treat cells in vitro, and the modified cells can optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues. 
     Viral vectors can include lentivirus (e.g., HIV and FIV-based vectors), Adenovirus (e.g., AD100), Retrovirus (e.g., Maloney murine leukemia virus, MML-V), herpesvirus vectors (e.g., HSV-2), and Adeno-associated viruses (AAVs), or other plasmid or viral vector types, in particular, using formulations and doses from, for example, U.S. Pat. No. 8,454,972 (formulations, doses for adenovirus), U.S. Pat. No. 8,404,658 (formulations, doses for AAV) and U.S. Pat. No. 5,846,946 (formulations, doses for DNA plasmids) and from clinical trials and publications regarding the clinical trials involving lentivirus, AAV and adenovirus. For example, for AAV, the route of administration, formulation and dose can be as in U.S. Pat. No. 8,454,972 and as in clinical trials involving AAV. For Adenovirus, the route of administration, formulation and dose can be as in U.S. Pat. No. 8,404,658 and as in clinical trials involving adenovirus. For plasmid delivery, the route of administration, formulation and dose can be as in U.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids. Doses can be based on or extrapolated to an average 70 kg individual (e.g. a male adult human), and can be adjusted for patients, subjects, mammals of different weight and species. Frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed. The viral vectors can be injected into the tissue of interest. For cell-type specific base editing, the expression of the base editor and optional guide nucleic acid can be driven by a cell-type specific promoter. 
     The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (See, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700). 
     Retroviral vectors, especially lentiviral vectors, can require polynucleotide sequences smaller than a given length for efficient integration into a target cell. For example, retroviral vectors of length greater than 9 kb can result in low viral titers compared with those of smaller size. In some aspects, a base editor of the present disclosure is of sufficient size so as to enable efficient packaging and delivery into a target cell via a retroviral vector. In some embodiments, a base editor is of a size so as to allow efficient packing and delivery even when expressed together with a guide nucleic acid and/or other components of a targetable nuclease system. 
     In applications where transient expression is preferred, adenoviral based systems can be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus (“AAV”) vectors can also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (See, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). The construction of recombinant AAV vectors is described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &amp; Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989). 
     AAV is a small, single-stranded DNA dependent virus belonging to the parvovirus family. The 4.7 kb wild-type (wt) AAV genome is made up of two genes that encode four replication proteins and three capsid proteins, respectively, and is flanked on either side by 145-bp inverted terminal repeats (ITRs). The virion is composed of three capsid proteins, Vp1, Vp2, and Vp3, produced in a 1:1:10 ratio from the same open reading frame but from differential splicing (Vp1) and alternative translational start sites (Vp2 and Vp3, respectively). Vp3 is the most abundant subunit in the virion and participates in receptor recognition at the cell surface defining the tropism of the virus. A phospholipase domain, which functions in viral infectivity, has been identified in the unique N terminus of Vp1. 
     Similar to wt AAV, recombinant AAV (rAAV) utilizes the cis-acting 145-bp ITRs to flank vector transgene cassettes, providing up to 4.5 kb for packaging of foreign DNA. Subsequent to infection, rAAV can express a fusion protein of the invention and persist without integration into the host genome by existing episomally in circular head-to-tail concatemers. Although there are numerous examples of rAAV success using this system, in vitro and in vivo, the limited packaging capacity has limited the use of AAV-mediated gene delivery when the length of the coding sequence of the gene is equal or greater in size than the wt AAV genome. 
     Viral vectors can be selected based on the application. For example, for in vivo gene delivery, AAV can be advantageous over other viral vectors. In some embodiments, AAV allows low toxicity, which can be due to the purification method not requiring ultra-centrifugation of cell particles that can activate the immune response. In some embodiments, AAV allows low probability of causing insertional mutagenesis because it doesn&#39;t integrate into the host genome. Adenoviruses are commonly used as vaccines because of the strong immunogenic response they induce. Packaging capacity of the viral vectors can limit the size of the base editor that can be packaged into the vector. 
     AAV has a packaging capacity of about 4.5 Kb or 4.75 Kb including two 145 base inverted terminal repeats (ITRs). This means disclosed base editor as well as a promoter and transcription terminator can fit into a single viral vector. Constructs larger than 4.5 or 4.75 Kb can lead to significantly reduced virus production. For example, SpCas9 is quite large, the gene itself is over 4.1 Kb, which makes it difficult for packing into AAV. Therefore, embodiments of the present disclosure include utilizing a disclosed base editor which is shorter in length than conventional base editors. In some examples, the base editors are less than 4 kb. Disclosed base editors can be less than 4.5 kb, 4.4 kb, 4.3 kb, 4.2 kb, 4.1 kb, 4 kb, 3.9 kb, 3.8 kb, 3.7 kb, 3.6 kb, 3.5 kb, 3.4 kb, 3.3 kb, 3.2 kb, 3.1 kb, 3 kb, 2.9 kb, 2.8 kb, 2.7 kb, 2.6 kb, 2.5 kb, 2 kb, or 1.5 kb. In some embodiments, the disclosed base editors are 4.5 kb or less in length. 
     An AAV can be AAV1, AAV2, AAV5 or any combination thereof. One can select the type of AAV with regard to the cells to be targeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combination thereof for targeting brain or neuronal cells; and one can select AAV4 for targeting cardiac tissue. AAV8 is useful for delivery to the liver. A tabulation of certain AAV serotypes as to these cells can be found in Grimm, D. et al, J. Virol. 82: 5887-5911 (2008)). 
     Lentiviruses are complex retroviruses that have the ability to infect and express their genes in both mitotic and post-mitotic cells. The most commonly known lentivirus is the human immunodeficiency virus (HIV), which uses the envelope glycoproteins of other viruses to target a broad range of cell types. 
     Lentiviruses can be prepared as follows. After cloning pCasES10 (which contains a lentiviral transfer plasmid backbone), HEK293FT at low passage (p=5) were seeded in a T-75 flask to 50% confluence the day before transfection in DMEM with 10% fetal bovine serum and without antibiotics. After 20 hours, media is changed to OptiMEM (serum-free) media and transfection was done 4 hours later. Cells are transfected with 10 μg of lentiviral transfer plasmid (pCasES10) and the following packaging plasmids: 5 μg of pMD2.G (VSV-g pseudotype), and 7.5 μg of psPAX2 (gag/pol/rev/tat). Transfection can be done in 4 mL OptiMEM with a cationic lipid delivery agent (50 μl Lipofectamine 2000 and 100 ul Plus reagent). After 6 hours, the media is changed to antibiotic-free DMEM with 10% fetal bovine serum. These methods use serum during cell culture, but serum-free methods are preferred. 
     Lentivirus can be purified as follows. Viral supernatants are harvested after 48 hours. Supernatants are first cleared of debris and filtered through a 0.45 μm low protein binding (PVDF) filter. They are then spun in an ultracentrifuge for 2 hours at 24,000 rpm. Viral pellets are resuspended in 50 μl of DMEM overnight at 4° C. They are then aliquoted and immediately frozen at −80° C. 
     In another embodiment, minimal non-primate lentiviral vectors based on the equine infectious anemia virus (EIAV) are also contemplated. In another embodiment, RetinoStat®, an equine infectious anemia virus-based lentiviral gene therapy vector that expresses angiostatic proteins endostatin and angiostatin that is contemplated to be delivered via a subretinal injection. In another embodiment, use of a self-inactivating lentiviral vector is contemplated. 
     Any RNA of the systems, for example a guide RNA or a base editor-encoding mRNA, can be delivered in the form of RNA. Base editor-encoding mRNA can be generated using in vitro transcription. For example, nuclease mRNA can be synthesized using a PCR cassette containing the following elements: T7 promoter, optional kozak sequence (GCCACC), nuclease sequence, and 3′ UTR such as a 3′ UTR from beta globin-polyA tail. The cassette can be used for transcription by T7 polymerase. Guide polynucleotides (e.g., gRNA) can also be transcribed using in vitro transcription from a cassette containing a T7 promoter, followed by the sequence “GG”, and guide polynucleotide sequence. 
     To enhance expression and reduce possible toxicity, the base editor-coding sequence and/or the guide nucleic acid can be modified to include one or more modified nucleoside e.g. using pseudo-U or 5-Methyl-C. 
     The small packaging capacity of AAV vectors makes the delivery of a number of genes that exceed this size and/or the use of large physiological regulatory elements challenging. These challenges can be addressed, for example, by dividing the protein(s) to be delivered into two or more fragments, wherein the N-terminal fragment is fused to a split intein-N and the C-terminal fragment is fused to a split intein-C. These fragments are then packaged into two or more AAV vectors. As used herein, “intein” refers to a self-splicing protein intron (e.g., peptide) that ligates flanking N-terminal and C-terminal exteins (e.g., fragments to be joined). The use of certain inteins for joining heterologous protein fragments is described, for example, in Wood et al., J. Biol. Chem. 289(21); 14512-9 (2014). For example, when fused to separate protein fragments, the inteins IntN and IntC recognize each other, splice themselves out and simultaneously ligate the flanking N- and C-terminal exteins of the protein fragments to which they were fused, thereby reconstituting a full-length protein from the two protein fragments. Other suitable inteins will be apparent to a person of skill in the art. 
     A fragment of a fusion protein of the invention can vary in length. In some embodiments, a protein fragment ranges from 2 amino acids to about 1000 amino acids in length. In some embodiments, a protein fragment ranges from about 5 amino acids to about 500 amino acids in length. In some embodiments, a protein fragment ranges from about 20 amino acids to about 200 amino acids in length. In some embodiments, a protein fragment ranges from about 10 amino acids to about 100 amino acids in length. Suitable protein fragments of other lengths will be apparent to a person of skill in the art. 
     In one embodiment, dual AAV vectors are generated by splitting a large transgene expression cassette in two separate halves (5′ and 3′ ends, or head and tail), where each half of the cassette is packaged in a single AAV vector (of &lt;5 kb). The re-assembly of the full-length transgene expression cassette is then achieved upon co-infection of the same cell by both dual AAV vectors followed by: (1) homologous recombination (HR) between 5′ and 3′ genomes (dual AAV overlapping vectors); (2) ITR-mediated tail-to-head concatemerization of 5′ and 3′ genomes (dual AAV trans-splicing vectors); or (3) a combination of these two mechanisms (dual AAV hybrid vectors). The use of dual AAV vectors in vivo results in the expression of full-length proteins. The use of the dual AAV vector platform represents an efficient and viable gene transfer strategy for transgenes of &gt;4.7 kb in size. 
     Inteins 
     In some embodiments, a portion or fragment of a nuclease (e.g., Cas9) is fused to an intein. The nuclease can be fused to the N-terminus or the C-terminus of the intein. In some embodiments, a portion or fragment of a fusion protein is fused to an intein and fused to an AAV capsid protein. The intein, nuclease and capsid protein can be fused together in any arrangement (e.g., nuclease-intein-capsid, intein-nuclease-capsid, capsid-intein-nuclease, etc.). In some embodiments, the N-terminus of an intein is fused to the C-terminus of a fusion protein and the C-terminus of the intein is fused to the N-terminus of an AAV capsid protein. 
     Inteins (intervening protein) are auto-processing domains found in a variety of diverse organisms, which carry out a process known as protein splicing. Protein splicing is a multi-step biochemical reaction comprised of both the cleavage and formation of peptide bonds. While the endogenous substrates of protein splicing are proteins found in intein-containing organisms, inteins can also be used to chemically manipulate virtually any polypeptide backbone. 
     In protein splicing, the intein excises itself out of a precursor polypeptide by cleaving two peptide bonds, thereby ligating the flanking extein (external protein) sequences via the formation of a new peptide bond. This rearrangement occurs post-translationally (or possibly co-translationally). Intein-mediated protein splicing occurs spontaneously, requiring only the folding of the intein domain. 
     About 5% of inteins are split inteins, which are transcribed and translated as two separate polypeptides, the N-intein and C-intein, each fused to one extein. Upon translation, the intein fragments spontaneously and non-covalently assemble into the canonical intein structure to carry out protein splicing in trans. The mechanism of protein splicing entails a series of acyl-transfer reactions that result in the cleavage of two peptide bonds at the intein-extein junctions and the formation of a new peptide bond between the N- and C-exteins. This process is initiated by activation of the peptide bond joining the N-extein and the N-terminus of the intein. Virtually all inteins have a cysteine or serine at their N-terminus that attacks the carbonyl carbon of the C-terminal N-extein residue. This N to O/S acyl-shift is facilitated by a conserved threonine and histidine (referred to as the TXXH motif), along with a commonly found aspartate, which results in the formation of a linear (thio)ester intermediate. Next, this intermediate is subject to trans-(thio)esterification by nucleophilic attack of the first C-extein residue (+1), which is a cysteine, serine, or threonine. The resulting branched (thio)ester intermediate is resolved through a unique transformation: cyclization of the highly conserved C-terminal asparagine of the intein. This process is facilitated by the histidine (found in a highly conserved HNF motif) and the penultimate histidine and may also involve the aspartate. This succinimide formation reaction excises the intein from the reactive complex and leaves behind the exteins attached through a non-peptidic linkage. This structure rapidly rearranges into a stable peptide bond in an intein-independent fashion. 
     In some embodiments, an N-terminal fragment of a base editor (e.g., ABE, CBE) is fused to a split intein-N and a C-terminal fragment is fused to a split intein-C. These fragments are then packaged into two or more AAV vectors. The use of certain inteins for joining heterologous protein fragments is described, for example, in Wood et al., J. Biol. Chem. 289(21); 14512-9 (2014). For example, when fused to separate protein fragments, the inteins IntN and IntC recognize each other, splice themselves out and simultaneously ligate the flanking N- and C-terminal exteins of the protein fragments to which they were fused, thereby reconstituting a full-length protein from the two protein fragments. Other suitable inteins will be apparent to a person of skill in the art. 
     In some embodiments, an ABE was split into N- and C-terminal fragments at Ala, Ser, Thr, or Cys residues within selected regions of SpCas9. These regions correspond to loop regions identified by Cas9 crystal structure analysis. The N-terminus of each fragment is fused to an intein-N and the C-terminus of each fragment is fused to an intein C at amino acid positions S303, T310, T313, S355, A456, S460, A463, T466, S469, T472, T474, C574, S577, A589, and S590, which are indicated in Bold Capitals in the sequence below. 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 1) 
                   
               
            
           
           
               
               
               
            
               
                    1  
                 mdkkysigld igtnsvgwav itdeykvpsk kfkvlgntdr hsikknliga llfdsgetae 
                   
               
               
                   
               
               
                   61  
                 atrlkrtarr rytrrknric ylqeifsnem akvddsffhr leesflveed kkherhpifg 
               
               
                   
               
               
                  121  
                 nivdevayhe kyptiyhlrk klvdstdkad lrliylalah mikfrghfli egdlnpdnsd 
               
               
                   
               
               
                  181  
                 vdklfiqlvq tynqlfeenp inasgvdaka ilsarlsksr rlenliaqlp gekknglfgn 
               
               
                   
               
               
                  241  
                 lialslgltp nfksnfdlae daklqlskdt ydddldnlla qigdqyadlf laaknlsdai 
               
               
                   
               
               
                  301  
                 ll S dilrvn T  ei T kaplsas mikrydehhq dltllkalvr qqlpekykei ffdq S kngya 
               
               
                   
               
               
                  361 
                 gyidggasqe efykfikpil ekmdgteell vklnredllr kqrtfdngsi phqihlgelh 
               
               
                   
               
               
                  421 
                 ailrrqedfy pflkdnreki ekiltfripy yvgpl A rgn S  rf A wm T rk S e e T i T pwnfee 
               
               
                   
               
               
                  481 
                 vvdkgasaqs fiermtnfdk nlpnekvlpk hsllyeyftv yneltkvkyv tegmrkpafl 
               
               
                   
               
               
                  541 
                 sgeqkkaivd llfktnrkvt vkqlkedyfk kie C fd S vei sgvedrfn AS  lgtyhdllki 
               
               
                   
               
               
                  601 
                 ikdkdfldne enedilediv ltltlfedre mieerlktya hlfddkvmkq lkrrrytgwg 
               
               
                   
               
               
                  661 
                 rlsrklingi rdkqsgktil dflksdgfan rnfmqlihdd sltfkediqk aqvsgqgdsl 
               
               
                   
               
               
                  721 
                 hehianlags paikkgilqt vkvvdelvkv mgrhkpeniv iemarenqtt qkgqknsrer 
               
               
                   
               
               
                  781 
                 mkrieegike lgsqilkehp ventqlqnek lylyylqngr dmyvdqeldi nrlsdydvdh 
               
               
                   
               
               
                  841 
                 ivpqsflkdd sidnkvltrs dknrgksdnv pseevvkkmk nywrqllnak litqrkfdnl 
               
               
                   
               
               
                  901 
                 tkaergglse ldkagfikrq lvetrqitkh vaqildsrmn tkydendkli revkvitlks 
               
               
                   
               
               
                  961 
                 klvsdfrkdf qfykvreinn yhhahdayln avvgtalikk ypklesefvy gdykvydvrk 
               
               
                   
               
               
                 1021  
                 miakseqeig katakyffys nimnffktei tlangeirkr plietngetg eivwdkgrdf 
               
               
                   
               
               
                 1081  
                 atvrkvlsmp qvnivkktev qtggfskesi lpkrnsdkli arkkdwdpkk yggfdsptva 
               
               
                   
               
               
                 1141 
                 ysvlvvakve kgkskklksv kellgitime rssfeknpid fleakgykev kkdliiklpk 
               
               
                   
               
               
                 1201 
                 yslfelengr krmlasagel qkgnelalps kyvnflylas hyeklkgspe dneqkqlfve 
               
               
                   
               
               
                 1261 
                 qhkhyldeii eqisefskrv iladanldkv lsaynkhrdk pireqaenii hlftltnlga 
               
               
                   
               
               
                 1321 
                 paafkyfdtt idrkrytstk evldatlihq sitglyetri dlsqlggd 
               
            
           
         
       
     
     Use of Nucleobase Editors to Target Mutations 
     The suitability of nucleobase editors that targets a mutation is evaluated as described herein. In one embodiment, a single cell of interest is transduced with a base editing system together with a small amount of a vector encoding a reporter (e.g., GFP). These cells can be any cell line known in the art, including immortalized human cell lines, such as 293T, K562 or U20S. Alternatively, primary cells (e.g., human) may be used. Such cells may be relevant to the eventual cell target. 
     Delivery may be performed using a viral vector. In one embodiment, transfection may be performed using lipid transfection (such as Lipofectamine or Fugene) or by electroporation. Following transfection, expression of GFP can be determined either by fluorescence microscopy or by flow cytometry to confirm consistent and high levels of transfection. These preliminary transfections can comprise different nucleobase editors to determine which combinations of editors give the greatest activity. 
     The activity of the nucleobase editor is assessed as described herein, i.e., by sequencing the genome of the cells to detect alterations in a target sequence. For Sanger sequencing, purified PCR amplicons are cloned into a plasmid backbone, transformed, miniprepped and sequenced with a single primer. Sequencing may also be performed using next generation sequencing techniques. When using next generation sequencing, amplicons may be 300-500 bp with the intended cut site placed asymmetrically. Following PCR, next generation sequencing adapters and barcodes (for example Illumina multiplex adapters and indexes) may be added to the ends of the amplicon, e.g., for use in high throughput sequencing (for example on an Illumina MiSeq). 
     The fusion proteins that induce the greatest levels of target specific alterations in initial tests can be selected for further evaluation. 
     In particular embodiments, the nucleobase editors are used to target polynucleotides of interest. In one embodiment, a nucleobase editor of the invention is delivered to cells (e.g., hematopoietic cells or their progenitors, hematopoietic stem cells, and/or induced pluripotent stem cells) in conjunction with a guide RNA that is used to target a mutation of interest within the genome of a cell, thereby altering the mutation. In some embodiments, a base editor is targeted by a guide RNA to introduce one or more edits to the sequence of a gene of interest. 
     In one embodiment, a nucleobase editor is used to target a regulatory sequence, including but not limited to splice sites, enhancers, and transcriptional regulatory elements. The effect of the alteration on the expression of a gene controlled by the regulatory element is then assayed using any method known in the art. 
     In still other embodiments, a nucleobase editor of the invention is used to target polynucleotides of interest within the genome of an organism. In one embodiment, a nucleobase editor of the invention is delivered to cells in conjunction with a library of guide RNAs that are used to tile a variety of sequences within the genome of a cell, thereby systematically altering sequences throughout the genome. 
     The system can comprise one or more different vectors. In an aspect, the base editor is codon optimized for expression the desired cell type, preferentially a eukaryotic cell, preferably a mammalian cell or a human cell. 
     In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/(visited Jul. 9, 2002), and these tables can be adapted in a number of ways. See, Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding an engineered nuclease correspond to the most frequently used codon for a particular amino acid. 
     Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and psi.2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA can be packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line can also be infected with adenovirus as a helper. The helper virus can promote replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid in some cases is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. 
     Applications for Multi-Effector Nucleobase Editors 
     The multi-effector nucleobase editors can be used to target polynucleotides of interest to create alterations that modify protein expression. In one embodiment, a multi-effector nucleobase editor is used to modify a non-coding or regulatory sequence, including but not limited to splice sites, enhancers, and transcriptional regulatory elements. The effect of the alteration on the expression of a gene controlled by the regulatory element is then assayed using any method known in the art. In a particular embodiment, a multi-effector nucleobase editor is able to substantially alter a regulatory sequence, thereby abolishing its ability to regulate gene expression. Advantageously, this can be done without generating double-stranded breaks in the genomic target sequence, in contrast to other RNA-programmable nucleases. 
     The multi-effector nucleobase editors can be used to target polynucleotides of interest to create alterations that modify protein activity. In the context of mutagenesis, for example, multi-effector nucleobase editors have a number of advantages over error-prone PCR and other polymerase-based methods. Because multi-effector nucleobase editors of the invention create alterations at multiple bases in a target region, such mutations are more likely to be expressed at the protein level relative to mutations introduced by error-prone PCR, which are less likely to be expressed at the protein level given that a single nucleotide change in a codon may still encode the same amino acid (e.g., codon degeneracy). Unlike error-prone PCR, which induces random alterations throughout a polynucleotide, multi-effector nucleobase editors of the invention can be used to target specific amino acids within a small or defined region of a protein of interest. 
     In other embodiments, a multi-effector nucleobase editor of the invention is used to target a polynucleotide of interest within the genome of an organism. In one embodiment, the organism is a bacteria of the microbiome (e.g., Bacteriodetes, Verrucomicrobia, Firmicutes; Gammaproteobacteria, Alphaproteobacteria, Bacteriodetes, Clostridia, Erysipelotrichia, Bacilli; Enterobacteriales, Bacteriodales, Verrucomicrobiales, Clostridiales, Erysiopelotrichales, Lactobacillales; Enterobacteriaceae, Bacteroidaceae, Erysiopelotrichaceae, Prevotellaceae, Coriobacteriaceae, and Alcaligenaceae,  Escherichia, Bacteroides, Alistipes, Akkermansia, Clostridium, Lactobacillus ). In another embodiment, the organism is an agriculturally important animal (e.g., cow, sheep, goat, horse, chicken, turkey) or plant (e.g., soybeans, wheat, corn, rice, tobacco, apples, grapes, peaches, plums, cherries). In one embodiment, a multi-effector nucleobase editor of the invention is delivered to cells in conjunction with a library of guide RNAs that are used to tile a variety of sequences within the genome of a cell, thereby systematically altering sequences throughout the genome. 
     Mutations may be made in any of a variety of proteins to facilitate structure function analysis or to alter the endogenous activity of the protein. Mutations may be made, for example, in an enzyme (e.g., kinase, phosphatase, carboxylase, phosphodiesterase) or in an enzyme substrate, in a receptor or in its ligand, and in an antibody and its antigen. In one embodiment, a multi-effector nucleobase editor targets a nucleic acid molecule encoding the active site of the enzyme, the ligand binding site of a receptor, or a complementarity determining region (CDR) of an antibody. In the case of an enzyme, inducing mutations in the active site could increase, decrease, or abolish the enzyme&#39;s activity. The effect of mutations on the enzyme is characterized in an enzyme activity assay, including any of a number of assays known in the art and/or that would be apparent to the skilled artisan. In the case of a receptor, mutations made at the ligand binding site could increase, decrease or abolish the receptors affinity for its ligand. The effect of such mutations is assayed in a receptor/ligand binding assay, including any of a number of assays known in the art and/or that would be apparent to the skilled artisan. 
     Pharmaceutical Compositions 
     Other aspects of the present disclosure relate to pharmaceutical compositions comprising any of the base editors, fusion proteins, or the fusion protein-guide polynucleotide complexes described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g., for specific delivery, increasing half-life, or other therapeutic compounds). 
     Suitable pharmaceutically acceptable carriers generally comprise inert substances that aid in administering the pharmaceutical composition to a subject, aid in processing the pharmaceutical compositions into deliverable preparations, or aid in storing the pharmaceutical composition prior to administration. Pharmaceutically acceptable carriers can include agents that can stabilize, optimize or otherwise alter the form, consistency, viscosity, pH, pharmacokinetics, solubility of the formulation. 
     Some nonlimiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer&#39;s solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Buffering agents, wetting agents, emulsifying agents, diluents, encapsulating agents, skin penetration enhancers, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. For example, carriers can include, but are not limited to, saline, buffered saline, dextrose, arginine, sucrose, water, glycerol, ethanol, sorbitol, dextran, sodium carboxymethyl cellulose, and combinations thereof. 
     Pharmaceutical compositions can comprise one or more pH buffering compounds to maintain the pH of the formulation at a predetermined level that reflects physiological pH, such as in the range of about 5.0 to about 8.0. The pH buffering compound used in the aqueous liquid formulation can be an amino acid or mixture of amino acids, such as histidine or a mixture of amino acids such as histidine and glycine. Alternatively, the pH buffering compound is preferably an agent which maintains the pH of the formulation at a predetermined level, such as in the range of about 5.0 to about 8.0, and which does not chelate calcium ions. Illustrative examples of such pH buffering compounds include, but are not limited to, imidazole and acetate ions. The pH buffering compound may be present in any amount suitable to maintain the pH of the formulation at a predetermined level. 
     Pharmaceutical compositions can also contain one or more osmotic modulating agents, i.e., a compound that modulates the osmotic properties (e.g., tonicity, osmolality, and/or osmotic pressure) of the formulation to a level that is acceptable to the blood stream and blood cells of recipient individuals. The osmotic modulating agent can be an agent that does not chelate calcium ions. The osmotic modulating agent can be any compound known or available to those skilled in the art that modulates the osmotic properties of the formulation. One skilled in the art may empirically determine the suitability of a given osmotic modulating agent for use in the inventive formulation. Illustrative examples of suitable types of osmotic modulating agents include, but are not limited to: salts, such as sodium chloride and sodium acetate; sugars, such as sucrose, dextrose, and mannitol; amino acids, such as glycine; and mixtures of one or more of these agents and/or types of agents. The osmotic modulating agent(s) may be present in any concentration sufficient to modulate the osmotic properties of the formulation. 
     In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, e.g., for gene editing. In some embodiments, administration of the pharmaceutical compositions contemplated herein may be carried out using conventional techniques including, but not limited to, infusion, transfusion, or parenterally. In some embodiments, parenteral administration includes infusing or injecting intravascularly, intravenously, intramuscularly, intraarterially, intrathecally, intratumorally, intradermally, intraperitoneally, transtracheally, subcutaneously, subcuticularly, intraarticularly, subcapsularly, subarachnoidly and intrasternally. In some embodiments, suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration. 
     In some embodiments, the pharmaceutical composition described herein is administered locally to a diseased site (e.g., tumor site). In some embodiments, the pharmaceutical composition described herein is administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber. 
     In other embodiments, the pharmaceutical composition described herein is delivered in a controlled release system. In one embodiment, a pump can be used (see, e.g., Langer, 1990, Science 249: 1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al, 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used. (See, e.g., Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25:351; Howard et ah, 1989, J. Neurosurg. 71: 105.) Other controlled release systems are discussed, for example, in Langer, supra. 
     In some embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human. In some embodiments, pharmaceutical composition for administration by injection are solutions in sterile isotonic use as solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration. 
     A pharmaceutical composition for systemic administration can be a liquid, e.g., sterile saline, lactated Ringer&#39;s or Hank&#39;s solution. In addition, the pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated. The pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration. The particles can be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein. Compounds can be entrapped in “stabilized plasmid-lipid particles” (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol %) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating (Zhang Y. P. et ah, Gene Ther. 1999, 6: 1438-47). Positively charged lipids such as N-[1-(2,3-dioleoyloxi)propyl]-N,N,N-trimethyl-amoniummethylsulfate, or “DOTAP,” are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g., U.S. Pat. Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757; each of which is incorporated herein by reference. 
     The pharmaceutical composition described herein can be administered or packaged as a unit dose, for example. The term “unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle. 
     Further, the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a compound of the invention in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile used for reconstitution or dilution of the lyophilized compound of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. 
     In another aspect, an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease described herein and can have a sterile access port. For example, the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The active agent in the composition is a compound of the invention. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer&#39;s solution, or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. 
     In some embodiments, any of the fusion proteins, gRNAs, and/or complexes described herein are provided as part of a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises any of the fusion proteins provided herein. In some embodiments, the pharmaceutical composition comprises any of the complexes provided herein. In some embodiments, the pharmaceutical composition comprises a ribonucleoprotein complex comprising an RNA-guided nuclease (e.g., Cas9) that forms a complex with a gRNA and a cationic lipid. In some embodiments pharmaceutical composition comprises a gRNA, a nucleic acid programmable DNA binding protein, a cationic lipid, and a pharmaceutically acceptable excipient. Pharmaceutical compositions can optionally comprise one or more additional therapeutically active substances. 
     In some embodiments, compositions provided herein are administered to a subject, for example, to a human subject, in order to effect a targeted genomic modification within the subject. In some embodiments, cells are obtained from the subject and contacted with any of the pharmaceutical compositions provided herein. In some embodiments, cells removed from a subject and contacted ex vivo with a pharmaceutical composition are re-introduced into the subject, optionally after the desired genomic modification has been effected or detected in the cells. Methods of delivering pharmaceutical compositions comprising nucleases are known, and are described, for example, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of which are incorporated by reference herein in their entireties. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals or organisms of all sorts, for example, for veterinary use. 
     Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, domesticated animals, pets, and commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys. 
     Formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient(s) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit. Pharmaceutical formulations can additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington&#39;s The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams &amp; Wilkins, Baltimore, Md., 2006; incorporated in its entirety herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. See also PCT application PCT/US2010/055131 (Publication number WO2011/053982 A8, filed Nov. 2, 2010), incorporated in its entirety herein by reference, for additional suitable methods, reagents, excipients and solvents for producing pharmaceutical compositions comprising a nuclease. 
     Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure. 
     The compositions, as described above, can be administered in effective amounts. The effective amount will depend upon the mode of administration, the particular condition being treated, and the desired outcome. It may also depend upon the stage of the condition, the age and physical condition of the subject, the nature of concurrent therapy, if any, and like factors well-known to the medical practitioner. For therapeutic applications, it is that amount sufficient to achieve a medically desirable result. 
     In some embodiments, compositions in accordance with the present disclosure can be used for treatment of any of a variety of diseases, disorders, and/or conditions. 
     Methods of Treatment 
     Provided also are methods of treating a disease or condition and/or the genetic mutations that cause the disease or condition. These methods comprise administering to a subject (e.g., a mammal, such as a human) a therapeutically effective amount of a pharmaceutical composition that comprises a polynucleotide encoding a base editor system (e.g., base editor and gRNA) described herein. In some embodiments, the base editor is a fusion protein that comprises a napDNAbp domain and an adenosine deaminase domain or a cytidine deaminase domain. A cell of the subject is transduced with the base editor and one or more guide polynucleotides that target the base editor to effect an A•T to G•C alteration (if the cell is transduced with an adenosine deaminase domain) or a C•G to U•A alteration (if the cell is transduced with a cytidine deaminase domain) of a nucleic acid sequence containing mutations in a gene of interest. 
     The methods herein include administering to the subject (including a subject identified as being in need of such treatment, or a subject suspected of being at risk of disease and in need of such treatment) an effective amount of a composition described herein. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method). 
     The therapeutic methods, in general, comprise administration of a therapeutically effective amount of a pharmaceutical composition comprising, for example, a vector encoding a base editor and a gRNA that targets the gene of interest of a subject (e.g., a human patient) in need thereof. Such treatment will be suitably administered to a subject, particularly a human subject, suffering from, having, susceptible to, or at risk for the disease or condition. 
     In one embodiment, the invention provides a method of monitoring treatment progress is provided. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., SNP associated with the disease or condition) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disease, disorder, or symptoms thereof in which the subject has been administered a therapeutic amount of a composition herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject&#39;s disease status. In preferred embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment. 
     In some embodiments, cells are obtained from the subject and contacted with a pharmaceutical composition as provided herein. In some embodiments, cells removed from a subject and contacted ex vivo with a pharmaceutical composition are re-introduced into the subject, optionally after the desired genomic modification has been effected or detected in the cells. Methods of delivering pharmaceutical compositions comprising nucleases are described, for example, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of all of which are incorporated by reference herein in their entireties. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals or organisms of all sorts, for example, for veterinary use. 
     Provided herein are methods of treating a disease or disorder with a composition or system, e.g., a base editor system or a base editor protein, described herein. Optionally, the base editor system described herein can be combined with one or more other treatments. In one aspect, provided herein is a method of treating Stargardt Disease (SD) in a subject in need thereof by administering a base editor described herein. In one aspect, provided herein is a method of treating Parkinson&#39;s Disease (PD) in a subject in need thereof by administering a base editor described herein. In one aspect, provided herein is a method of treating Rett Syndrome (RTT) in a subject in need thereof by administering a base editor described herein. In one aspect, provided herein is a method of treating Hurler Syndrome (HS) in a subject in need thereof by administering a base editor described herein. 
     The response in individual subjects can be characterized as a complete response, a partial response, or stable disease. In some embodiments, the response is a partial response (PR). In some embodiments, the response is a complete response (CR). In some embodiments, the response results in progression-free survival of the subject (e.g., stable disease). 
     In some embodiments, the treatment results in an increased survival time of the human subject as compared to the expected survival time of the human subject if the human subject was not treated with the compound, e.g. the base editor system. In some embodiments increased survival time comprises slower progression of the disease compared to a subject treated with a base editor comprising a ABET base editor, e.g. ABE7.10. 
     In some embodiments, the human subject to be treated with the described methods is a child (e.g., 0-18 years of age). In other embodiments, the human subject to be treated with the described methods is an adult (e.g., 18+ years of age). 
     Stargardt Disease (SD) 
     Stargardt disease (also known as Stargardt macular dystrophy, juvenile macular degeneration, or fundus flavimaculatus) is an inherited disorder of the retina (i.e., the tissue at the back of the eye that senses light). Stargardt disease is one of several genetic disorders that cause macular degeneration. The disease generally causes vision loss during childhood or adolescence; although vision loss may not be noticed until later in adulthood in some cases. It is rare for the disease to progress to complete blindness. Generally, vision loss progresses slowly over time to 20/200 or worse as progressive damage (degeneration) of the macula occurs over time. In one instance, the Stargardt disease to be treated with the methods described herein comprises juvenile Stargardt disease. In another instance, the Stargardt disease to be treated with the methods described herein comprises late onset Stargardt disease. In another instance, the Stargardt disease to be treated with the methods described herein comprises Stargardt-type Dominant macular dystrophy. In another instance, the Stargardt disease to be treated with the methods described herein comprises Dominant Stargardt-like macular dystrophy. 
     Progression of symptoms in Stargardt disease may differ for each patient. Patients with an earlier onset of disease generally tend to have more rapid vision loss. Vision loss may decrease slowly at first, then worsen rapidly until it levels off. Most patients with Stargardt disease will end up with 20/200 vision or worse. People with Stargardt disease may also begin to lose some of their peripheral (side) vision as they get older. 
     In some embodiments, a pathogenic SNP is associated with Stargardt disease; optionally, the pathogenic SNP is in an ABCA4 gene; and optionally, the pathogenic mutation comprises A1038V, L541P, G1961E, or a combination thereof. In some embodiments, the pathogenic SNP is associated with  Pseudoxanthoma elasticum ; optionally, the pathogenic SNP is in an ABCC6 gene; and optionally, the pathogenic mutation comprises R1141* (a nonsense mutation) In some embodiments, the pathogenic SNP is associated with medium-chain acyl-CoA dehydrogenase deficiency; optionally, the pathogenic SNP is in an ACADM gene; and optionally, the pathogenic mutation comprises K329E. In some embodiments, the pathogenic SNP is associated with severe combined immunodeficiency; optionally, the pathogenic SNP is in an ADA gene; and optionally, the pathogenic mutation comprises G216R, Q3*, or a combination thereof. 
     An exemplary amino acid sequence of the ABCA4 polypeptide is provided below: 
     
       
         
           
               
               
            
               
                 &gt;sp|P78363|ABCA4_HUMAN Retinal-specific phospholipid-transporting ATPase 
                   
               
               
                 ABCA4 OS =  Homo sapiens  OX = 9606 GN = ABCA4 PE = 1 SV = 3 
               
               
                 (SEQ ID NO: 6) 
                   
               
               
                 MGFVRQIQLLLWKNWTLRKRQKIRFVVELVWPLSLFLVLIWLRNANPLYSHH 
                   
               
               
                   
               
               
                 ECHFPNKAMPSAGMLPWLQGIFCNVNNPCFQSPTPGESPGIVSNYNNSILARVYRDF 
               
               
                   
               
               
                 QELLMNAPESQHLGRIWTELHILSQFMDTLRTHPERIAGRGIRIRDILKDEETLTLFLIK 
               
               
                   
               
               
                 NIGLSDSVVYLLINSQVRPEQFAHGVPDLALKDIACSEALLERFIIFSQRRGAKTVRYA 
               
               
                   
               
               
                 LCSLSQGTLQWIEDTLYANVDFFKLFRVLPTLLDSRSQGINLRSWGGILSDMSPRIQEF 
               
               
                   
               
               
                 IHRPSMQDLLWVTRPLMQNGGPETFTKLMGILSDLLCGYPEGGGSRVLSFNWYEDN 
               
               
                   
               
               
                 NYKAFLGIDSTRKDPIYSYDRRTTSFCNALIQSLESNPLTKIAWRAAKPLLMGKILYTP 
               
               
                   
               
               
                 DSPAARRILKNANSTFEELEHVRKLVKAWEEVGPQIWYFFDNSTQMNMIRDTLGNP 
               
               
                   
               
               
                 TVKDFLNRQLGEEGITAEAILNFLYKGPRESQADDMANFDWRDIFNITDRTLRLVNQ 
               
               
                   
               
               
                 YLECLVLDKFESYNDETQLTQRALSLLEENMFWAGVVFPDMYPWTSSLPPHVKYKI 
               
               
                   
               
               
                 RMDIDVVEKTNKIKDRYWDSGPRADPVEDFRYIWGGFAYLQDMVEQGITRSQVQA 
               
               
                   
               
               
                 EAPVGIYLQQMPYPCFVDDSFMIILNRCFPIFMVLAWIYSVSMTVKSIVLEKELRLKE 
               
               
                   
               
               
                 TLKNQGVSNAVIWCTWFLDSFSIMSMSIFLLTIFIMHGRILHYSDPFILFLFLLAFSTATI 
               
               
                   
               
               
                 MLCFLLSTFFSKASLAAACSGVIYFTLYLPHILCFAWQDRMTAELKKAVSLLSPVAFG 
               
               
                   
               
               
                 FGTEYLVRFEEQGLGLQWSNIGNSPTEGDEFSFLLSMQMMLLDAAVYGLLAWYLDQ 
               
               
                   
               
               
                 VFPGDYGTPLPWYFLLQESYWLGGEGCSTREERALEKTEPLTEETEDPEHPEGIHDSF 
               
               
                   
               
               
                 FEREHPGWVPGVCVKNLVKIFEPCGRPAVDRLNITFYENQITAFLGHNGAGKTTTLSI 
               
               
                   
               
               
                 LTGLLPPTSGTVLVGGRDIETSLDAVRQSLGMCPQHNILFHHLTVAEHMLFYAQLKG 
               
               
                   
               
               
                 KSQEEAQLEMEAMLEDTGLHHKRNEEAQDLSGGMQRKLSVAIAFVGDAKVVILDEP 
               
               
                   
               
               
                 TSGVDPYSRRSIWDLLLKYRSGRTIIMSTHHMDEADLLGDRIAIIAQGRLYCSGTPLFL 
               
               
                   
               
               
                 KNCFGTGLYLTLVRKMKNIQSQRKGSEGTCSCSSKGFSTTCPAHVDDLTPEQVLDGD 
               
               
                   
               
               
                 VNELMDVVLHHVPEAKLVECIGQELIFLLPNKNFKHRAYASLFRELEETLADLGLSSF 
               
               
                   
               
               
                 GISDTPLEEIFLKVTEDSDSGPLFAGGAQQKRENVNPRHPCLGPREKAGQTPQDSNVC 
               
               
                   
               
               
                 SPGAPAAHPEGQPPPEPECPGPQLNTGTQLVLQHVQALLVKRFQHTIRSHKDFLAQIV 
               
               
                   
               
               
                 LPATFVFLALMLSIVIPPFGEYPALTLHPWIYGQQYTFFSMDEPGSEQFTVLADVLLN 
               
               
                   
               
               
                 KPGFGNRCLKEGWLPEYPCGNSTPWKTPSVSPNITQLFQKQKWTQVNPSPSCRCSTR 
               
               
                   
               
               
                 EKLTMLPECPEGAGGLPPPQRTQRSTEILQDLTDRNISDFLVKTYPALIRSSLKSKFWV 
               
               
                   
               
               
                 NEQRYGGISIGGKLPVVPITGEALVGFLSDLGRIMNVSGGPITREASKEIPDFLKHLET 
               
               
                   
               
               
                 EDNIKVWFNNKGWHALVSFLNVAHNAILRASLPKDRSPEEYGITVISQPLNLTKEQLS 
               
               
                   
               
               
                 EITVLTTSVDAVVAICVIFSMSFVPASFVLYLIQERVNKSKHLQFISGVSPTTYWVTNF 
               
               
                   
               
               
                 LWDIMNYSVSAGLVVGIFIGFQKKAYTSPENLPALVALLLLYGWAVIPMMYPASFLF 
               
               
                   
               
               
                 DVPSTAYVALSCANLFIGINSSAITFILELFENNRTLLRFNAVLRKLLIVFPHFCLGRGL 
               
               
                   
               
               
                 IDLALSQAVTDVYARFGEEHSANPFHWDLIGKNLFAMVVEGVVYFLLTLLVQRHFF 
               
               
                   
               
               
                 LSQWIAEPTKEPIVDEDDDVAEERQRIITGGNKTDILRLHELTKIYPGTSSPAVDRLCV 
               
               
                   
               
               
                 GVRPGECFGLLGVNGAGKTTTFKMLTGDTTVTSGDATVAGKSILTNISEVHQNMGY 
               
               
                   
               
               
                 CPQFDAIDELLTGREHLYLYARLRGVPAEEIEKVANWSIKSLGLTVYADCLAGTYSG 
               
               
                   
               
               
                 GNKRKLSTAIALIGCPPLVLLDEPTTGMDPQARRMLWNVIVSIIREGRAVVLTSHSME 
               
               
                   
               
               
                 ECEALCTRLAIMVKGAFRCMGTIQHLKSKFGDGYIVTMKIKSPKDDLLPDLNPVEQF 
               
               
                   
               
               
                 FQGNFPGSVQRERHYNMLQFQVSSSSLARIFQLLLSHKDSLLIEEYSVTQTTLDQVFV 
               
               
                   
               
               
                 NFAKQQTESHDLPLHPRAAGASRQAQD 
               
               
                   
               
               
                 Guide RNA sequences target ABCA4 gene at sequence 
               
               
                 (SEQ ID NO: 291) 
                   
               
               
                 GCTGTGTGTCGAAGTTCGCCCTGGAG AGG TG 
                   
               
               
                 or 
               
               
                   
               
               
                 (SEQ ID NO: 292) 
                   
               
               
                 GCTGTGTGTCGGAGTTCGCCCTGGAG AGG TG, where the PAM sequence is underlined. 
                   
               
               
                   
               
               
                 The guide RNA comprises a sequence 
               
               
                 (SEQ ID NO: 293) 
                   
               
               
                 CACCUCUCCAGGGCGAACUUCGACACACAGC 
                   
               
               
                 or 
               
               
                   
               
               
                 (SEQ ID NO: 294) 
                   
               
               
                 CACCUCUCCAGGGCGAACUCCGACACACAGC. 
                   
               
            
           
         
       
     
     One or more symptoms of Stargardt disease include, but are not limited to, variable, slow loss of central vision in both eyes&#39; gray, black, or hazy spots in the center of vision; that it takes longer than usual for eyes to adjust when moving from light to dark environments; eyes may be more sensitive to bright light; color blindness later in the disease, accumulation of toxic lipofuscin pigments such as A2E in cells of the retinal pigment epithelium (RPE), photoreceptor death, increased synthesis of 11-cis-retinaldehyde (11cRAL or retinal), increased regeneration of rhodopsin, lipofuscin accumulation, formation of the lipofuscin pigment, retinal degeneration, production of waste products, formation of A2E (and A2E-related molecules), accumulation of A2E (and A2E-related molecules), choroidal neovascularization, chorioretinal atrophy, or a combination thereof. The subject may exhibit an improvement in one or more of the symptoms of Stargardt Disease. In one embodiment, the improvement in one or more of the symptoms is at least 5%. In another embodiment, the improvement in one or more of the symptoms is at least 10%. In another embodiment, the improvement in one or more of the symptoms is at least 15%. In another embodiment, the improvement in one or more of the symptoms is at least 20%. In another embodiment, the improvement in one or more of the symptoms is at least 25%. In another embodiment, the improvement in one or more of the symptoms is at least 30%. In another embodiment, the improvement in one or more of the symptoms is at least 35%. In another embodiment, the improvement in one or more of the symptoms is at least 40%. In another embodiment, the improvement in one or more of the symptoms is at least 50%. In another embodiment, the improvement in one or more of the symptoms is at least 60%. In another embodiment, the improvement in one or more of the symptoms is at least 70%. In another embodiment, the improvement in one or more of the symptoms is at least 75%. In another embodiment, the improvement in one or more of the symptoms is at least 80%. In another embodiment, the improvement in one or more of the symptoms is at least 85%. In another embodiment, the improvement in one or more of the symptoms is at least 90%. In another embodiment, the improvement in one or more of the symptoms is at least 95%. 
     Parkinson&#39;s Disease (PD) 
     Parkinson disease (PD) is the most common movement disorder, affecting over 6 million people worldwide. PD can present with a juvenile or early onset, but it predominantly afflicts individuals over the age of 55 and the incidence of disease sharply rises after the age of 65. The clinical features of PD include bradykinesia, postural instability, resting tremor, and rigidity which are predominantly associated with the progressive loss of dopaminergic neurons in the substantia nigra (SN) pars compacta. It is believed that during normal aging approximately 0.1-0.2% of the dopaminergic neurons in this area are lost per year, but this rate is greatly accelerated in patients with PD and symptoms manifests when ˜70-80% of these neurons have been lost. Another pathological hallmark of PD is the presence of α-synuclein proteiniacous inclusions, known as Lewy bodies (LBs) in the remaining dopaminergic neurons. 
     Although the majority of PD cases are idiopathic, ˜10% of cases report with a family history, and a growing number of mutations have been associated with familial and sporadic forms of the disease. The autosomal dominant G2019S mutation in leucine-rich repeat kinase 2 (LRRK2) is the most common known cause of familial and sporadic patients with PD. 
     An exemplary amino acid sequence of the LRRK2 polypeptide is provided below: 
     &gt;sp|Q5S007|LRRK2_HUMAN Leucine-rich repeat serine/threonine-protein kinase 2 OS═ Homo sapiens  OX=9606 GN=LRRK2 PE=1 SV=2 
     MASGSCQGCEEDEETLKKLIVRLNNVQEGKQIETLVQILEDLLVFTYSERASK LFQGKNIHVPLLIVLDSYMRVASVQQVGWSLLCKLIEVCPGTMQSLMGPQDVGND WEVLGVHQLILKMLTVHNASVNLSVIGLKTLDLLLTSGKITLLILDEESDIFMLIFDA MHSFPANDEVQKLGCKALHVLFERVSEEQLTEFVENKDYMILLSALTNFKDEEEIVL HVLHCLHSLAIPCNNVEVLMSGNVRCYNIVVEAMKAFPMSERIQEVSCCLLHRLTLG NFFNILVLNEVHEFVVKAVQQYPENAALQISALSCLALLTETIFLNQDLEEKNENQEN DDEGEEDKLFWLEACYKALTWHRKNKHVQEAACWALNNLLMYQNSLHEKIGDED GHFPAHREVMLSMLMHSSSKEVFQASANALSTLLEQNVNFRKILLSKGIHLNVLELM QKHIHSPEVAESGCKMLNHLFEGSNTSLDIMAAVVPKILTVMKRHETSLPVQLEALR AILHFIVPGMPEESREDTEFHHKLNMVKKQCFKNDIHKLVLAALNRFIGNPGIQKCGL KVISSIVHFPDALEMLSLEGAMDSVLHTLQMYPDDQEIQCLGLSLIGYLITKKNVFIGT GHLLAKILVSSLYRFKDVAEIQTKGFQTILAILKLSASFSKLLVHHSFDLVIFHQMSSNI MEQKDQQFLNLCCKCFAKVAMDDYLKNVMLERACDQNNSIMVECLLLLGADANQ AKEGSSLICQVCEKESSPKLVELLLNSGSREQDVRKALTISIGKGDSQIISLLLRRLALD VANNSICLGGFCIGKVEPSWLGPLFPDKTSNLRKQTNIASTLARMVIRYQMKSAVEE GTASGSDGNFSEDVLSKFDEWTFIPDSSMDSVFAQSDDLDSEGSEGSFLVKKKSNSIS VGEFYRDAVLQRCSPNLQRHSNSLGPIFDHEDLLKRKRKILSSDDSLRSSKLQSHMRH SDSISSLASEREYITSLDLSANELRDIDALSQKCCISVHLEHLEKLELHQNALTSFPQQL CETLKSLTHLDLHSNKFTSFPSYLLKMSCIANLDVSRNDIGPSVVLDPTVKCPTLKQF NLSYNQLSFVPENLTDVVEKLEQLILEGNKISGICSPLRLKELKILNLSKNHISSLSENF LEACPKVESFSARMNFLAAMPFLPPSMTILKLSQNKFSCIPEAILNLPHLRSLDMSSND IQYLPGPAHWKSLNLRELLFSHNQISILDLSEKAYLWSRVEKLHLSHNKLKEIPPEIGC LENLTSLDVSYNLELRSFPNEMGKLSKIWDLPLDELHLNFDFKHIGCKAKDIIRFLQQ RLKKAVPYNRMKLMIVGNTGSGKTTLLQQLMKTKKSDLGMQSATVGIDVKDWPIQ IRDKRKRDLVLNVWDFAGREEFYSTHPHFMTQRALYLAVYDLSKGQAEVDAMKPW LFNIKARASSSPVILVGTHLDVSDEKQRKACMSKITKELLNKRGFPAIRDYHFVNATE ESDALAKLRKTIINESLNFKIRDQLVVGQLIPDCYVELEKIILSERKNVPIEFPVIDRKR LLQLVRENQLQLDENELPHAVHFLNESGVLLHFQDPALQLSDLYFVEPKWLCKIMA QILTVKVEGCPKHPKGIISRRDVEKFLSKKRKFPKNYMSQYFKLLEKFQIALPIGEEYL LVPSSLSDHRPVIELPHCENSEIIIRLYEMPYFPMGFWSRLINRLLEISPYMLSGRERAL RPNRMYWRQGIYLNWSPEAYCLVGSEVLDNHPESFLKITVPSCRKGCILLGQVVDHI DSLMEEWFPGLLEIDICGEGETLLKKWALYSFNDGEEHQKILLDDLMKKAEEGDLLV NPDQPRLTIPISQIAPDLILADLPRNIMLNNDELEFEQAPEFLLGDGSFGSVYRAAYEG EEVAVKIFNKHTSLRLLRQELVVLCHLHHPSLISLLAAGIRPRMLVMELASKGSLDRL LQQDKASLTRTLQHRIALHVADGLRYLHSAMITYRDLKPHNVLLFTLYPNAAIIAKIA DYGIAQYCCRMGIKTSEGTPGFRAPEVARGNVIYNQQADVYSFGLLLYDILTTGGRI VEGLKFPNEFDELEIQGKLPDPVKEYGCAPWPMVEKLIKQCLKENPQERPTSAQVFDI LNSAELVCLTRRILLPKNVIVECMVATHHNSRNASIWLGCGHTDRGQLSFLDLNTEG YTSEEVADSRILCLALVHLPVEKESWIVSGTQSGTLLVINTEDGKKRHTLEKMTDSVT CLYCNSFSKQSKQKNFLLVGTADGKLAIFEDKTVKLKGAAPLKILNIGNVSTPLMCL SESTNSTERNVMWGGCGTKIFSFSNDFTIQKLIETRTSQLFSYAAFSDSNIITVVVDTA LYIAKQNSPVVEVWDKKTEKLCGLIDCVHFLREVMVKENKESKHKMSYSGRVKTL CLQKNTALWIGTGGGHILLLDLSTRRLIRVIYNFCNSVRVMMTAQLGSLKNVMLVL GYNRKNTEGTQKQKEIQSCLTVWDINLPHEVQNLEKHIEVRKELAEKMRRTSVE (SEQ ID NO: 3) Parkinson&#39;s disease is a progressive nervous system disorder that affects movement. Symptoms typically start gradually, sometimes starting with a barely noticeable tremor in just one hand. Tremors may be common, but the disorder also commonly causes stiffness or slowing of movement. In the early stages of Parkinson&#39;s disease, a subject&#39;s face may show little or no expression. The patient&#39;s arms may not swing when while walking. Speech may become soft or slurred. Parkinson&#39;s disease symptoms generally worsen over time. 
     Signs and symptoms of Parkinson&#39;s disease vary between patients. Early signs may be mild and go unnoticed. Symptoms often begin on one side of the body and usually remain worse on that side, even after symptoms begin to affect both sides. Signs and symptoms of Parkinson&#39;s disease include, but are not limited to one or more of tremors, slowed movement (bradykinesia); rigid muscles; impaired posture and balance; loss of automatic movements (e.g., a decreased ability to perform unconscious movements, including blinking, smiling or swinging the arms while walking); speech changes (e.g., speaking softly, quickly, slurring, or hesitating before talking; speech may be more of a monotone rather than with the usual inflections); writing changes (e.g., it may become hard to write, and writing may appear small); blood pressure changes (e.g., a patient may feel dizzy or lightheaded when standing due to a sudden drop in blood pressure (orthostatic hypotension)); smell dysfunction (e.g., a patient may experience problems with sense of smell.); fatigue (e.g., many patients with Parkinson&#39;s disease lose energy and experience fatigue, especially later in the day): pain (e.g., some patients with Parkinson&#39;s disease experience pain, either in specific areas of their bodies or throughout their bodies); sexual dysfunction (e.g., some patients with Parkinson&#39;s disease notice a decrease in sexual desire or performance); or a combination thereof. 
     The subject may exhibit an improvement in one or more of the symptoms of Parkinson&#39;s Disease. In one embodiment, the improvement in one or more of the symptoms is at least 5%. In another embodiment, the improvement in one or more of the symptoms is at least 10%. In another embodiment, the improvement in one or more of the symptoms is at least 15%. In another embodiment, the improvement in one or more of the symptoms is at least 20%. In another embodiment, the improvement in one or more of the symptoms is at least 25%. In another embodiment, the improvement in one or more of the symptoms is at least 30%. In another embodiment, the improvement in one or more of the symptoms is at least 35%. In another embodiment, the improvement in one or more of the symptoms is at least 40%. In another embodiment, the improvement in one or more of the symptoms is at least 50%. In another embodiment, the improvement in one or more of the symptoms is at least 60%. In another embodiment, the improvement in one or more of the symptoms is at least 70%. In another embodiment, the improvement in one or more of the symptoms is at least 75%. In another embodiment, the improvement in one or more of the symptoms is at least 80%. In another embodiment, the improvement in one or more of the symptoms is at least 85%. In another embodiment, the improvement in one or more of the symptoms is at least 90%. In another embodiment, the improvement in one or more of the symptoms is at least 95%. 
     Provided also are methods of treating PD and/or the genetic mutations in LRRK2 that cause PD that comprise administering to a subject (e.g., a mammal, such as a human) a therapeutically effective amount of a pharmaceutical composition that comprises a polynucleotide encoding a base editor system (e.g., base editor and gRNA) described herein. In some embodiments, the base editor is a fusion protein that comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain or a cytidine deaminase domain. A cell of the subject is transduced with the base editor and one or more guide polynucleotides that target the base editor to effect an A•T to G•C alteration (if the cell is transduced with an adenosine deaminase domain) or a C•G to U•A alteration (if the cell is transduced with a cytidine deaminase domain) of a nucleic acid sequence containing mutations in the LRRK2 gene. 
     The methods herein include administering to the subject (including a subject identified as being in need of such treatment, or a subject suspected of being at risk of disease and in need of such treatment) an effective amount of a composition described herein. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method). 
     The therapeutic methods, in general, comprise administration of a therapeutically effective amount of a pharmaceutical composition comprising, for example, a vector encoding a base editor and a gRNA that targets the LRRK2 gene of a subject (e.g., a human patient) in need thereof. Such treatment will be suitably administered to a subject, particularly a human subject, suffering from, having, susceptible to, or at risk for PD. The compositions herein may be also used in the treatment of any other disorders in which PD may be implicated. 
     In some embodiments, the guide RNA targets LRRK gene at a target sequence 
     
       
         
           
               
            
               
                 (SEQ ID NO: 295) 
               
               
                 GCTCGCCCTTCTTCTTCCCCTGTGA, 
               
               
                   
               
               
                 (SEQ ID NO: 296) 
               
               
                 GTCTTTCCCTCCAGGCTCGCCCTTCTTCTTCCCCTGTGA, 
               
               
                   
               
               
                 (SEQ ID NO: 297) 
               
               
                 TCACAGGGGAAGAAGAAGGGCGAGC, 
               
               
                 or 
               
               
                   
               
               
                 (SEQ ID NO: 298) 
               
               
                 TCACAGGGGAAGAAGAAGGGCGAGCCTGGAGGGAAAGAC. 
               
               
                 In some embodiments, the guide RNA comprises 
               
               
                 sequence 
               
               
                   
               
               
                 (SEQ ID NO: 299) 
               
               
                 GCUCGCCCUUCUUCUUCCCCUGUGA, 
               
               
                   
               
               
                 (SEQ ID NO: 300) 
               
               
                 GUCUUUCCCUCCAGGCUCGCCCUUCUUCUUCCCCUGUGA, 
               
               
                   
               
               
                 (SEQ ID NO: 301) 
               
               
                 UCACAGGGGAAGAAGAAGGGCGAGC, 
               
               
                 or 
               
               
                   
               
               
                 (SEQ ID NO: 302) 
               
               
                 UCACAGGGGAAGAAGAAGGGCGAGCCUGGAGGGAAAGAC. 
               
               
                 In some embodiments, the guide RNA comprises 
               
               
                 sequence 
               
               
                   
               
               
                 (SEQ ID NO: 303) 
               
               
                 UCCGACUAUAUGAAAUGCCUUAUUUUCCAAUGGGAUUUUGG 
               
               
                 or 
               
               
                   
               
               
                 (SEQ ID NO: 304) 
               
               
                 UUGCAAAGAUUGCUGACUAGGGCAUUGCUCAGUACUGCUGUAGAAUGG. 
               
            
           
         
       
     
     In one embodiment, a method of monitoring treatment progress is provided. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., SNP associated with PD) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with PD in which the subject has been administered a therapeutic amount of a composition herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject&#39;s disease status. In preferred embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment. 
     In some embodiments, compositions provided herein are administered to a subject, for example, to a human subject, in order to effect a targeted genomic modification within the subject. In some embodiments, cells are obtained from the subject and contacted with any of the pharmaceutical compositions provided herein. In some embodiments, cells removed from a subject and contacted ex vivo with a pharmaceutical composition are re-introduced into the subject, optionally after the desired genomic modification has been effected or detected in the cells. Methods of delivering pharmaceutical compositions comprising nucleases are known, and are described, for example, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of all of which are incorporated by reference herein in their entireties. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals or organisms of all sorts, for example, for veterinary use. 
     Hurler Syndrome (HS) 
     Hurler Syndrome is the most severe form of mucopolysaccharidosis type 1 (MPS1), a rare autosomal recessive lysosomal storage disorder that occurs in about one in 200,000 births. MPS1 is characterized by skeletal abnormalities, cognitive impairment, heart disease, respiratory problems, enlarged liver and spleen, and reduced life expectancy. MPS1 is caused by mutations in alpha-L-iduronidase (IDUA) gene, leading to deficiency of alpha-L-iduronidase, which is essential for the breakdown of glycosaminoglycans in lysosomes. The standard of care for patients with Hurler Syndrome is bone marrow transplantation, but this treatment cannot correct any damage already incurred, especially in the central nervous system, and there are risks of mortality associated with transplantation. Therefore, there is a need for novel compositions and methods for treating patients with Hurler Syndrome. 
     The present disclosure provides methods for the treatment of Hurler Syndrome that are associated with or caused by a point mutation that can be corrected and/or symptoms can be treated or ameliorated by base editor mediated gene editing. 
     Provided also are methods of treating Hurler Syndrome and/or the genetic mutations in IDUA gene that cause Hurler Syndrome that comprise administering to a subject (e.g., a mammal, such as a human) a therapeutically effective amount of a pharmaceutical composition that comprises a polynucleotide encoding a base editor system (e.g., ABE8 base editor and gRNA) described herein. In some embodiments, the base editor is a fusion protein that comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain. A cell of the subject is transduced with the base editor and one or more guide polynucleotides that target the base editor to effect an A•T to G•C alteration of a nucleic acid sequence containing mutations in the IDUA gene. 
     An exemplary amino acid sequence of the IDUA polypeptide is provided below: 
     
       
         
           
               
            
               
                 &gt;sp|P35475|IDUA_HUMAN Alpha-L-iduronidase OS = 
               
               
                   Homo sapiens  OX = 9606 GN = IDUA PE = 1 SV = 2 
               
               
                 (SEQ ID NO: 4) 
               
               
                 MRPLRPRAALLALLASLLAAPPVAPAEAPHLVHVDAARALWPLRRFWRS 
               
               
                   
               
               
                 TGFCPPLPHSQADQYVLSWDQQLNLAYVGAVPHRGIKQVRTHWLLELVT 
               
               
                   
               
               
                 TRGSTGRGLSYNFTHLDGYLDLLRENQLLPGFELMGSASGHFTDFEDKQ 
               
               
                   
               
               
                 QVFEWKDLVSSLARRYIGRYGLAHVSKWNFETWNEPDHHDFDNVSMTMQ 
               
               
                   
               
               
                 GFLNYYDACSEGLRAASPALRLGGPGDSFHTPPRSPLSWGLLRHCHDGT 
               
               
                   
               
               
                 NFFTGEAGVRLDYISLHRKGARSSISILEQEKVVAQQIRQLFPKFADTP 
               
               
                   
               
               
                 IYNDEADPLVGWSLPQPWRADVTYAAMVVKVIAQHQNLLLANTTSAFPY 
               
               
                   
               
               
                 ALLSNDNAFLSYHPHPFAQRTLTARFQVNNTRPPHVQLLRKPVLTAMGL 
               
               
                   
               
               
                 LALLDEEQLWAEVSQAGTVLDSNHTVGVLASAHRPQGPADAWRAAVLIY 
               
               
                   
               
               
                 ASDDTRAHPNRSVAVTLRLRGVPPGPGLVYVTRYLDNGLCSPDGEWRRL 
               
               
                   
               
               
                 GRPVFPTAEQFRRMRAAEDPVAAAPRPLPAGGRLTLRPALRLPSLLLVH 
               
               
                   
               
               
                 VCARPEKPPGQVTRLRALPLTQGQLVLVWSDEHVGSKCLWTYEIQFSQD 
               
               
                   
               
               
                 GKAYTPVSRKPSTFNLFVFSPDTGAVSGSYRVRALDYWARPGPFSDPVP 
               
               
                   
               
               
                 YLEVPVPRGPPSPGNP. 
               
            
           
         
       
     
     The methods herein include administering to the subject (including a subject identified as being in need of such treatment, or a subject suspected of being at risk of disease and in need of such treatment) an effective amount of a composition described herein. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method). 
     The therapeutic methods, in general, comprise administration of a therapeutically effective amount of a pharmaceutical composition comprising, for example, a vector encoding a base editor and a gRNA that targets the IDUA gene of a subject (e.g., a human patient) in need thereof. Such treatment will be suitably administered to a subject, particularly a human subject, suffering from, having, susceptible to, or at risk for Hurler Syndrome. The compositions herein may be also used in the treatment of any other disorders in which Hurler Syndrome may be implicated. The guide RNA sequences may comprise a spacer sequence CTTTTCACTTTTCCTGCCGGGG (SEQ ID NO: 305) (R255X), AGCTTCCATGTCCAGCCTTC (SEQ ID NO: 306) (R106W), ACCATGAAGTCAAAATCATT (SEQ ID NO: 307) (T158M), or GCTTTCAGCCCCGTTTCTTG (SEQ ID NO: 308) (R270X). 
     In one embodiment, the invention provides a method of monitoring treatment progress is provided. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., SNP associated with Hurler Syndrome) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with Hurler Syndrome in which the subject has been administered a therapeutic amount of a composition herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject&#39;s disease status. In preferred embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment. 
     In some embodiments, cells are obtained from the subject and contacted with a pharmaceutical composition as provided herein. In some embodiments, cells removed from a subject and contacted ex vivo with a pharmaceutical composition are re-introduced into the subject, optionally after the desired genomic modification has been affected or detected in the cells. Methods of delivering pharmaceutical compositions comprising nucleases are described, for example, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of all of which are incorporated by reference herein in their entireties. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals or organisms of all sorts, for example, for veterinary use. 
     One or more symptoms of Hurler Syndrome to be treated include, but are not limited to, coarse facial features, conical clouding, hepatomegaly, kyphosis/gibbus, hernias, airway-related symptoms, such as sleep disturbances/snoring, splenomegaly, cardiac valve abnormalities, cognitive impairment, dystosis multiplex, enlarged tongue, joint contractures, enlarged tonsils; or a combination thereof. 
     The subject may exhibit an improvement in one or more of the symptoms of Hurler Syndrome. In one embodiment, the improvement in one or more of the symptoms is at least 5%. In another embodiment, the improvement in one or more of the symptoms is at least 10%. In another embodiment, the improvement in one or more of the symptoms is at least 15%. In another embodiment, the improvement in one or more of the symptoms is at least 20%. In another embodiment, the improvement in one or more of the symptoms is at least 25%. In another embodiment, the improvement in one or more of the symptoms is at least 30%. In another embodiment, the improvement in one or more of the symptoms is at least 35%. In another embodiment, the improvement in one or more of the symptoms is at least 40%. In another embodiment, the improvement in one or more of the symptoms is at least 50%. In another embodiment, the improvement in one or more of the symptoms is at least 60%. In another embodiment, the improvement in one or more of the symptoms is at least 70%. In another embodiment, the improvement in one or more of the symptoms is at least 75%. In another embodiment, the improvement in one or more of the symptoms is at least 80%. In another embodiment, the improvement in one or more of the symptoms is at least 85%. In another embodiment, the improvement in one or more of the symptoms is at least 90%. In another embodiment, the improvement in one or more of the symptoms is at least 95%. 
     Rett Syndrome (RTT) 
     Provided also are methods of treating Rett Syndrome (RTT) and/or the genetic mutations in Mecp2 that cause RTT that comprise administering to a subject (e.g., a mammal, such as a human) a therapeutically effective amount of a pharmaceutical composition that comprises a polynucleotide encoding a base editor system (e.g., base editor and gRNA) described herein. In some embodiments, the base editor is a fusion protein that comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain or a cytidine deaminase domain. A cell of the subject is transduced with the base editor and one or more guide polynucleotides that target the base editor to effect an A•T to G•C alteration (if the cell is transduced with an adenosine deaminase domain) or a C•G to U•A alteration (if the cell is transduced with a cytidine deaminase domain) of a nucleic acid sequence containing mutations in the Mecp2 gene. 
     An exemplary amino acid sequence of the MECP2 polypeptide is provided below: 
     
       
         
           
               
            
               
                 &gt;sp|P51608|MECP2_HUMAN Methyl-CpG-binding protein  
               
               
                 2 OS =  Homo sapiens  OX = 9606 GN = MECP2 PE = 1 
               
               
                 SV = 1 
               
               
                 (SEQ ID NO: 5) 
               
               
                 MVAGMLGLREEKSEDQDLQGLKDKPLKFKKVKKDKKEEKEGKHEPVQPS 
               
               
                   
               
               
                 AHHSAEPAEAGKAETSEGSGSAPAVPEASASPKQRRSIIRDRGPMYDDP 
               
               
                   
               
               
                 TLPEGWTRKLKQRKSGRSAGKYDVYLINPQGKAFRSKVELIAYFEKVGD 
               
               
                   
               
               
                 TSLDPNDFDFTVTGRGSPSRREQKPPKKPKSPKAPGTGRGRGRPKGSGT 
               
               
                   
               
               
                 TRPKAATSEGVQVKRVLEKSPGKLLVKMPFQTSPGGKAEGGGATTSTQV 
               
               
                   
               
               
                 MVIKRPGRKRKAEADPQAIPKKRGRKPGSVVAAAAAEAKKKAVKESSIR 
               
               
                   
               
               
                 SVQETVLPIKKRKTRETVSIEVKEVVKPLLVSTLGEKSGKGLKTCKSPG 
               
               
                   
               
               
                 RKSKESSPKGRSSSASSPPKKEHHHHHHHSESPKAPVPLLPPLPPPPPE 
               
               
                   
               
               
                 PESSEDPTSPPEPQDLSSSVCKEEKMPRGGSLESDGCPKEPAKTQPAVA 
               
               
                   
               
               
                 TAATAAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPVTERVS. 
               
            
           
         
       
     
     The guide RNA sequences may comprise a spacer 
     
       
         
           
               
               
            
               
                   
                 (R255X) 
               
               
                   
                 (SEQ ID NO: 305) 
               
               
                   
                 CTTTTCACTTTTCCTGCCGGGG, 
               
               
                   
                   
               
               
                   
                 (R106W) 
               
               
                   
                 (SEQ ID NO: 306) 
               
               
                   
                 AGCTTCCATGTCCAGCCTTC, 
               
               
                   
                   
               
               
                   
                 (T158M) 
               
               
                   
                 (SEQ ID NO: 307) 
               
               
                   
                 ACCATGAAGTCAAAATCATT, 
               
               
                   
                 or 
               
               
                   
                   
               
               
                   
                 (R270X) 
               
               
                   
                 (SEQ ID NO: 308) 
               
               
                   
                 GCTTTCAGCCCCGTTTCTTG. 
               
            
           
         
       
     
     The methods herein include administering to the subject (including a subject identified as being in need of such treatment, or a subject suspected of being at risk of disease and in need of such treatment) an effective amount of a composition described herein. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method). 
     The therapeutic methods, in general, comprise administration of a therapeutically effective amount of a pharmaceutical composition comprising, for example, a vector encoding a base editor and a gRNA that targets the Mecp2 gene of a subject (e.g., a human patient) in need thereof. Such treatment will be suitably administered to a subject, particularly a human subject, suffering from, having, susceptible to, or at risk for RTT. The compositions herein may be also used in the treatment of any other disorders in which RTT may be implicated. 
     In one embodiment, the invention provides a method of monitoring treatment progress is provided. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., SNP associated with RTT) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with RTT in which the subject has been administered a therapeutic amount of a composition herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject&#39;s disease status. In preferred embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment. 
     In some embodiments, cells are obtained from the subject and contacted with a pharmaceutical composition as provided herein. In some embodiments, cells removed from a subject and contacted ex vivo with a pharmaceutical composition are re-introduced into the subject, optionally after the desired genomic modification has been effected or detected in the cells. Methods of delivering pharmaceutical compositions comprising nucleases are described, for example, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of all of which are incorporated by reference herein in their entireties. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals or organisms of all sorts, for example, for veterinary use. 
     One or more symptoms of Rett Syndrome include, but are not limited to, bedtime resistance, sleep onset delay, sleep duration, sleep anxiety, night wakings, parasomnias, sleep disordered breathing, daytime sleepiness, hand function, walking, verbal and non-verbal communication, comprehension, attention, behavior problems, mood, seizure activity, behavior and emotional features, or a combination thereof. 
     The subject may exhibit an improvement in one or more of the symptoms of Rett Syndrome. In one embodiment, the improvement in one or more of the symptoms is at least 5%. In another embodiment, the improvement in one or more of the symptoms is at least 10%. In another embodiment, the improvement in one or more of the symptoms is at least 15%. In another embodiment, the improvement in one or more of the symptoms is at least 20%. In another embodiment, the improvement in one or more of the symptoms is at least 25%. In another embodiment, the improvement in one or more of the symptoms is at least 30%. In another embodiment, the improvement in one or more of the symptoms is at least 35%. In another embodiment, the improvement in one or more of the symptoms is at least 40%. In another embodiment, the improvement in one or more of the symptoms is at least 50%. In another embodiment, the improvement in one or more of the symptoms is at least 60%. In another embodiment, the improvement in one or more of the symptoms is at least 70%. In another embodiment, the improvement in one or more of the symptoms is at least 75%. In another embodiment, the improvement in one or more of the symptoms is at least 80%. In another embodiment, the improvement in one or more of the symptoms is at least 85%. In another embodiment, the improvement in one or more of the symptoms is at least 90%. In another embodiment, the improvement in one or more of the symptoms is at least 95%. 
     Kits 
     Various aspects of this disclosure provide kits comprising a base editor system. In one embodiment, the kit comprises a nucleic acid construct comprising a nucleotide sequence encoding a nucleobase editor fusion protein. The fusion protein comprises a deaminase (e.g., cytidine deaminase or adenine deaminase) and a nucleic acid programmable DNA binding protein (napDNAbp). In some embodiments, the kit comprises at least one guide RNA capable of targeting a nucleic acid molecule of interest. In some embodiments, the kit comprises a nucleic acid construct comprising a nucleotide sequence encoding at least one guide RNA. 
     The kit provides, in some embodiments, instructions for using the kit to edit one or more mutations. The instructions will generally include information about the use of the kit for editing nucleic acid molecules. In other embodiments, the instructions include at least one of the following: precautions; warnings; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. In a further embodiment, a kit can comprise instructions in the form of a label or separate insert (package insert) for suitable operational parameters. In yet another embodiment, the kit can comprise one or more containers with appropriate positive and negative controls or control samples, to be used as standard(s) for detection, calibration, or normalization. The kit can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as (sterile) phosphate-buffered saline, Ringer&#39;s solution, or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. 
     The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow. 
     The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. 
     EXAMPLES 
     Example 1: Adenosine Base Editors with Increased Editing Efficiency 
     Base editing systems that include a Tad7.10-dCas9 fusion proteins are capable of editing a target polynucleotide with approximately 10-20% efficiency, but for uses requiring higher efficiency their use may be limited. In an effort to identify adenine base editors having increased efficiency and specificity, constructs comprising the adenosine deaminase TadA 7.10 were mutagenized by error prone PCR and subsequently cloned into an expression vector adjacent to a nucleic acid sequence encoding dCas9, a nucleic acid programmable DNA binding protein ( FIG.  1 A ). The expression vectors comprising the adenosine deaminase variants were co-transformed into competent bacterial cells with a selection plasmid encoding chloramphenicol resistance (CamR) and spectinomycin resistance (SpectR) and having a kanamycin resistance gene that was rendered nonfunctional by two point mutations (evolution round 7 strategy) ( FIG.  1 B ). The cells were selected for restoration of kanamycin resistance, which was a read out for adenosine deaminase activity. In subsequent rounds of selection, the expression vectors were co-transformed into competent cells with a plasmid encoding chloramphenicol resistance (CamR) and spectinomycin resistance (SpectR) and having a kanamycin resistance gene that was rendered nonfunctional by three point mutations (evolution round 8 strategy) ( FIG.  1 C ). An inactivated kanamycin resistance gene nucleic acid sequence is provided below: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 309) 
               
               
                 ccggaattgccagctggggcgccctctggtaaggttgggaagccctgca 
               
               
                   
               
               
                 aagtaaactggatggctttcttgccgccaaggatctgatggcgcagggg 
               
               
                   
               
               
                 atcaagatctgatcaagagacaggatgaggat cct   ttcgcATGATCGAA   
               
               
                   
               
               
                   TAAGAT GGATTGCACGCAGGTTCTCCGGCC GCTTAGGTGGAGCGCCTAT   
               
               
                   
               
               
                   T   CGG CTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGT 
               
               
                   
               
               
                 GTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGAC 
               
               
                   
               
               
                 CTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGT 
               
               
                   
               
               
                 GGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCAC 
               
               
                   
               
               
                 TGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGAT 
               
               
                   
               
               
                 CTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTG 
               
               
                   
               
               
                 ATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGA 
               
               
                   
               
               
                 CCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCC 
               
               
                   
               
               
                 GGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGC 
               
               
                   
               
               
                 CAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGA 
               
               
                   
               
               
                 TCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAA 
               
               
                   
               
               
                 AATGGCCGCTTTTCTGGATTCATTAACTGTGGCCGGCTGGGTGTGGCGG 
               
               
                   
               
               
                 ACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCT 
               
               
                   
               
               
                 TGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCT 
               
               
                   
               
               
                 CCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCT  
               
               
                   
               
               
                 AA 
               
            
           
         
       
     
     In the above sequence, lower case denotes the kanamycin resistance promoter region, bold sequence indicates targeted inactivation portion (Q4* and W15*), the italicized sequence denotes the targeted inactive site of kanamycin resistance gene (D208N), and the underlined sequences denote the PAM sequences. 
     Again, the cells were plated onto a series of agarose plates with increasing kanamycin concentration. As shown in  FIG.  2   , adenosine deaminase variants having efficient base editing activity were able to correct the mutations present in the kanamycin resistance gene and were selected for further analysis. Adenosine deaminase variant base editors showing efficient base editing in bacterial cells are described in Table 13. Mammalian expression vectors encoding base editors comprising the selected adenosine deaminase variants were generated. 
     Hek293T cells expressing a β-globin protein associated with sickle cell disease that contains an E6V (also termed E7V) mutation were used to test the editing efficiency of the adenosine deaminase variants ( FIGS.  3 A and  3 B ). These cells termed “Hek293T/HBBE6V” cells were transduced using lentiviral vectors expressing a base editing system that includes a fusion protein comprising the ABE8 base editors listed in Table 13. The ABE8 base editors were generated by cloning an adenosine deaminase variant into a scaffold that included a circular permutant Cas9 and a bipartite nuclear localization sequence. Circular permutant Cas9s are known in the art and described, for example, in Oakes et al., Cell 176, 254-267, 2019. These sequences are provided herein below. 
     Upregulation of fetal hemoglobin is a therapeutic approach to overcoming sickle cell disease.  FIG.  3 A  shows a therapeutically relevant site for upregulation of fetal hemoglobin. Editing adenosines at residues 5 and 8 can significantly reduce BCL11A binding, thereby increasing expression of fetal hemoglobin. Referring to  FIG.  3 A , the ABE8 base editors exhibited approximately 2-3 fold more base editing activity than the ABE7.10 base editor. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Novel Adenine Base Editors ABE8 
               
            
           
           
               
               
               
            
               
                 plasmid ID 
                 description 
                 function 
               
               
                   
               
            
           
           
               
               
               
            
               
                 280 
                 ABE8.1 
                 monomer_TadA*7.10 + Y147T 
               
               
                 281 
                 ABE8.2 
                 monomer_TadA*7.10 + Y147R 
               
               
                 282 
                 ABE8.3 
                 monomer_TadA*7.10 + Q154S 
               
               
                 283 
                 ABE8.4 
                 monomer_TadA*7.10 + Y123H 
               
               
                 284 
                 ABE8.5 
                 monomer_TadA*7.10 + V82S 
               
               
                 285 
                 ABE8.6 
                 monomer_TadA*7.10 + T166R 
               
               
                 286 
                 ABE8.7 
                 monomer_TadA*7.10 + Q154R 
               
               
                 287 
                 ABE8.8 
                 monomer_Y147R_Q154R_Y123H 
               
               
                 288 
                 ABE8.9 
                 monomer_Y147R_Q154R_I76Y 
               
               
                 289 
                 ABE8.10 
                 monomer_Y147R_Q154R_T166R 
               
               
                 290 
                 ABE8.11 
                 monomer_Y147T_Q154R 
               
               
                 291 
                 ABE8.12 
                 monomer_Y147T_Q154S 
               
               
                 292 
                 ABE8.13 
                 monomer_H123Y123H_Y147R_Q154R_I76Y 
               
               
                 293 
                 ABE8.14 
                 heterodimer_TadA*7.10 + Y147T 
               
               
                 294 
                 ABE8.15 
                 heterodimer_TadA*7.10 + Y147R 
               
               
                 295 
                 ABE8.16 
                 heterodimer_TadA*7.10 + Q154S 
               
               
                 296 
                 ABE8.17 
                 heterodimer_TadA*7.10 + Y123H 
               
               
                 297 
                 ABE8.18 
                 heterodimer_TadA*7.10 + V82S 
               
               
                 298 
                 ABE8.19 
                 heterodimer_TadA*7.10 + T166R 
               
               
                 299 
                 ABE8.20 
                 heterodimer_TadA*7.10 + Q154R 
               
               
                 300 
                 ABE8.21 
                 heterodimer_Y147R_Q154R_Y123H 
               
               
                 301 
                 ABE8.22 
                 heterodimer_Y147R_Q154R_I76Y 
               
               
                 302 
                 ABE8.23 
                 heterodimer_Y147R_Q154R_T166R 
               
               
                 303 
                 ABE8.24 
                 heterodimer_Y147T_Q154R 
               
               
                 304 
                 ABE8.25 
                 heterodimer_Y147T_Q154S 
               
               
                   
               
            
           
         
       
     
     Referring to  FIG.  4   , the ABE8 base editors were introduced into Hek293T/HBBE6V cells along with 18, 19, 20, 21, or 22 nucleotide guide RNAs targeting the polynucleotide encoding HBB E6V. The ABE8 editors showed increased editing efficiency when fused to circular permutant (Cp)-Cas9. In total, 40 different ABE8 constructs (Table 14) and three ABE7.10 constructs were tested for editing activity in Hek293T/HBBE6V cells. The sequence of exemplary constructs follows. To evaluate the specificity of editing, target and unintended or bystander mutations were monitored ( FIG.  5   ). Unintended editing of an adenosine in codon 5 was silent. However, unintended editing of codon 9 resulted in a serine to proline mutation. Referring again to  FIG.  5   , multiple ABE8 base editors showed increased editing efficiency and specificity compared to the ABE7.10 editors, and none of the editors had significant bystander editing that led to the serine to proline missense mutation. 
     Further analysis of selected ABE8 base editors and an ABE7.10 base editor control was carried out in fibroblast cells containing the sickle cell mutation. As shown in  FIG.  6   , the ABE8 editors had increased base editing activity compared to the ABE7.10. ABE8.18 showed approximately 70% efficiency. The selected ABE8 editors also displayed unprecedented specificity. Importantly, the average INDEL formation for all ABE8 editors was less than 0.1%. 
     
       
         
           
               
               
               
             
               
                 TABLE 14 
               
               
                   
               
               
                 plasmid ID 
                 description 
                 function 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 335 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.1 
                 monomer_TadA*7.10 + Y147T 
               
               
                 336 
                 NGC PAM CP5 variant ( S. pyogrouns  Cas9)_ABE8.2 
                 monomer_TadA*7.10 + Y147R 
               
               
                 337 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.3 
                 monomer_TadA*7.10 + Q154S 
               
               
                 338 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.4 
                 monomer_TadA*7.10 + Y123H 
               
               
                 339 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.5 
                 monomer_TadA*7.10 + V82S 
               
               
                 340 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.6 
                 monomer_TadA*7.10 + T166R 
               
               
                 341 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.7 
                 monomer_TadA*7.10 + Q154R 
               
               
                 342 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.8 
                 monomer_Y147R_Q154R_Y123H 
               
               
                 343 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.9 
                 monomer_Y147R_Q154R_I76Y 
               
               
                 344 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.10 
                 monomer_Y147R_Q154R_T166R 
               
               
                 345 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.11 
                 monomer_Y147T_Q154R 
               
               
                 346 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.12 
                 monomer_Y147T_Q154S 
               
               
                 347 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.13 
                 monomer_H123Y123H_Y147R_Q154R_I76Y 
               
               
                 348 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE7.10 
                 monomer_deletion at TadA7.10* residue 149 
               
               
                 349 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE7.10 
                 monomer_deletion at TadA7.10* residue 150 
               
               
                 350 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE7.10 
                 monomer_deletion at TadA7.10* residue 151 
               
               
                 351 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE7.10 
                 monomer_deletion at TadA7.10* residue 152 
               
               
                 352 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE7.10 
                 monomer_deletion at TadA7.10* residue 153 
               
               
                 353 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE7.10 
                 monomer_deletion at TadA7.10* residue 154 
               
               
                 354 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE7.10 
                 monomer_deletion at TadA7.10* residue 155 
               
               
                 355 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE7.10 
                 monomer_deletion at TadA7.10* residue 156 
               
               
                 356 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE7.10 
                 monomer_deletion at TadA7.10* residue 157 
               
               
                 357 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.14 
                 heterodimer_TadA*7.10 + Y147T 
               
               
                 358 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.15 
                 heterodimer_TadA*7.10 + Y147R 
               
               
                 359 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.16 
                 heterodimer_TadA*7.10 + Q154S 
               
               
                 360 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.17 
                 heterodimer_TadA*7.10 + Y123H 
               
               
                 361 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.18 
                 heterodimer_TadA*7.10 + V82S 
               
               
                 362 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.19 
                 heterodimer_TadA*7.10 + T166R 
               
               
                 363 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.20 
                 heterodimer_TadA*7.10 + Q154R 
               
               
                 364 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.21 
                 heterodimer_Y147R_Q154R_Y123H 
               
               
                 365 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.22 
                 heterodimer_Y147R_Q154R_I76Y 
               
               
                 366 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.23 
                 heterodimer_Y147R_Q154R_T166R 
               
               
                 367 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.24 
                 heterodimer_Y147T_Q154R 
               
               
                 368 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.25 
                 heterodimer_Y147T_Q154S 
               
               
                 369 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE8.26 
                 heterodimer_H123Y123H_Y147R_Q154R_I76Y 
               
               
                 370 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE7.10 
                 heterodimer_deletion at TadA7.10* residue 149 
               
               
                 371 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE7.10 
                 heterodimer_deletion at TadA7.10* residue 150 
               
               
                 372 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE7.10 
                 heterodimer_deletion at TadA7.10* residue 151 
               
               
                 373 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE7.10 
                 heterodimer_deletion at TadA7.10* residue 152 
               
               
                 374 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE7.10 
                 heterodimer_deletion at TadA7.10* residue 153 
               
               
                 375 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE7.10 
                 heterodimer_deletion at TadA7.10* residue 154 
               
               
                 376 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE7.10 
                 heterodimer_deletion at TadA7.10* residue 155 
               
               
                 377 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE7.10 
                 heterodimer_deletion at TadA7.10* residue 156 
               
               
                 378 
                 NGC PAM CP5 variant ( S. pyogenes  Cas9)_ABE7.10 
                 heterodimer_deletion at TadA7.10* residue 157 
               
               
                   
               
            
           
         
       
     
     Example 2: Codon Optimization and NLS Choice for ABE8 Design 
     It has been established that Cas9 codon usage and nuclear localization sequence can dramatically alter genome editing efficiencies in eukaryotes (see e.g., Kim, S. et al., Rescue of high-specificity Cas9 variants using sgRNAs with matched 5′ nucleotides. Genome Biol 18, 218, doi:10.1186/s13059-017-1355-3 (2017); Mikami, M. et al., Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice. Plant Mol Biol 88, 561-572, doi:10.1007/s11103-015-0342-x (2015); Jinek, M. et al., RNA-programmed genome editing in human cells. Elife 2, e00471, doi:10.7554/eLife.00471 (2013)). The original Cas9n component of base editors contains six potential polyadenylation sites, leading to poor expression in eukaryotes (see e.g., Kim, S. et al., Rescue of high-specificity Cas9 variants using sgRNAs with matched 5′ nucleotides. Genome Biol 18, 218, doi:10.1186/s13059-017-1355-3 (2017); Komor, A. C. et al., Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420-424, doi:10.1038/nature17946 (2016); Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464-471, doi:10.1038/nature24644 (2017)). Replacing this with an extensively optimized codon sequence improves base editing efficiencies (see e.g., Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823, doi:10.1126/science.1231143 (2013); Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat Biotechnol, doi:10.1038/nbt.4172 (2018); Zafra, M. P. et al. Optimized base editors enable efficient editing in cells, organoids and mice. Nat Biotechnol, doi:10.1038/nbt.4194 (2018)). 
     DNA on-target ( FIG.  9 A,  9 B ), DNA off-target ( FIG.  9 C,  9 D ) and RNA off-target ( FIG.  9 E ) base editing frequencies associated with four ABE constructs were assessed, all of which contain a codon-optimized Cas9(D10A): i) ABE7.10, which has a single C-terminal BP-SV40 NLS; ii) monoABE7.10 which lacks the 5′ TadA wild-type portion of ABE7.10; iii) ABEmax, which contains codon-optimized TadA regions and two BPNLS sequences; and iv) ABEmax(-BPNLS), which has the TadA codon optimization as ABEmax but contains a single C-terminal BP-SV40 NLS. 
     All four constructs displayed remarkably similar on-target editing efficiencies, indicating that the NLS architecture and TadA codon optimization are not determining for on-target editing efficiency ( FIG.  9 A,  9 B ). The off-target profiles were also highly similar, but ABEmax displayed significantly greater DNA off-target editing (p=0.00027, Students&#39; two-tailed T test) at one site when compared to ABE7.10 ( FIG.  9 C,  9 D ). ABEmax(-NBPNLS) displayed a 1.6-fold greater mean frequency of RNA off-target editing than ABE7.10 ( FIG.  9 E ). 
     Example 3: Superior Adenine Base Editors with Expanded Targeting Range 
     ABE is a molecular machine comprising an evolved  E. coli  tRNAARG modifying enzyme, TadA, covalently fused to a catalytically-impaired Cas9 protein (D10A nickase Cas9, nCas9) ( FIGS.  7 A and  7 B ). To overcome limitation of prior adenine base editors, the stringency of the bacterial selection system was increased by designing ABEs that must make three concurrent A•T to G•C reversion edits to survive antibiotic selection. In the prior ABE evolutions, TadA libraries were created via error-prone PCR. Contrastingly, a synthetic library of TadA alleles was utilized containing all 20 canonical amino acid substitutions at each position of TadA, with an average frequency of 1-2 nucleotide substitution mutations per library member. This chemical library enabled access to a greater sequence space than is achievable with error-prone PCR techniques. 
     About 300 clones were isolated and subsequently sequenced. From the resultant sequencing data, eight mutations were identified within TadA* that were enriched with high frequency (Tables 7 and 9). Six of the eight identified amino acid mutations required at least two nucleobase changes per codon, which were unobserved with the previous TadA error-prone libraries. Two of the enriched mutations alter residues proximal to the active site of adenine deamination (176 and V82) ( FIG.  7 C ). In addition to the four previously reported mutations in the C-terminal alpha helix of TadA*7.10, two new mutations were observed within the same alpha-helix (Y147R and Q154R) ( FIG.  7 C ). This highly mutated alpha-helix is necessary for robust product formation because upon truncation, base editing efficiency was substantially reduced ( FIGS.  10 A and  10 B ). 
     To test the activity of TadA* variants in mammalian cells, BE codon optimization and NLS orientation was utilized with the most favorable on- and off-target profile (see Example 2;  FIGS.  9 A- 9 E ). The eight enriched TadA* mutations were incorporated into ABE7.10 in various combinations, yielding forty new ABE8 variants (Tables 7 and 9). ABE8 constructs were made where the TadA region of ABE is either heterodimeric fusion of an inactive (wild-type) and active (evolved) TadA* protomer or a single protomer of an engineered TadA*, resulting in about a 500 base-pair smaller editor. These architectural variants are referred to as ABE8.x-d and ABE8.x-m respectively (Tables 7 and 9). 
     First, these forty constructs were evaluated for their on-target DNA editing efficiencies relative to ABE7.10 across eight genomic sites containing target A bases in positions which range from 2 to 20 (where NGG PAM=positions 21, 22, 23) within the canonical 20-nt  S. pyogenes  protospacer ( FIG.  11   ). The N-terminal wild-type TadA construct was not necessary for robust DNA editing using ABE8. Indeed, constructs containing the N-terminal, wild-type TadA (ABE8.x-d) perform similarly in terms of both editing window preference, total DNA editing outcome, and INDEL frequency relative to its economized architecture (ABE8.x-m) ( FIG.  7 D ,  FIG.  11   ,  FIG.  12   ). Although intra-construct, TadA(wt):TadA*8 dimerization may not be necessary for ABE8 activity, it does not preclude the possibility of in trans TadA*8:TadA*8 dimerization occurring between ABE8 expressed base editors. 
     Across all sites tested, ABE8s result in about 1.5× higher editing at canonical positions (A5-A7) in the protospacer and about 3.2× higher editing at non-canonical positions (A3-A4, A8-A10) compared with ABE7.10 ( FIG.  13   ). Fold differences vary between sequence of the target, position of the “A” within the target window and ABE8 construct identity ( FIG.  7 D ,  FIG.  11   ,  FIG.  13   ). Overall, the median change in editing across all positions, in all sites tested is 1.94-fold relative to ABE7.10 (range 1.34-4.49). 
     Next, from the large ABE8 pool of forty constructs, a sub-set of ABE8 constructs (ABE8.8-m, ABE8.13-m, ABE8.17-m, ABE8.20-m, ABE8.8-d, ABE8.13-m, ABE8.17-d and ABE8.20-d) were selected to evaluate in greater detail. These constructs represent ABE8s with distinct differences in editing performance amongst the 8 genomic sites as determined through a hierarchical clustering analysis ( FIG.  14   ). These ABE8s all significantly outperform ABE7.10 at all genomic sites tested (P-value=0.0006871, two-tailed Wilcoxon rank sum test) and encompass a variety of combinations of mutations identified from the ABE8 directed evolution campaign ( FIG.  15    and  FIG.  16   ). 
     Although ABE variants recognizing non-NGG PAMs have been described, editing efficiencies of these constructs are decreased in many instances when compared to outcomes observed with  S. pyogenes  Cas9 targeting NGG PAM sequences (see e.g., Huang, T. P. et al. Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors. Nat Biotechnol 37, 626-631, doi:10.1038/s41587-019-0134-y (2019); Hua, K. et al., Expanding the base editing scope in rice by using Cas9 variants. Plant Biotechnol J, doi:10.1111/pbi.12993 (2018); Yang, L. et al., Increasing targeting scope of adenosine base editors in mouse and rat embryos through fusion of TadA deaminase with Cas9 variants. Protein Cell 9, 814-819, doi:10.1007/s13238-018-0568-x (2018)). To determine whether evolved deaminase also increases the editing efficiencies at target sites bearing non-NGG PAMs, ABE8 editors were created that replace  S. pyogenes  Cas9 with an engineered  S. py . variant, NG-Cas9 (PAM: NG) (Nishimasu, H. et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science (2018)) or  Staphylococcus aureus  Cas9 (SaCas9, PAM: NNGRRT) (Ran, F. A. et al. In vivo genome editing using  Staphylococcus aureus  Cas9. Nature 520, 186-191, doi:10.1038/nature14299 (2015)). Median increases were observed in A•T to G•C editing frequencies of 1.6- and 2.0-fold, respectively, when comparing ABE8 variants to ABE7.10 for both SpCas9-NG (NG-ABE8.x-m/d) and SaCas9 (Sa-ABE8.x-m/d) ( FIGS.  8 A,  8 B, and  17 - 20   ). Similar to SpCas9-ABE8, the greatest differences in editing efficiencies between ABE7.10 and ABE8 constructs for the non-NGG PAM variants are observed at target A positions located at the periphery of the preferred position in the editing window ( S. pyogenes : positions 4-8 ; S. aureus : positions 6-13; see also Rees, H. A. &amp; Liu, D. R., Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet 19, 770-788, doi:10.1038/s41576-018-0059-1 (2018)). ABE8 orthologs utilizing non-NGG PAMs broaden the targeting scope for efficient A base editing. 
     For applications where minimizing indel formation is necessary, the effect of replacing the catalytically impaired D10A nickase mutant of Cas9 with a catalytically “dead” version of Cas9 (D10A+H840A) (see Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821, doi:10.1126/science.1225829 (2012)) was explored in the core eight ABE8 constructs (“dC9-ABE8.x-m/d”). By replacing the nickase with dead Cas9 in ABEs, a &gt;90% reduction in indel frequency was observed for dC9-ABE8 variants relative to ABE7.10 while maintaining a significantly higher (2.1-fold), on-target DNA editing efficiency ( FIGS.  8 C,  21 ,  22 ,  23 A, and  23 B ). Although indels above background are observed, frequencies ranged only from 0.3-0.8% at sites tested. Encouragingly, dC9-ABE8 variants only have a median 14% reduction in on-target DNA editing efficiencies relative to canonical ABE8s. 
     Another class of undesired ABE-mediated genome edit at an on-target locus is an ABE-dependent cytosine to uracil (C•G to T•A) conversion (see Grunewald, J. et al., CRISPR DNA base editors with reduced RNA off-target and self-editing activities. Nat Biotechnol 37, 1041-1048, doi:10.1038/s41587-019-0236-6 (2019); Lee, C. et al. CRISPR-Pass: Gene Rescue of Nonsense Mutations Using Adenine Base Editors. Mol Ther 27, 1364-1371, doi:10.1016/j.ymthe.2019.05.013 (2019)). At the eight target sites tested, the 95 th  percentile of C-to-T editing was measured to be 0.45% with ABE8 variants and 0.15% with ABE7.10-d or -m, indicating that on-target cytosine deamination with ABEs can occur but the frequencies are generally very low ( FIG.  24   ). Together, these data indicate that ABE8s retain high specificity for A-to-G conversion compared to other, often undesirable byproducts. 
     Example 4: DNA On-Target and sgRNA-Dependent DNA Off-Target Editing by ABE8 Constructs Improve Specificity for DNA 
     As with all base editors, ABE8s have the potential to act at off-target loci in the genome and transcriptome (see e.g., Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464-471, doi:10.1038/nature24644 (2017); Komor, A. C., et al., Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420-424, doi:10.1038/nature17946 (2016); Grunewald, J. et al. CRISPR DNA base editors with reduced RNA off-target and self-editing activities. Nat Biotechnol 37, 1041-1048, doi:10.1038/s41587-019-0236-6 (2019); Rees, H. A., et al., Analysis and minimization of cellular RNA editing by DNA adenine base editors. Sci Adv 5, eaax5717, doi:10.1126/sciadv.aax5717 (2019); Rees, H. A. et al. Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat Commun 8, 15790, doi:10.1038/ncomms15790 (2017); Jin, S. et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364, 292-295, doi:10.1126/science.aaw7166 (2019); Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289-292, doi:10.1126/science.aav9973 (2019); Lee, H. K., et al., Cytosine but not adenine base editor generates mutations in mice. Biorxiv, doi:doi.org/10.1101/731927 (2019); Grunewald, J. et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433-437, doi:10.1038/s41586-019-1161-z (2019); Zhou, C. et al. Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature 571, 275-278, doi:10.1038/s41586-019-1314-0 (2019)). 
     Base editing at four on-target ( FIGS.  25 A and  25 B ) and twelve previously identified sgRNA-associated off-target loci in genomic DNA (Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 33, 187-197, doi:10.1038/nbt.3117 (2015)) ( FIGS.  25 E and  25 F ) were measured, all of which were confirmed to be true Cas9 off-target loci in HEK293T cells ( FIG.  26   ). As expected from their increased activity at on-target loci, ABE8 constructs exhibit 3-6-fold greater DNA off-target editing frequencies than ABE7.10. Whilst this is a caveat for use of ABE8 constructs, careful choice and analysis of the sgRNA can substantially decrease sgRNA-dependent off-target editing (see Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 33, 187-197, doi:10.1038/nbt.3117 (2015); Yeh, W. H., et al., In vivo base editing of post-mitotic sensory cells. Nat Commun 9, 2184, doi:10.1038/s41467-018-04580-3 (2018)). For applications requiring use of promiscuous sgRNAs, installation of the DNA- and RNA-specificity enhancing V106W mutation (Rees, H. A., Wilson, C., Doman, J. L. &amp; Liu, D. R. Analysis and minimization of cellular RNA editing by DNA adenine base editors. Sci Adv 5, eaax5717, doi:10.1126/sciadv.aax5717 (2019)) into the TadA domain of ABE8.17m can decrease the DNA off-target editing 2.6-fold while maintaining levels of on-target editing exceeding those of ABE7.10 ( FIGS.  25 C,  25 D,  25 G and  25 H ). 
     To measure the sgRNA-independent off-target activity of ABE8s, targeted amplification and high throughput sequencing of cellular RNAs was performed in HEK293T cells treated with ABEs (see Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464-471, doi:10.1038/nature24644 (2017); Rees, H. A., et al., Analysis and minimization of cellular RNA editing by DNA adenine base editors. Sci Adv 5, eaax5717, doi:10.1126/sciadv.aax5717 (2019)). In this assay, ABE8s displayed between 2.3-5.3-fold greater mean frequencies of cellular RNA adenosine deamination as compared to ABE7.10 ( FIG.  25 A ). 
     To mitigate spurious RNA off target editing, previously published mutations (Grunewald, J. et al. CRISPR DNA base editors with reduced RNA off-target and self-editing activities. Nat Biotechnol 37, 1041-1048, doi:10.1038/s41587-019-0236-6 (2019); Rees, H. A., et al., Analysis and minimization of cellular RNA editing by DNA adenine base editors. Sci Adv 5, eaax5717, doi:10.1126/sciadv.aax5717 (2019); Grunewald, J. et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433-437, doi:10.1038/s41586-019-1161-z (2019); Zhou, C. et al. Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature 571, 275-278, doi:10.1038/s41586-019-1314-0 (2019)) were installed into the TadA portion of the deaminase enzyme into ABE8.17-m to evaluate reductions in off-target editing frequencies. All of these mutations decreased the on-target editing frequencies of ABE8.17-m to differing extents, with V106W and F148A impairing ABE8 the least ( FIGS.  25 C and  25 D ). Of these, only V106W was able to substantially reduce the level of off-target RNA and DNA editing ( FIG.  25 B ). Thus, the inclusion of the V106W mutation to ABE8 is applicable where transient perturbation of the cellular transcriptome must be avoided, or for use with promiscuous sgRNAs. 
     Example 5: Adenine Base Editors for the Treatment of Hematological Disorders 
     ABE8 constructs were evaluated in human hematopoietic stem cells (HSC). Ex vivo manipulation and/or editing of HSCs prior to administration to patients as a cell therapy is a promising approach for the treatment of hematological disorders. It has been previously demonstrated that ABEs can introduce a T to C substitution at the −198 position of the promoter region of HBG1/2 (Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464-471, doi:10.1038/nature24644 (2017)). This naturally occurring allele yields Hereditary Persistence of Fetal Hemoglobin (HPFH) resulting in increased levels of γ-globin into adulthood, which can mitigate the defects in β-globin seen in sickle cell disease and β-thalassemia (Wienert, B. et al. KLF1 drives the expression of fetal hemoglobin in British HPFH. Blood 130, 803-807, doi:10.1182/blood-2017-02-767400 (2017)). With the goal of reproducing the HPFH phenotype and evaluating the clinical relevance of ABE8, CD34+ hematopoietic stem cells were isolated from two donors and transfected with mRNA encoding ABE8 editors and end-modified sgRNA placing the target A at position 7 within the protospacer. 
     The average ABE8 editing efficiencies at the −198 HBG1/2 promoter target site were 2-3× higher than either ABE7.10 construct at early time points (48 h), and 1.3-2-fold higher than either ABE7.10 at the later time (144 h) ( FIG.  27 A ,  FIG.  28   ,  FIG.  29   ). These kinetic distinctions are clinically important for ex vivo therapies in which cell culturing must be kept to a minimum prior to administration of cell therapy. 
     Next, the amount of γ-globin protein produced following ABE treatment and erythrocyte differentiation was quantified by UPLC ( FIGS.  30 - 50   ). A 3.5-fold average increase in % γ-globin/α-globin expression in erythrocytes derived from the ABE8 treatment groups was observed when compared to mock treated cells and about a 1.4-fold increase when comparing ABE8.13-d to levels achieved with ABE7.10-m/d ( FIG.  27 B ). It is predicted that 20% HbF is required to ameliorate symptoms of sickle cell disease and β-thalassemia patients likely require even higher minimum levels (see e.g., Canver, M. C. &amp; Orkin, S. H. Customizing the genome as therapy for the beta-hemoglobinopathies. Blood 127, 2536-2545, doi:10.1182/blood-2016-01-678128 (2016); Fitzhugh, C. D. et al. At least 20% donor myeloid chimerism is necessary to reverse the sickle phenotype after allogeneic HSCT. Blood 130, 1946-1948, doi:10.1182/blood-2017-03-772392 (2017)). The γ-globin levels observed following ABE8 treatment surpassed this threshold. 
     Overall, ABE8s recreated a naturally occurring hereditary persistence of fetal hemoglobin (HPFH) allele at the promoter of the γ-globin genes HBG1 and HBG2, achieving editing efficiencies of up to 60% in human CD34+ cell cultures and a corresponding upregulation of gamma globin expression in differentiated erythrocytes. 
     Example 6: Complementary Base Editing Approaches for the Treatment of Sickle Cell Disease and Beta Thalassemia 
     Sickle cell disease (SCD) and Beta thalassemia are disorders of beta globin production and function that lead to severe anemia and significant disease complications across a multitude of organ systems. Autologous transplantation of hematopoietic stem cells engineered through the upregulation of fetal hemoglobin (HbF) or correction of the beta globin gene have the potential to reduce disease burden in patients with beta hemoglobinopathies. Base editing is a recently developed technology that enables precise modification of the genome without the introduction of double strand DNA breaks. 
     Gamma globin gene promoters were comprehensively screened with cytosine and adenine base editors (ABE) for the identification of alterations that would derepress HbF. Three regions were identified that significantly upregulated HbF, and the most effective nucleotide residue conversions are supported by natural variation seen in patients with hereditary persistence of fetal hemoglobin (HPFH). ABEs have been developed that significantly increase the level of HbF following nucleotide conversion at key regulatory motifs within the HBG1 and HBG2 promoters. CD34+ hematopoietic stem and progenitor cells (HSPC) were purified at clinical scale and edited using a process designed to preserve self-renewal capacity. Editing at two independent sites with different ABEs reached 94 percent and resulted in up to 63 percent gamma globin by UPLC ( FIGS.  51 A- 51 E ). The levels of HbF observed should afford protection to the majority of SCD and Beta thalassemia patients based on clinical observations of HPFH and non-interventional therapy that links higher HbF dosage with milder disease (Ngo et al., 2011 Brit J Hem; Musallam et al., 2012 Blood). 
     Directly correcting the Glu6Val mutation of SCD has been a recent goal of genetic therapies designed for the SCD population. Current base editing technology cannot yet convert mutations like those that result from the A-T transversion in sickle beta globin; however, ABE variants have been designed to recognize and edit the opposite stranded adenine residue of valine. This results in the conversion of valine to alanine and the production of a naturally occurring variant known as Hb G-Makassar. Beta globin with alanine at this position does not contribute to polymer formation, and patients with Hb G-Makassar present with normal hematological parameters and red blood cell morphology. SCD patient fibroblasts edited with these ABE variants achieve up to 70 percent conversion of the target adenine ( FIG.  52 A ). CD34 cells from healthy donors were then edited with a lead ABE variant, targeting a synonymous mutation in an adjacent proline that resides within the editing window and serves as a proxy for editing the SCD mutation. The average editing frequency was 40 percent ( FIG.  52 B ). Donor myeloid chimerism documented at these levels in the allogeneic transplant setting exceeds the 20 percent that is required for reversing the sickle phenotype (Fitzhugh et al, 2017 Blood). 
     Example 7: Materials and Methods 
     General Methods: 
     All cloning was conducted via USER enzyme (New England Biolabs) cloning methods (see Geu-Flores et al., USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Res 35, e55, doi:10.1093/nar/gkm106 (2007)) and templates for PCR amplification were purchased as bacterial or mammalian codon optimized gene fragments (GeneArt). Vectors created were transformed into Mach T1 R  Competent Cells (Thermo Fisher Scientific) and maintained at −80 C for long-term storage. All primers used in this work were purchased from Integrated DNA Technologies and PCRS were carried out using either Phusion U DNA Polymerase Green MultiPlex PCR Master Mix (ThermoFisher) or Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs). All plasmids used in this work were freshly prepared from 50 mL of Machl culture using ZymoPURE Plasmid Midiprep (Zymo Research Corporation) which involves an endotoxin removal procedure. Molecular biology grade, Hyclone water (GE Healthcare Life Sciences) was used in all assays, transfections, and PCR reactions to ensure exclusion of DNAse activity. 
     Amino acid sequences of sgRNAs used for Hek293T mammalian cell transfection are provided in Table 15 below. The 20-nt target protospacer is shown in bold font. When a target DNA sequence did not start with a ‘G,’ a ‘G’ was added to the 5′ end of the primer since it has been established that the human U6 promoter prefers a ‘G’ at the transcription start site (see Cong, L. et al., Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823, doi:10.1126/science.1231143 (2013)). The pFYF sgRNA plasmid described previously was used as a template for PCR amplification. 
                     TABLE 15                  Sequences of sgRNAs used for Hek293T mammalian cell transfection.                             Site   RNA protospacer sequence   Cas9 scaffold   PAM                1   GAACACAAAGCAUAGACUGC (SEQ     A pyogenes     NGG           ID NO: 310)                        2   GGGAAAGACCCAGCAUCCGU (SEQ     S. pyogenes     NGG           ID NO: 311)                        3   GCUCCCAUCACAUCAACCGG(SEQ     S. pyogenes     NGG           ID NO: 312)                        4   GGUGAGUGAGUGUGUGCGUG (SEQ     S. pyogenes     NGG           ID NO: 313)                        5   GGCUUCAGGUUCUAAAUGAG (SEQ     S. pyogenes     NGG           ID NO: 314)                        6   GCAGAGAGUCGCCGUCUCCA (SEQ     S. pyogenes     NGG           ID NO: 315)                        7   GUGUAAGACCUCAAAAGCAC (SEQ     S. pyogenes     NGG           ID NO: 316)                        8   GAUGAGAAGGAGAAGUUCUU (SEQ     5. pyogenes     NGG           ID NO: 317)                        9   GAGGACAAAGUACAAACGGC (SEQ     S. pyogenes     AGA           ID NO: 318)                       10   GCCACCACAGGGAAGCUGGG (SEQ     S. pyogenes     TGA           ID NO: 319)                       11   GCUCUCAGGCCCUGUCCGCA (SEQ     S. pyogenes     CGT           ID NO: 320)                       12   GAGCAAAUACCAGAGAUAAG (SEQ     S. pyogenes     AGA           ID NO: 321)                       13   GAUCAGGAAAUAGAGCCACA (SEQ     S. pyogenes     GGC           ID NO: 322)                       14   GCCCAUCCCUGAGUCCAGCG(SEQ     S. pyogenes     AGC           ID NO: 323)                       15   GAACACGAAGACAUCUGAAGGUA     S. aureus     TTGAAT           (SEQ ID NO: 324)                       16   GAUUUACAGCCUGGCCUUUGGGG     S. aureus     TCGGGT           (SEQ ID NO: 325)                       17   GGAGAGAAAGAGAAGUUGAUUG     S. aureus     ATGGGT           (SEQ ID NO: 326)                       18   GAGGGUGAGGGAUGAGAUAAUG     S. aureus     ATGAGT           (SEQ ID NO: 327)                       19   GGUGGAGGAGGGUGCAUGGGGU     S. aureus     CAGAAT           (SEQ ID NO: 328)                       20   GCUGUUGCAUGAGGAAAGGGAC     S. aureus     TAGAGT           (SEQ ID NO: 329)                       HEK2   GAACACAAAGCAUAGACUGC (SEQ     S. pyogenes     CGG           ID NO: 310)                       HEK3   GGCCCAGACUGAGCACGUGA (SEQ     S. pyogenes     TGG           ID NO: 330)                       HEK4   GGCACUGCGGCUGGAGGUGG (SEQ     S. pyogenes     GGG           ID NO: 331)                       LDLR   GCAGAGCACUGGAAUUCGUCA     S. pyogenes     GGG           (SEQ ID NO: 332)                    
sgRNA scaffold sequences are as follows:
 
     
       
         
           
               
            
               
                   S. pyogenes . 
               
               
                 (SEQ ID NO: 333) 
               
               
                 GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA 
               
               
                 CUUGAAAAAGUGGCACCGAGUCGGUGC 
               
               
                   
               
               
                   S. aureus . 
               
               
                 (SEQ ID NO: 334) 
               
               
                 GUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACUAAAACAAGGCAA 
               
               
                 AAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGA 
               
            
           
         
       
     
     Generation of Input Bacterial TadA* Libraries for Directed Evolution 
     The TadA*8.0 library was designed to encode all 20 amino acids at each amino acid position in the TadA*7.10 open reading frame (Gaudelli, N. M. et al., Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464-471, doi:10.1038/nature24644 (2017)). Each TadA*8.0 library member contained about 1-2 new coding mutations and was chemically synthesized and purchased from Ranomics Inc (Toronto, Canada). The TadA*8.0 library was PCR amplified with Phusion U Green MultiPlex PCR Master Mix and USER-assembled into a bacterial vector optimized for ABE directed evolution (Gaudelli, N. M. et al., Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464-471, doi:10.1038/nature24644 (2017)). 
     Bacterial Evolution of TadA Variants 
     Directed evolution of ABE containing the TadA*8 library was conducted as previously described (Gaudelli, N. M. et al., Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464-471, doi:10.1038/nature24644 (2017)) with the following changes: i)  E. coli  10 betas (New England Biolabs) were used as the evolution host; and ii) survival on kanamycin relied on correction of three genetic inactivating components (e.g. survival required reversion of two stop mutations and one active site mutation in kanamycin). The kanamycin resistance gene sequence contains selection mutations for ABE8 evolution. After overnight co-culturing of selection plasmid and editor in 10 beta host cells, the library cultures were plated on 2×YT-agar medium supplemented with plasmid maintenance antibiotic and increasing concentrations of selection antibiotic, kanamycin (64-512 μg/mL). Bacteria were allowed to grow for 1 day and the TadA*8 portion of the surviving clones were Sanger sequenced after enrichment. Identified TadA*8 mutations of interest were then were then incorporated into mammalian expression vector via USER assembly. 
     General HEK293T and RPMI-8226 Mammalian Culture Conditions 
     Cells were cultured at 37° C. with 5% CO 2 . HEK293T cells [CLBTx013, American Type Cell Culture Collection (ATCC)] were cultured in Dulbecco&#39;s modified Eagles medium plus Glutamax (10566-016, Thermo Fisher Scientific) with 10% (v/v) fetal bovine serum (A31606-02, Thermo Fisher Scientific). RPMI-8226 (CCL-155, ATCC) cells were cultured in RPMI-1640 medium (Gibco) with 10% (v/v) fetal bovine serum (Gibco). Cells were tested negative for  mycoplasma  after receipt from supplier. 
     Hek293T Plasmid Transfection and gDNA Extraction 
     HEK293T cells were seeded onto 48-well Poly-D-Lysine treated BioCoat plates (Corning) at a density of 35,000 cells/well and transfected 18-24 hours after plating. Cells were counted using a NucleoCounter NC-200 (Chemometec). To these cells were added 750 ng of base editor or nuclease control, 250 ng of sgRNA, and 10 ng of GFP-max plasmid (Lonza) diluted to 12.5 μL total volume in Opti-MEM reduced serum media (ThermoFisher Scientific). The solution was combined with 1.5 μl of Lipofectamine 2000 (ThermoFisher) in 11 μl of Opti-MEM reduced serum media and left to rest at room temperature for 15 min. The entire 25 μl mixture was then transferred to the pre-seeded Hek293T cells and left to incubate for about 120 h. Following incubation, media was aspirated and cells were washed two times with 250 μL of 1×PBS solution (ThermoFisher Scientific) and 100 μl of freshly prepared lysis buffer was added (100 mM Tris-HCl, pH 7.0, 0.05% SDS, 25 μg/mL Proteinase K (Thermo Fisher Scientific). Transfection plates containing lysis buffer were incubated at 37° C. for 1 hour and the mixture was transferred to a 96-well PCR plate and heated at 80° C. for 30 min. 
     Analysis of DNA and RNA Off-Target Editing for ABE Architecture and ABE8 Constructs 
     HEK293T cells were plated on 48-well poly-D-lysine coated plates (Corning) 16 to 20 hours before lipofection at a density of 30,000 cells per well in DMEM+Glutamax medium (Thermo Fisher Scientific) without antibiotics. 750 ng nickase or base editor expression plasmid DNA was combined with 250 ng of sgRNA expression plasmid DNA in 15 μl OPTIMEM+Glutamax. This was combined with 10 μl of lipid mixture, comprising 1.5 μl Lipofectamine 2000 and 8.5 μl OPTIMEM+Glutamax per well. Cells were harvested 3 days after transfection and either DNA or RNA was harvested. For DNA analysis, cells were washed once in 1×PBS, and then lysed in 100 μl QuickExtract™ Buffer (Lucigen) according to the manufacturer&#39;s instructions. For RNA harvest, the MagMAX™ mirVana™ Total RNA Isolation Kit (Thermo Fisher Scientific) was used with the KingFisher™ Flex Purification System according to the manufacturer&#39;s instructions. 
     Targeted RNA sequencing was performed largely as previously described (see Rees, H. A. et al., Analysis and minimization of cellular RNA editing by DNA adenine base editors. Sci Adv 5, eaax5717, doi:10.1126/sciadv.aax5717 (2019)). cDNA was prepared from the isolated RNA using the SuperScript IV One-Step RT-PCR System with EZDnase (Thermo Fisher Scientific) according to the manufacturer&#39;s instructions. The following program was used: 58° C. for 12 min; 98° C. for 2 min; followed by PCR cycles which varied by amplicon: for CTNNB1 and IP90: 32 cycles of [98° C. for 10 sec; 60° C. for 10 sec; 72° C. for 30 sec] and for RSL1D135 cycles of [98° C. for 10 sec; 58° C. for 10 sec; 72° C. for 30 sec]. No RT controls were run concurrently with the samples. Following the combined RT-PCR, amplicons were barcoded and sequenced using an Illumina Miseq as described above. The first 125 nt in each amplicon, beginning at the first base after the end of the forward primer in each amplicon, was aligned to a reference sequence and used for analysis of mean and maximum A-to-I frequencies in each amplicon ( FIGS.  53 A and  53 B ). 
     Off-target DNA sequencing was performed using previously published primers (see Komor, A. C. et al., Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420-424, doi:10.1038/nature17946 (2016); Rees, H. A. et al., Analysis and minimization of cellular RNA editing by DNA adenine base editors. Sci Adv 5, eaax5717, doi:10.1126/sciadv.aax5717 (2019)) listed in Table 16 below using a two-step PCR and barcoding method to prepare samples for sequencing using Illumina Miseq sequencers as above. 
                     TABLE 16                  HTS Primers used to amplify genomic sites:                     Primer Name   Sequence               fwd_site_1   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCAGCCCCATCTGTCAAACT           (SEQ ID NO: 335)               rev_site_1   TGGAGTTCAGACGTGTGCTCTTCCGATCTTGAATGGATTCCTTGGAAACAATGA           (SEQ ID NO: 336)               fwd_site_2   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGAGGGAGAGCCGTGTAGTT           (SEQ ID NO: 337)               rev_site_2   TGGAGTTCAGACGTGTGCTCTTCCGATCTGCCTCTCAAAGTGCTGGGAT (SEQ ID           NO: 338)               fwd_site_3   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCATCAGGCTCTCAGCTCAG           (SEQ ID NO: 339)               rev_site_3   TGGAGTTCAGACGTGTGCTCTTCCGATCTCTCGTGGGTTTGTGGTTGC (SEQ ID           NO: 340)               fwd_site_4   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCCCATTCCCTCTTTAGCCA           (SEQ ID NO: 341)               rev_site_4   TGGAGTTCAGACGTGTGCTCTTCCGATCTGAGCCGTTCCCTCTTTGCTA (SEQ ID           NO: 342)               fwd_site_5   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAACCTGTGTGACACTTGGCA           (SEQ ID NO: 343)               rev_site_5   TGGAGTTCAGACGTGTGCTCTTCCGATCTGTCTGGCCCAAGATCACACA (SEQ ID           NO: 344)               fwd_site_6   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNCACGGATAAAGACGCTGGGA           (SEQ ID NO: 345)               rev_site_6   TGGAGTTCAGACGTGTGCTCTTCCGATCTGGGGTCCCAGGTGCTGAC (SEQ ID           NO: 346)               fwd_site_7   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNTTGATTGTCTCCTTTGCCGC           (SEQ ID NO: 347)               rev_site_7   TGGAGTTCAGACGTGTGCTCTTCCGATCTTGACCCAGTGTTTGATAGATCAGT           (SEQ ID NO: 348)               fwd_site_8   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNCACCCCTTCAGTCCATGCTT           (SEQ ID NO: 349)               rev_site_8   TGGAGTTCAGACGTGTGCTCTTCCGATCTTCTGATGGGGAGGAACGAGT (SEQ ID           NO: 350)               fwd_site_9   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCAGCTCAGCCTGAGTGTTGA           (SEQ ID NO: 351)               rev_site_9   TGGAGTTCAGACGTGTGCTCTTCCGATCTGCCCACCCTAGTCATTGGAG (SEQ ID           NO: 352)               fwd_site_10   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTCAGAGGGACACACTGTGG           (SEQ ID NO: 353)               rev_site_10   TGGAGTTCAGACGTGTGCTCTTCCGATCTCACACTCACTCACCCACACA (SEQ ID           NO: 354)               fwd_site_11   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGTGTGGGTGAGTGAGTGTG           (SEQ ID NO: 355)               rev_site_11   TGGAGTTCAGACGTGTGCTCTTCCGATCTCACCAAGGTTCACAGCCTGA (SEQ ID           NO: 356)               fwd_site_12   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTGTCTCTGCCTGTAGCTGC           (SEQ ID NO: 357)               rev_site_12   TGGAGTTCAGACGTGTGCTCTTCCGATCTCGCTCTGGGCTTCATCTTCA (SEQ ID           NO: 358)               fwd_site_13   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGGGATTATGGGTGTGAGCC           (SEQ ID NO: 359)               rev_site_13   TGGAGTTCAGACGTGTGCTCTTCCGATCTTGCCTTCCTCCTCTCTCTCC (SEQ ID           NO: 360)               fwd_site_14   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCAGACCAGATTCGGAGAA           (SEQ ID NO: 361)               rev_site_14   TGGAGTTCAGACGTGTGCTCTTCCGATCTGTTCAGTTTCCAGGGGGTCC (SEQ ID           NO: 362)               fwd_site_15   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCGCACAGCCTTAGTTCAA           (SEQ ID NO: 363)               rev_site_15   TGGAGTTCAGACGTGTGCTCTTCCGATCTAACTTGAAGAGACGGCAGCA (SEQ ID           NO: 364)               fwd_site_16   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCCCCAGCTACAGAAAGGTC           (SEQ ID NO: 365)               rev_site_16   TGGAGTTCAGACGTGTGCTCTTCCGATCTATTTCCACCGCAAAATGGCC (SEQ ID           NO: 366)               fwd_site_17   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCACTTCAGCCCAGGAGTAT           (SEQ ID NO: 367)               rev_site_17   TGGAGTTCAGACGTGTGCTCTTCCGATCTTGTGTATGGTGAGAGGTAGGGA (SEQ           ID NO: 368)               fwd_site_18   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTCTGAGGTCACACAGTGGG           (SEQ ID NO: 369)               rev_site_18   TGGAGTTCAGACGTGTGCTCTTCCGATCTCTGAGAGCAGGGACCACATC (SEQ ID           NO: 370)               fwd_site_19   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGGAGGTGGAGAGAGGATGT           (SEQ ID NO: 371)               rev_site_19   TGGAGTTCAGACGTGTGCTCTTCCGATCTACTCTTCCTGAGGTCTAGGAACCCG           (SEQ ID NO: 372)               fwd_site_20   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCCTGTTCCTAAAGCCCACC           (SEQ ID NO: 373)               rev_site_20   TGGAGTTCAGACGTGTGCTCTTCCGATCTACTCTCTGGTTCTGTTTGTGGCCA           (SEQ ID NO: 374)               fwd_CTNNB1   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNATTTGATGGAGTTGGACATG           GCC (SEQ ID NO: 375)               rev_CTNNB1   TGGAGTTCAGACGTGTGCTCTCCAGCTACTTGTTCTTGAGTGAAGG (SEQ ID           NO: 376)               fwd_RSL1D1   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGGCTTTCCAAATCAGTGGG           TC (SEQ ID NO: 377)               rev_RSL1D1   TGGAGTTCAGACGTGTGCTCTTCCGATCTCTCATAAGCTTAGACCAACAAGC (SEQ           ID NO: 378)               fwd_IP90   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCTGGTTGACCAATCTGTGGT           G (SEQ ID NO: 379)               rev_IP90   TGGAGTTCAGACGTGTGCTCTCTGCGTCTGGATCAGGTACG (SEQ ID NO:           380)               fwd_HEK293_   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTGTGGAGAGTGAGTAAGCC       site2_off1   A (SEQ ID NO: 381)               rev_HEK293_   TGGAGTTCAGACGTGTGCTCTTCCGATCTACGGTAGGATGATTTCAGGCA (SEQ       siite2_off1   ID NO: 382)               fwd_HEK293_   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCACAAAGCAGTGTAGCTCAG       site2_off2   G (SEQ ID NO: 383)               rev_HEK293_   TGGAGTTCAGACGTGTGCTCTTCCGATCTTTTTTGGTACTCGAGTGTTATTCAG       site2_off2   (SEQ ID NO: 384)               fwd_HEK293_   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCCCTGTTGACCTGGAGAA       site3_off1   (SEQ ID NO: 385)               rev_HEK293_   TGGAGTTCAGACGTGTGCTCTTCCGATCTCACTGTACTTGCCCTGACCA (SEQ ID       site3_off1   NO: 386)               fwd_HEK293_   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTGGTGTTGACAGGGAGCAA       site3_off2   (SEQ ID NO: 387)               rev_HEK293_   TGGAGTTCAGACGTGTGCTCTTCCGATCTCTGAGATGTGGGCAGAAGGG (SEQ ID       site3_off2   NO: 388)               fwd_HEK293_   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGAGAGGGAACAGAAGGGCT       site3_off3   (SEQ ID NO: 389)               rev_HEK293_   TGGAGTTCAGACGTGTGCTCTTCCGATCTGTCCAAAGGCCCAAGAACCT (SEQ ID       site3_off3   NO: 390)               fwd_HEK293_   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCTAGCACTTTGGAAGGTC       site3_off4   G (SEQ ID NO: 391)               rev_HEK293_   TGGAGTTCAGACGTGTGCTCTTCCGATCTGCTCATCTTAATCTGCTCAGCC (SEQ       site3_off4   ID NO: 392)               fwd_HEK293_   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAAAGGAGCAGCTCTTCCTGG       site3_off5   (SEQ ID NO: 393)               rev_HEK293_   TGGAGTTCAGACGTGTGCTCTTCCGATCTGTCTGCACCATCTCCCACAA (SEQ ID       site3_off5   NO: 394)               fwd_HEK293_   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGCATGGCTTCTGAGACTCA       site4_off1   (SEQ ID NO: 395)               rev_HEK293_   TGGAGTTCAGACGTGTGCTCTTCCGATCTGTCTCCCTTGCACTCCCTGTCTTT       site4_off1   (SEQ ID NO: 396)               fwd_HEK293_   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTTGGCAATGGAGGCATTGG       site4_off2   (SEQ ID NO: 397)               rev_HEK293_   TGGAGTTCAGACGTGTGCTCTTCCGATCTGAAGAGGCTGCCCATGAGAG (SEQ ID       site4_off2   NO: 398)               fwd_HEK293_   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGTCTGAGGCTCGAATCCTG       site4_off3   (SEQ ID NO: 399)               rev_HEK293_   TGGAGTTCAGACGTGTGCTCTTCCGATCTCTGTGGCCTCCATATCCCTG (SEQ ID       site4_off3   NO: 400)               fwd_HEK293_   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTTCCACCAGAACTCAGCCC       site4_off4   (SEQ ID NO: 401)               rev_HEK293_   TGGAGTTCAGACGTGTGCTCTTCCGATCTCCTCGGTTCCTCCACAACAC (SEQ ID       site4_off4   NO: 402)               fwd_HEK293_   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCACGGGAAGGACAGGAGAAG       site4_off5   (SEQ ID NO: 403)               rev_HEK293_   TGGAGTTCAGACGTGTGCTCTTCCGATCTGCAGGGGAGGGATAAAGCAG (SEQ ID       site4_off5   NO: 404)               fwd_HEK_   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGAAACGCCCATGCAATTAG       site_3   TC (SEQ ID NO: 405)               rev_HEK_   TGGAGTTCAGACGTGTGCTCTTCCGATCTCTTGTCAACCAGTATCCCGGTG (SEQ       site_3   ID NO: 406)               fwd_HEK_   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGAATGGATTCCTTGGAAAC       site_2   AATG (SEQ ID NO: 407)               rev_HEK_   TGGAGTTCAGACGTGTGCTCTTCCGATCTCCAGCCCCATCTGTCAAACT (SEQ ID       site_2   NO: 408)               fwd_HEK_   TGGAGTTCAGACGTGTGCTCTTCCGATCTTCCTTTCAACCCGAACGGAG (SEQ ID       site_4   NO: 409)               rev_HEK_   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCTGGTCTTCTTTCCCCTCC       site_4   (SEQ ID NO: 410)               fwd_LDLR   ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCCCTGCTTCTTTTTCTCTG           GT (SEQ ID NO: 411)               rev_LDLR   TGGAGTTCAGACGTGTGCTCTTCCGATCTACCATTAACGCAGCCAACTTCA (SEQ           ID NO: 412)               fwd_TRAC   ACACTCTTTCCCTACACGACGCTCTTCCGATCTCATGAGGTCTATGGACTTCAAGAG           CAA (SEQ ID NO: 413)               Rev_TRAC   TGGAGTTCAGACGTGTGCTCTTCCGATCTCATCATTGACCAGAGCTCTGGGCAGAA           (SEQ ID NO: 414)               fwd_CBLB   ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCACTTACCAGCATTACTTCCTAA           ACC (SEQ ID NO: 415)               Rev_CBLB   TGGAGTTCAGACGTGTGCTCTTCCGATCTATGGGCTCCACTTTTCAGCTCTGTAA           (SEQ ID NO: 416)               fwd_CD7   ACACTCTTTCCCTACACGACGCTCTTCCGATCTCAGTTCAGGCACATGTAGGAGGGA           (SEQ ID NO: 417)               Rev_CD7   TGGAGTTCAGACGTGTGCTCTTCCGATCTACCGCCTGCAGCTGTCGGACACTGGCA           (SEQ ID NO: 418)               fwd_B2M   ACACTCTTTCCCTACACGACGCTCTTCCGATCTAAAAGATGAGTATGCCTGCCGTG           (SEQ ID NO: 419)               Rev_B2M   TGGAGTTCAGACGTGTGCTCTTCCGATCTCAGATTGTTTATATCAGATGGGATGGG           (SEQ ID NO: 420)               fwd_CIITA   ACACTCTTTCCCTACACGACGCTCTTCCGATCTATGCAAGTTTGGTCCTGAGCCCTC           CC (SEQ ID NO: 421)               Rev_CIITA   TGGAGTTCAGACGTGTGCTCTTCCGATCTGATGTGGGTTCCCTGCGCTCTGCA           (SEQ ID NO: 422)               fwd_PDCD1   ACACTCTTTCCCTACACGACGCTCTTCCGATCTCCAGGGACTGAGGGTGGAAGGTCC           (SEQ ID NO: 423)               Rev_PDCD1   TGGAGTTCAGACGTGTGCTCTTCCGATCTACCTCCGCCTGAGCAGTGGAGAA (SEQ           ID NO: 424)                    
mRNA Production for ABE Editors Used in CD34+ Cells
 
     Editors were cloned into a plasmid encoding a dT7 promoter followed by a 5′UTR, Kozak sequence, ORF, and 3′UTR. The dT7 promoter carries an inactivating point mutation within the T7 promoter that prevents transcription from circular plasmid. This plasmid templated a PCR reaction (Q5 Hot Start 2X Master Mix), in which the forward primer corrected the SNP within the T7 promoter and the reverse primer appended a 120A tail (SEQ ID NO: 425) to the 3′ UTR. The resulting PCR product was purified on a Zymo Research 25 μg DCC column and used as mRNA template in the subsequent in vitro transcription. The NEB HiScribe High-Yield Kit was used as per the instruction manual but with full substitution of N1-methyl-pseudouridine for uridine and co-transcriptional capping with CleanCap AG (Trilink). Reaction cleanup was performed by lithium chloride precipitation. Primers used for amplification can be found in Table 17. 
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Primers used for ABE8 T7 in vitro transcription reactions 
               
            
           
           
               
               
            
               
                 Name 
                 Sequence 
               
               
                   
               
               
                 fwd_IVT 
                 TCGAGCTCGGTACCTAATACGACTCAC (SEQ ID NO: 426) 
               
               
                   
               
               
                 rev_IVT 
                 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT 
               
               
                   
                 TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT 
               
               
                   
                 CTTCCTACTCAGGCTTTATTCAAAGACCA (SEQ ID NO: 427) 
               
               
                   
               
            
           
         
       
     
     CD34+ Cell Preparation 
     Mobilized peripheral blood was obtained and enriched for Human CD34+ HSPCs (HemaCare, M001F-GCSF/MOZ-2). The CD34+ cells were thawed and put into X-VIVO 10 (Lonza) containing 1% Glutamax (Gibco), 100 ng/mL of TPO (Peprotech), SCF (Peprotech) and Flt-3 (Peprotech) at 48 hours prior to electroporation 
     Electroporation of CD34+ Cells 
     48 hours post thaw, the cells were spun down to remove the X-VIVO 10 media and washed in MaxCyte buffer (HyClone) with 0.1% HSA (Akron Biotechnologies). The cells were then resuspended in cold MaxCyte buffer at 1,250,000 cells per mL and split into multiple 20 μL aliquots. The ABE mRNA (0.15 μM) and −198 HBG1/2 sgRNA (4.05 μM) were then aliquoted as per the experimental conditions and raised to a total of 54 in MaxCyte buffer. The 20 μL of cells was the added into the 5 μL RNA mixture in groups of 3 and loaded into each chamber of an OC25×3 MaxCyte cuvette for electroporation. After receiving the charge, 25 μL was collected from the chambers and placed in the center of the wells in a 24-well untreated culture plate. The cells recovered for 20 minutes in an incubator (37° C., 5% CO 2 ). After the 20 minutes recovery, X-VIVO 10 containing 1% Glutamax, 100 ng/mL of TPO, SCF and Flt-3 was added to the cells for a concentration of 1,000,000 cells per mL. The cells were then left to further recover in an incubator (37° C., 5% CO 2 ) for 48 hrs. 
     Erythrocyte Differentiation Post ABE Electroporation 
     Following 48 h post electroporation rest (day 0 of culture), the cells were spun down and moved to “Phase 1” IMDM media (ATCC) containing 5% human serum, 330 μg/mL transferrin (Sigma), 10 μg/mL human insulin (Sigma), 2 U/mL heparin sodium (Sigma), 3 U/mL EPO (Peprotech), 100 ng/mL SCF (Peprotech), 5 μg/mL IL3 and 50 μM hydrocortisone (Sigma) at 20,000 cells per mL. On day 4 of culture, the cells were fed 4× volume of the same media. On day 7, the cells were spun down and moved to “Phase 2” IMDM media containing 5% human serum (Sigma), 330 μg/mL transferrin, 10 μg/mL human insulin, 2 U/mL heparin sodium, 3 U/mL EPO and 100 ng/mL SCF at 200,000 cells per mL. On day 11, cells were spun down and moved to “Phase 3” IMDM media containing 5% human serum, 330 μg/mL of transferrin, 10 μg/mL human insulin, 2 U/mL of heparin sodium and 3 U/mL of EPO at 1,000,000 cells per mL. On day 14, the cells were spun down and resuspended in the same media as day 11 but at 5,000,000 cells per mL. On day 18, the differentiated red blood cells were collected in 500,000 cell aliquots, washed once in 500 μL DPBS (Gibco) and frozen at −80° C. for 24 hours before UHPLC processing. 
     Preparation of Red Blood Cell Sample for UHPLC Analysis 
     Frozen red blood cell pellets were thawed at room temperature. Pellets were diluted to a final concentration of 5×10 4  cells/μL with ACK lysis buffer. Samples were mixed by pipette and incubated at room temperate for 5 min. Samples were then frozen in at −80° C. for 5 min, allowed to thaw, and mixed by pipette prior to centrifugation at 6,700g for 10 min. The supernatant was carefully removed (without disturbing cell debris pellet), transferred to a new plate and diluted to 5×10 3  cells/μL in ultrapure water for UHPLC analysis. 
     Ultra-High Performance Liquid Chromatography (UHPLC) Analysis 
     Reverse-phase separation of globin chains was performed on a UHPLC system configured with a binary pump and UV detector (Thermo Fisher Scientific, Vanquish Horizon). The stationary phase consisted of an ACQUITY Peptide BEH C18 Column (2.1×150 mm, 1.7 μm beads, 300A pores) after an AQUITY Peptide BEH C18 VanGuard pre-column (2.1×5 mm, 1.7 μm beads, 300A pores) (both Waters Corp) with a column temperature of 60° C. Elution was preformed using 0.1% trifluoroacetic acid (TFA) in water (A) and 0.08% TFA in acetonitrile (B) with a flow rate of 0.25 mL/min. Separation of the globin chains was achieved using a linear gradient of 40-52% B from 0-10 min; 52-40% B from 10-10.5 min; 40% B to 12 min. Sample injection volume was 10 μL, UV spectra at a wavelength of 220 nm with a data rate of 5 Hz was collected throughout the analysis. Globin chain identities were confirmed through LC/MS analysis of hemoglobin standards. 
     Genomic DNA Extraction for CD34+ Cells 
     Following ABE electroporation (48 h later), an aliquot of cells was cultured in X-VIVO 10 media (Lonza) containing 1% Glutamax (Gibco), 100 ng/mL of TPO (Peprotech), SCF (Peprotech) and Flt-3 (Peprotech). Following 48 h and 144 h post culturing, 100,000 cells were collected and spun down. 50 μL of Quick Extract (Lucigen) was added to the cell pellet and the cell mixture was transferred to a 96-well PCR plate (Bio-Rad). The lysate was heated for 15 minutes at 65° C. followed by 10 minutes at 98° C. The cell lysates were stored at −20° C. 
     Sequences 
     In the following sequence, lower case denotes the kanamycin resistance promoter region, bold sequence indicates targeted inactivation portion (Q4* and W15*), the italicized sequence denotes the targeted inactive site of kanamycin resistance gene (D208N), and the underlined sequences denote the PAM sequences. 
     Inactivated Kanamycin Resistance Gene: 
       
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 309) 
                   
               
               
                 ccggaattgccagctggggcgccctctggtaaggttgggaagccctgcaaagtaaactggatgg 
                   
               
               
                   
               
               
                 ctttcttgccgccaaggatctgatggcgcaggggatcaagatctgatcaagagacaggatgagg 
               
               
                   
               
               
                 at cct   ttcgcATGATCGAATAAGAT GGATTGCACGCAGGTTCTCCGGCC GCTTAGGTGGAGCGC   
               
               
                   
               
               
                   CTATT   CGG CTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGT 
               
               
                   
               
               
                 CAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCA 
               
               
                   
               
               
                 GGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGAC 
               
               
                   
               
               
                 GTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGT 
               
               
                   
               
               
                 CATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATAC 
               
               
                   
               
               
                 GCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACT 
               
               
                   
               
               
                 CGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAG 
               
               
                   
               
               
                 CCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGG 
               
               
                   
               
               
                 CGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATTAACTGTGGC 
               
               
                   
               
               
                 CGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGC 
               
               
                   
               
               
                 TTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCG 
               
               
                   
               
               
                 CATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTAA 
               
            
           
         
       
     
     In the following sequences, the plain text denotes an adenosine deaminase sequence, bold sequence indicates sequence derived from Cas9, the italicized sequence denotes a linker sequence, underlined sequence denotes a bipartite nuclear localization sequence, and double underlined sequence indicates mutations. 
     
       
         
           
               
               
            
               
                 CP5 (with MSP “NGC” PID and “D10A” nickase): 
                   
               
               
                 (SEQ ID NO: 38) 
                   
               
               
                 
                   EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQ 
                 
                   
               
               
                   
               
               
                 VNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFMQPTVAYSVLVVAKVEKGKSK 
               
               
                   
               
               
                 
                   KLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKF 
                 
               
               
                   
               
               
                 
                   LQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILA 
                 
               
               
                   
               
               
                 
                   DANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIARKEYRSTKEVLDATL 
                 
               
               
                   
               
               
                 
                   IHQSITGLYETRIDLSQLGGD 
                   GGSGGSGGSGGSGGSGGSGGM 
                   DKKYSIGLAIGTNSVGWAVITD 
                 
               
               
                   
               
               
                 
                   EYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFS 
                 
               
               
                   
               
               
                 
                   NEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL 
                 
               
               
                   
               
               
                 
                   RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSAR 
                 
               
               
                   
               
               
                 
                   LSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLL 
                 
               
               
                   
               
               
                 
                   AQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL 
                 
               
               
                   
               
               
                 
                   PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDN 
                 
               
               
                   
               
               
                 
                   GSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEE 
                 
               
               
                   
               
               
                 
                   TITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMR 
                 
               
               
                   
               
               
                 
                   KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLK 
                 
               
               
                   
               
               
                 
                   IIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS 
                 
               
               
                   
               
               
                 
                   RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL 
                 
               
               
                   
               
               
                 
                   AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKEL 
                 
               
               
                   
               
               
                 
                   GSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK 
                 
               
               
                   
               
               
                 
                   VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR 
                 
               
               
                   
               
               
                 
                   QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHH 
                 
               
               
                   
               
               
                 
                   AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ 
                   EGADKRTADGSEFESPKKKR 
                 
               
               
                   
               
               
                   KV * 
               
               
                   
               
               
                 ABE8.1_Y147T_CP5_NGC PAM_monomer 
               
               
                 (SEQ ID NO: 254) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD     
               
               
                   
               
               
                 
                   
                   EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVR 
                 
               
               
                   
               
               
                 
                   KVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFMQPTVAYSVLVVAK 
                 
               
               
                   
               
               
                 
                   VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR 
                 
               
               
                   
               
               
                 
                   MLASAKFLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEF 
                 
               
               
                   
               
               
                 
                   SKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIARKEYRSTK 
                 
               
               
                   
               
               
                 
                   EVLDATLIHQSITGLYETRIDLSQLGGD 
                   
                   
                   DKKYSIGLAIGTNSV 
                 
               
               
                   
               
               
                 
                   GWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC 
                 
               
               
                   
               
               
                 
                   YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVD 
                 
               
               
                   
               
               
                 
                   STDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA 
                 
               
               
                   
               
               
                 
                   KAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYD 
                 
               
               
                   
               
               
                 
                   DDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLK 
                 
               
               
                   
               
               
                 
                   ALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
                 
               
               
                   
               
               
                 
                   KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW 
                 
               
               
                   
               
               
                 
                   MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVK 
                 
               
               
                   
               
               
                 
                   YVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLG 
                 
               
               
                   
               
               
                 
                   TYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY 
                 
               
               
                   
               
               
                 
                   TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL 
                 
               
               
                   
               
               
                 
                   HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRI 
                 
               
               
                   
               
               
                 
                   EEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK 
                 
               
               
                   
               
               
                 
                   DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD 
                 
               
               
                   
               
               
                 
                   KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR 
                 
               
               
                   
               
               
                 
                   EINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ 
                   EGADKRTADGSEE 
                 
               
               
                   
               
               
                   ESPKKKRKV * 
               
               
                   
               
               
                 pNMG-B335 ABE8.1_Y147T_CP5_NGC PAM_monomer 
               
               
                 (SEQ ID NO: 254) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTDSGGSSG GSSGSEPPGPSESATPESS   
               
               
                   
               
               
                 
                   GGSSGGS 
                   EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVR 
                 
               
               
                   
               
               
                 
                   KVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFMQPTVAYSVLVVAK 
                 
               
               
                   
               
               
                 
                   VEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR 
                 
               
               
                   
               
               
                 
                   MLASAKFLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEF 
                 
               
               
                   
               
               
                 
                   SKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIARKEYRSTK 
                 
               
               
                   
               
               
                 
                   EVLDATLIHQSITGLYETRIDLSQLGGD 
                   GGSGGSGGSGGSGGSGGSGGM 
                   DKKYSIGLAIGTNSV 
                 
               
               
                   
               
               
                 
                   GWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC 
                 
               
               
                   
               
               
                 
                   YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVD 
                 
               
               
                   
               
               
                 
                   STDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA 
                 
               
               
                   
               
               
                 
                   KAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYD 
                 
               
               
                   
               
               
                 
                   DDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLK 
                 
               
               
                   
               
               
                 
                   ALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
                 
               
               
                   
               
               
                 
                   KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW 
                 
               
               
                   
               
               
                 
                   MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVK 
                 
               
               
                   
               
               
                 
                   YVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLG 
                 
               
               
                   
               
               
                 
                   TYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY 
                 
               
               
                   
               
               
                 
                   TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL 
                 
               
               
                   
               
               
                 
                   HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRI 
                 
               
               
                   
               
               
                 
                   EEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK 
                 
               
               
                   
               
               
                 
                   DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD 
                 
               
               
                   
               
               
                 
                   KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR 
                 
               
               
                   
               
               
                 
                   EINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ 
                   EGADKRTADGSEE 
                 
               
               
                   
               
               
                   ESPKKKRKV * 
               
               
                   
               
               
                 pNMG-357_ABE8.14 withNGC PAM CP5 
               
               
                 (SEQ ID NO: 255) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDGGSSGGS SGSEPPGPSESATPESSG   
               
               
                   
               
               
                   GSSGGS MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHA 
               
               
                   
               
               
                 EIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLH 
               
               
                   
               
               
                 YPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD SGGSSGGSSGSEPPGPSES   
               
               
                   
               
               
                 
                   ATPESSGGSSGGS 
                 
               
               
                   
               
               
                 
                   EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQ 
                 
               
               
                   
               
               
                 
                   VNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFMQPTVAYSVLVVAKVEKGKSK 
                 
               
               
                   
               
               
                 
                   KLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKF 
                 
               
               
                   
               
               
                 
                   LQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILA 
                 
               
               
                   
               
               
                 
                   DANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIARKEYRSTKEVLDATL 
                 
               
               
                   
               
               
                 
                   IHQSITGLYETRIDLSQLGGD 
                   GGSGGSGGSGGSGGSGGSGGM 
                   DKKYSIGLAIGTNSVGWAVITD 
                 
               
               
                   
               
               
                 
                   EYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFS 
                 
               
               
                   
               
               
                 
                   NEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL 
                 
               
               
                   
               
               
                 
                   RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSAR 
                 
               
               
                   
               
               
                 
                   LSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLL 
                 
               
               
                   
               
               
                 
                   AQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL 
                 
               
               
                   
               
               
                 
                   PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDN 
                 
               
               
                   
               
               
                 
                   GSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEE 
                 
               
               
                   
               
               
                 
                   TITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMR 
                 
               
               
                   
               
               
                 
                   KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLK 
                 
               
               
                   
               
               
                 
                   IIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS 
                 
               
               
                   
               
               
                 
                   RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL 
                 
               
               
                   
               
               
                 
                   AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKEL 
                 
               
               
                   
               
               
                 
                   GSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNK 
                 
               
               
                   
               
               
                 
                   VLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR 
                 
               
               
                   
               
               
                 
                   QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHH 
                 
               
               
                   
               
               
                 
                   AHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ 
                   EGADKRTADGSEEESPKKKR 
                 
               
               
                   
               
               
                   KV * 
               
               
                   
               
               
                 ABE8.8-m 
               
               
                 (SEQ ID NO: 256) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLH H PGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLC R FFRMPR R VFNAQKKAQSSTD SGGSSGGSSGSETPGTSESATPESS   
               
               
                   
               
               
                 
                   GGSSGGS 
                   DKKYSIGL 
                   
                     A 
                   
                   IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
                 
               
               
                   
               
               
                 
                   AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
                 
               
               
                   
               
               
                 
                   VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
                 
               
               
                   
               
               
                 
                   QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
                 
               
               
                   
               
               
                 
                   NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
                 
               
               
                   
               
               
                 
                   APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
                 
               
               
                   
               
               
                 
                   ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
                 
               
               
                   
               
               
                 
                   ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
                 
               
               
                   
               
               
                 
                   VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
                 
               
               
                   
               
               
                 
                   KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
                 
               
               
                   
               
               
                 
                   RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
                 
               
               
                   
               
               
                 
                   DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
                 
               
               
                   
               
               
                 
                   MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
                 
               
               
                   
               
               
                 
                   ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
                 
               
               
                   
               
               
                 
                   LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
                 
               
               
                   
               
               
                 
                   VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
                 
               
               
                   
               
               
                 
                   RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
                 
               
               
                   
               
               
                 
                   VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV 
                 
               
               
                   
               
               
                 
                   AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
                 
               
               
                   
               
               
                 
                   KRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
                 
               
               
                   
               
               
                 
                   EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS 
                 
               
               
                   
               
               
                   TKEVLDATLIHQSITGLYETRIDLSQLGGD   EGADKRTADGSEFESPKKKRKV * 
               
               
                   
               
               
                 ABE8.8-d 
               
               
                 (SEQ ID NO: 257) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD SGGSSGGSSGSETPGTSESATPESS   
               
               
                   
               
               
                   GGSSGGS SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHA 
               
               
                   
               
               
                 EIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLH 
               
               
                   
               
               
                   H PGMNHRVEITEGILADECAALLC R FFRMPR R VFNAQKKAQSSTD SGGSSGGSSGSETPGTSES   
               
               
                   
               
               
                   ATPESSGGSSGGS   DKKYSIG L   A     IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL   
               
               
                   
               
               
                 
                   FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH 
                 
               
               
                   
               
               
                 
                   PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSD 
                 
               
               
                   
               
               
                 
                   VDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL 
                 
               
               
                   
               
               
                 
                   SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRV 
                 
               
               
                   
               
               
                 
                   NTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF 
                 
               
               
                   
               
               
                 
                   YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN 
                 
               
               
                   
               
               
                 
                   REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
                 
               
               
                   
               
               
                 
                   NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQL 
                 
               
               
                   
               
               
                 
                   KEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED 
                 
               
               
                   
               
               
                 
                   REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN 
                 
               
               
                   
               
               
                 
                   FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP 
                 
               
               
                   
               
               
                 
                   ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGR 
                 
               
               
                   
               
               
                 
                   DMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWR 
                 
               
               
                   
               
               
                 
                   QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK 
                 
               
               
                   
               
               
                 
                   LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGD 
                 
               
               
                   
               
               
                 
                   YKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDK 
                 
               
               
                   
               
               
                 
                   GRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVA 
                 
               
               
                   
               
               
                 
                   YSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF 
                 
               
               
                   
               
               
                 
                   ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE 
                 
               
               
                   
               
               
                 
                   IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID 
                 
               
               
                   
               
               
                   RKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD   EGADKRTADGSEFESPKKKRKV * 
               
               
                   
               
               
                 ABE8.13-m 
               
               
                 (SEQ ID NO: 258) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRL Y DATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLH H PGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLC R FFRMPR R VFNAQKKAQSSTD SGGSSGGSSGSETPGTSESATPESS   
               
               
                   
               
               
                 
                   GGSSGGS 
                   DKKYSIGL 
                   
                     A 
                   
                   IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
                 
               
               
                   
               
               
                 
                   AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
                 
               
               
                   
               
               
                 
                   VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
                 
               
               
                   
               
               
                 
                   QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
                 
               
               
                   
               
               
                 
                   NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
                 
               
               
                   
               
               
                 
                   APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
                 
               
               
                   
               
               
                 
                   ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
                 
               
               
                   
               
               
                 
                   ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
                 
               
               
                   
               
               
                 
                   VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
                 
               
               
                   
               
               
                 
                   KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
                 
               
               
                   
               
               
                 
                   RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
                 
               
               
                   
               
               
                 
                   DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
                 
               
               
                   
               
               
                 
                   MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
                 
               
               
                   
               
               
                 
                   ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
                 
               
               
                   
               
               
                 
                   LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
                 
               
               
                   
               
               
                 
                   VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
                 
               
               
                   
               
               
                 
                   RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
                 
               
               
                   
               
               
                 
                   VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV 
                 
               
               
                   
               
               
                 
                   AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
                 
               
               
                   
               
               
                 
                   KRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
                 
               
               
                   
               
               
                 
                   EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS 
                 
               
               
                   
               
               
                   TKEVLDATLIHQSITGLYETRIDLSQLGGD     E GADKRTADGSEFESPKKKRKV * 
               
               
                   
               
               
                 ABE8.13-d 
               
               
                 (SEQ ID NO: 259) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD SGGSSGGSSGSETPGTSESATPESS   
               
               
                   
               
               
                 GGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHA 
               
               
                   
               
               
                 EIMALRQGGLVMQNYRL Y DATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLH 
               
               
                   
               
               
                   H PGMNHRVEITEGILADECAALLC R FFRMPR R VFNAQKKAQSSTD SGGSSGGSSGSETPGTSES   
               
               
                   
               
               
                 
                   ATPESSGGSSGGS 
                   DKKYSIGL 
                   
                     A 
                   
                   IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL 
                 
               
               
                   
               
               
                 
                   FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH 
                 
               
               
                   
               
               
                 
                   PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSD 
                 
               
               
                   
               
               
                 
                   VDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL 
                 
               
               
                   
               
               
                 
                   SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRV 
                 
               
               
                   
               
               
                 
                   NTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF 
                 
               
               
                   
               
               
                 
                   YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN 
                 
               
               
                   
               
               
                 
                   REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
                 
               
               
                   
               
               
                 
                   NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQL 
                 
               
               
                   
               
               
                 
                   KEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED 
                 
               
               
                   
               
               
                 
                   REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN 
                 
               
               
                   
               
               
                 
                   FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP 
                 
               
               
                   
               
               
                 
                   ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGR 
                 
               
               
                   
               
               
                 
                   DMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWR 
                 
               
               
                   
               
               
                 
                   QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK 
                 
               
               
                   
               
               
                 
                   LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGD 
                 
               
               
                   
               
               
                 
                   YKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDK 
                 
               
               
                   
               
               
                 
                   GRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVA 
                 
               
               
                   
               
               
                 
                   YSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF 
                 
               
               
                   
               
               
                 
                   ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE 
                 
               
               
                   
               
               
                 
                   IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID 
                 
               
               
                   
               
               
                   RKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD   EGADKRTADGSEFESPKKKRKV * 
               
               
                   
               
               
                 ABE8.17-m 
               
               
                 (SEQ ID NO: 260) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLY S TFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPR R VFNAQKKAQSSTD SGGSSGGSSGSETPGTSESATPESS   
               
               
                   
               
               
                 
                   GGSSGGS 
                   DKKYSIGL 
                   
                     A 
                   
                   IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
                 
               
               
                   
               
               
                 
                   AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
                 
               
               
                   
               
               
                 
                   VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
                 
               
               
                   
               
               
                 
                   QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
                 
               
               
                   
               
               
                 
                   NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
                 
               
               
                   
               
               
                 
                   APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
                 
               
               
                   
               
               
                 
                   ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
                 
               
               
                   
               
               
                 
                   ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
                 
               
               
                   
               
               
                 
                   VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
                 
               
               
                   
               
               
                 
                   KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
                 
               
               
                   
               
               
                 
                   RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
                 
               
               
                   
               
               
                 
                   DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
                 
               
               
                   
               
               
                 
                   MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
                 
               
               
                   
               
               
                 
                   ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
                 
               
               
                   
               
               
                 
                   LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
                 
               
               
                   
               
               
                 
                   VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
                 
               
               
                   
               
               
                 
                   RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
                 
               
               
                   
               
               
                 
                   VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV 
                 
               
               
                   
               
               
                 
                   AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
                 
               
               
                   
               
               
                 
                   KRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
                 
               
               
                   
               
               
                 
                   EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS 
                 
               
               
                   
               
               
                   TKEVLDATLIHQSITGLYETRIDLSQLGGD   EGADKRTADGSEFESPKKKRKV * 
               
               
                   
               
               
                 ABE8.17-d 
               
               
                 (SEQ ID NO: 261) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD SGGSSGGSSGSETPGTSESATPESS   
               
               
                   
               
               
                   GGSSGGS SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHA 
               
               
                   
               
               
                 EIMALRQGGLVMQNYRLIDATLY S TFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLH 
               
               
                   
               
               
                 YPGMNHRVEITEGILADECAALLCYEFRMPR R VFNAQKKAQSSTD SGGSSGGSSGSETPGRSES   
               
               
                   
               
               
                 
                   ATRESSGGSSGGS 
                   DKKYSIGL 
                   
                     A 
                   
                   IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL 
                 
               
               
                   
               
               
                 
                   FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH 
                 
               
               
                   
               
               
                 
                   PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSD 
                 
               
               
                   
               
               
                 
                   VDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL 
                 
               
               
                   
               
               
                 
                   SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRV 
                 
               
               
                   
               
               
                 NTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF 
               
               
                   
               
               
                 
                   YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN 
                 
               
               
                   
               
               
                 
                   REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
                 
               
               
                   
               
               
                 
                   NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQL 
                 
               
               
                   
               
               
                 
                   KEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED 
                 
               
               
                   
               
               
                 
                   REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN 
                 
               
               
                   
               
               
                 
                   FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP 
                 
               
               
                   
               
               
                 
                   ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGR 
                 
               
               
                   
               
               
                 
                   DMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWR 
                 
               
               
                   
               
               
                 
                   QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK 
                 
               
               
                   
               
               
                 
                   LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGD 
                 
               
               
                   
               
               
                 
                   YKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDK 
                 
               
               
                   
               
               
                 
                   GRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVA 
                 
               
               
                   
               
               
                 
                   YSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF 
                 
               
               
                   
               
               
                 
                   ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE 
                 
               
               
                   
               
               
                 
                   IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID 
                 
               
               
                   
               
               
                   RKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD   EGADKRTADGSEFESPKKKRKV * 
               
               
                   
               
               
                 ABE8.20-m 
               
               
                 (SEQ ID NO: 262) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRL Y DATLY S TFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLH H PGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLC R FFRMPR R VFNAQKKAQSSTD SGGSSGGSSGSETPGTSESATPESS   
               
               
                   
               
               
                 
                   GGSSGGS 
                   DKKYSIGL 
                   
                     A 
                   
                   IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
                 
               
               
                   
               
               
                 
                   AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
                 
               
               
                   
               
               
                 
                   VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
                 
               
               
                   
               
               
                 
                   QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
                 
               
               
                   
               
               
                 
                   NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
                 
               
               
                   
               
               
                 
                   APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
                 
               
               
                   
               
               
                 
                   ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
                 
               
               
                   
               
               
                 
                   ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
                 
               
               
                   
               
               
                 
                   VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
                 
               
               
                   
               
               
                 
                   KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
                 
               
               
                   
               
               
                 
                   RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
                 
               
               
                   
               
               
                 
                   DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
                 
               
               
                   
               
               
                 
                   MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
                 
               
               
                   
               
               
                 
                   ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
                 
               
               
                   
               
               
                 
                   LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
                 
               
               
                   
               
               
                 
                   VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
                 
               
               
                   
               
               
                 
                   RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
                 
               
               
                   
               
               
                 
                   VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV 
                 
               
               
                   
               
               
                 
                   AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
                 
               
               
                   
               
               
                 
                   KRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
                 
               
               
                   
               
               
                 
                   EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTS 
                 
               
               
                   
               
               
                   TKEVLDATLIHQSITGLYETRIDLSQLGGD   EGADKRTADGSEFESPKKKRKV * 
               
               
                   
               
               
                 ABE8.20-d 
               
               
                 (SEQ ID NO: 263) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLSDFFRMRRQEIKAQKKAOSSTD SGGSSGGSSGSETPGTSESATPESS   
               
               
                   
               
               
                   GGSSGGS SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHA 
               
               
                   
               
               
                 EIMALRQGGLVMQNYRL Y DATLY S TFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLH 
               
               
                   
               
               
                   H PGMNHRVEITEGILADECAALLC R FFRMPR R VFNAQKKAQSSTD SGGSSGGSSGSETPGTSES   
               
               
                   
               
               
                 
                   ATPESSGGSSGGS 
                   DKKYSIGL 
                   
                     A 
                   
                   IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL 
                 
               
               
                   
               
               
                 
                   FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH 
                 
               
               
                   
               
               
                 
                   PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSD 
                 
               
               
                   
               
               
                 
                   VDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIAL 
                 
               
               
                   
               
               
                 
                   SLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRV 
                 
               
               
                   
               
               
                 
                   NTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEF 
                 
               
               
                   
               
               
                 
                   YKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN 
                 
               
               
                   
               
               
                 
                   REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
                 
               
               
                   
               
               
                 
                   NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQL 
                 
               
               
                   
               
               
                 
                   KEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED 
                 
               
               
                   
               
               
                 
                   REMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN 
                 
               
               
                   
               
               
                 
                   FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP 
                 
               
               
                   
               
               
                 
                   ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGR 
                 
               
               
                   
               
               
                 
                   DMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWR 
                 
               
               
                   
               
               
                 
                   QLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDK 
                 
               
               
                   
               
               
                 
                   LIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGD 
                 
               
               
                   
               
               
                 
                   YKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDK 
                 
               
               
                   
               
               
                 
                   GRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVA 
                 
               
               
                   
               
               
                 
                   YSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF 
                 
               
               
                   
               
               
                 
                   ELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE 
                 
               
               
                   
               
               
                 
                   IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTID 
                 
               
               
                   
               
               
                   RKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD     EGADKRTADGSEFESPKKKRKV   * 
               
               
                   
               
               
                 01. monoABE8.1_bpNLS + Y147T 
               
               
                 (SEQ ID NO: 264) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
               
               
                 02. monoABE8.1_bpNLS + Y147R 
               
               
                 (SEQ ID NO: 265) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCRFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
               
               
                 03. monoABE8.1_bpNLS + Q154S 
               
               
                 (SEQ ID NO: 266) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPRSVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDELEAKGYKEVKKDLIIKLPKYSLEELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
               
               
                 04. monoABE8.1_bpNLS + Y123H 
               
               
                 (SEQ ID NO: 267) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
               
               
                 05. monoABE8.1_bpNLS + V82S 
               
               
                 (SEQ ID NO: 268) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
               
               
                 06. monoABE8.1_bpNLS + T166R 
               
               
                 (SEQ ID NO: 269) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSRDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
               
               
                 07. monoABE8.1_bpNLS + Q154R 
               
               
                 (SEQ ID NO: 270) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
               
               
                 08. monoABE8.1_bpNLS + Y147R_Q154R_Y123H 
               
               
                 (SEQ ID NO: 271) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDELEAKGYKEVKKDLIIKLPKYSLEELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
               
               
                 09. monoABE8.1_bpNLS + Y147R_Q154R_I76Y 
               
               
                 (SEQ ID NO: 272) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
               
               
                 10. monoABE8.1_bpNLS + Y147R_Q154R_T166R 
               
               
                 (SEQ ID NO: 273) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSRDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
               
               
                 11. monoABE8.1_bpNLS + Y147T_Q154R 
               
               
                 (SEQ ID NO: 274) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCTFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
               
               
                 12. monoABE8.1_bpNLS + Y147T_Q154S 
               
               
                 (SEQ ID NO: 275) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCTFFRMPRSVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
               
               
                 13. monoABE8.1_bpNLS + H123Y123H_Y147R_Q154R_I76Y 
               
               
                 (SEQ ID NO: 276) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDELEAKGYKEVKKDLIIKLPKYSLEELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV 
               
               
                   
               
               
                 14. monoABE8.1_bpNLS + V82S + Q154R 
               
               
                 (SEQ ID NO: 277) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV  
               
            
           
         
       
     
     Example 8. Parkinson&#39;s Disease 
     Materials and Methods 
     The results provided in the Examples described herein were obtained using the following materials and methods. 
     The sequence of ABEs used in the Examples follows: 
     
       
         
           
               
               
            
               
                 01. monoABE8.1_bpNLS + Y147T 
                   
               
               
                 (SEQ ID NO: 264) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV  
               
               
                   
               
               
                 02. monoABE8.1_bpNLS + Y147R 
               
               
                 (SEQ ID NO: 265) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCRFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV  
               
               
                   
               
               
                 03. monoABE8.1_bpNLS + Q154S 
               
               
                 (SEQ ID NO: 266) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPRSVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV  
               
               
                   
               
               
                 04. monoABE8.1_bpNLS + Y123H 
               
               
                 (SEQ ID NO: 267) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEEVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV  
               
               
                   
               
               
                 05. monoABE8.1_bpNLS + V82S 
               
               
                 (SEQ ID NO: 268) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV  
               
               
                   
               
               
                 06. monoABE8.1_bpNLS + T166R 
               
               
                 (SEQ ID NO: 269) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSRDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV  
               
               
                   
               
               
                 07. monoABE8.1_bpNLS + Q154R 
               
               
                 (SEQ ID NO: 270) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV  
               
               
                   
               
               
                 08. monoABE8.1_bpNLS + Y147R_Q154R_Y123H 
               
               
                 (SEQ ID NO: 271) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV  
               
               
                   
               
               
                 09. monoABE8.1_bpNLS + Y147R_Q154R_I76Y 
               
               
                 (SEQ ID NO: 272) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLETLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV  
               
               
                   
               
               
                 10. monoABE8.1_bpNLS + Y147R_Q154R_T166R 
               
               
                 (SEQ ID NO: 273) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSRDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV  
               
               
                   
               
               
                 11. monoABE8.1_bpNLS + Y147T_Q154R 
               
               
                 (SEQ ID NO: 274) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCTFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESELVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV  
               
               
                   
               
               
                 12. monoABE8.1_bpNLS + Y147T_Q154S 
               
               
                 (SEQ ID NO: 275) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCTFFRMPRSVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV  
               
               
                   
               
               
                 513. monoABE8.1_bpNLS + H123Y123H_Y147R_Q154R_I76Y 
               
               
                 (SEQ ID NO: 276) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGEVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV  
               
               
                   
               
               
                 14. monoABE8.1_bpNLS + V82S + Q154R 
               
               
                 (SEQ ID NO: 277) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPRRVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESS 
               
               
                   
               
               
                 GGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET 
               
               
                   
               
               
                 AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI 
               
               
                   
               
               
                 VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFI 
               
               
                   
               
               
                 QLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITK 
               
               
                   
               
               
                 APLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP 
               
               
                   
               
               
                 ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK 
               
               
                   
               
               
                 KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH 
               
               
                   
               
               
                 DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE 
               
               
                   
               
               
                 MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
               
               
                   
               
               
                 LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV 
               
               
                   
               
               
                 RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT 
               
               
                   
               
               
                 VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVV 
               
               
                   
               
               
                 AKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGR 
               
               
                   
               
               
                 KRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS 
               
               
                   
               
               
                 EFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRS 
               
               
                   
               
               
                 TKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV  
               
            
           
         
       
     
     A modified SpCas9 including amino acid substitutions D1135M, S1136Q, G1218K, 35 E1219F, A1322R, D1332A, R1335E, and T1337R (MQKFRAER) and having specificity for the altered PAM 5′-NGC-3′ was used for correction of G2019S. A modified SpCas9-VRQR having specificity for altered PAM 5′-NGA-3 was used for correction of R1441C. 
     Cloning. 
     DNA sequences of target polynucleotides and gRNAs and primers used are described herein. For gRNAs, the following scaffold sequence is presented: GUUUUAGAGC 
     UAGAAAUAGC AAGUUAAAAU AAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU (SEQ ID NO: 101). This scaffold was used for the PAMs described in  FIGS.  57 A-C  and  FIGS.  58 A-C  (e.g., NGA and NGC PAMs, respectively). The gRNA encompasses the scaffold sequence and the spacer sequence (target sequence) for an LRRK2 gene comprising a pathogenic mutation as described herein or as determined based on the knowledge of the skilled practitioner and as would be understood to the skilled practitioner in the art. (See, e.g., Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), and Rees, H. A., et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 December; 19(12):770-788. doi: 10.1038/s41576-018-0059-1). 
     PCR was performed using VeraSeq ULtra DNA polymerase (Enzymatics), or Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs). Base Editor (BE) plasmids were constructed using USER cloning (New England Biolabs). Deaminase genes were synthesized as gBlocks Gene Fragments (Integrated DNA Technologies). Cas9 genes used are listed below. Cas9 genes were obtained from previously reported plasmids. Deaminase and fusion genes were cloned into pCMV (mammalian codon-optimized) or pET28b ( E. coli  codon-optimized) backbones. sgRNA expression plasmids were constructed using site-directed mutagenesis. 
     Briefly, the primers were 5′ phosphorylated using T4 Polynucleotide Kinase (New England Biolabs) according to the manufacturer&#39;s instructions. To amplify regions for Guides 1 and 2, the following primers were used: 
     
       
         
           
               
               
            
               
                 Guide 1 Primer  
                   
               
               
                 (oAM129; for 5′-AAGCGCAAGCCTGGAGGGAA-3′ (SEQ ID NO: 428)): 
               
               
                 (SEQ ID NO: 429) 
                   
               
               
                 5′-GAAGCGCAAGCCTGGAGGGAAGTTTTAGAGCTAGAAATAGCA-3′; 
                   
               
               
                   
               
               
                 Guide 2 Primer  
               
               
                 (oAM130; 5′-ACTACAGCATTGCTCAGTAC-3′ (SEQ ID NO: 430)): 
               
               
                 (SEQ ID NO: 431) 
                   
               
               
                 5′-GACTACAGCATTGCTCAGTACGTTTTAGAGCTAGAAATAGCA-3′; 
                   
               
               
                   
               
               
                 Shared Primer (oAM95): 
               
               
                 (SEQ ID NO: 432) 
                   
               
               
                 5′-GGTGTTTCGTCCTTTCCACAAG-3′. 
                   
               
            
           
         
       
     
     Next, PCR was performed using Q5 Hot Start High-Fidelity Polymerase (New England Biolabs) with the phosphorylated primers and the plasmid encoding a gene of interest as a template according to the manufacturer&#39;s instructions. PCR products were incubated with Dpnl (20 U, New England Biolabs) at 37° C. for 1 hour, purified on a QlAprep spin column (Qiagen), and ligated using QuickLigase (New England Biolabs) according to the manufacturer&#39;s instructions. DNA vector amplification was carried out using Machl competent cells (ThermoFisher Scientific). 
     In Vitro Deaminase Assay on ssDNA. 
     Sequences of all ssDNA substrates are provided below. All Cy3-labelled substrates were obtained from Integrated DNA Technologies (IDT). Deaminases were expressed in vitro using the TNT T7 Quick Coupled Transcription/Translation Kit (Promega) according to the manufacturer&#39;s instructions using 1 μg of plasmid. Following protein expression, 5 μl of lysate was combined with 35 μl of ssDNA (1.8 μM) and USER enzyme (1 unit) in CutSmart buffer (New England Biolabs) (50 mM potassium acetate, 29 mM Tris-acetate, 10 mM magnesium acetate, 100 μg ml-1 BSA, pH 7.9) and incubated at 37° C. for 2 h. Cleaved U-containing substrates were resolved from full-length unmodified substrates on a 10% TBE-urea gel (Bio-Rad). 
     Expression and Purification of His6-ABE8/PV1-28-Linker-dCas9 Fusions. 
       E. coli  BL21 STAR (DE3)-competent cells (ThermoFisher Scientific) were transformed with plasmids (e.g. plasmids encoding pET28b-His6-ABE8/PV1-28-linker-dCas9). The resulting expression strains were grown overnight in Luria-Bertani (LB) broth containing 100 μg ml-1 of kanamycin at 37° C. The cells were diluted 1:100 into the same growth medium and grown at 37° C. to OD600=—0.6. The culture was cooled to 4° C. over a period of 2 h, and isopropyl-β-d-1-thiogalactopyranoside (IPTG) was added at 0.5 mM to induce protein expression. After ˜16 h, the cells were collected by centrifugation at 4,000g and were resuspended in lysis buffer (50 mM tris(hydroxymethyl)-aminomethane (Tris)-HCl (pH 7.5), 1 M NaCl, 20% glycerol, 10 mM tris(2-carboxyethyl)phosphine (TCEP, Soltec Ventures)). The cells were lysed by sonication (20 s pulse-on, 20 s pulse-off for 8 min total at 6 W output) and the lysate supernatant was isolated following centrifugation at 25,000g for 15 minutes. The lysate was incubated with His-Pur nickel-nitriloacetic acid (nickel-NTA) resin (ThermoFisher Scientific) at 4° C. for 1 hour to capture the His-tagged fusion protein. The resin was transferred to a column and washed with 40 ml of lysis buffer. The His-tagged fusion protein was eluted in lysis buffer supplemented with 285 mM imidazole, and concentrated by ultrafiltration (Amicon-Millipore, 100-kDa molecular weight cut-off) to 1 ml total volume. The protein was diluted to 20 ml in low-salt purification buffer containing 50 mM tris(hydroxymethyl)-aminomethane (Tris)-HCl (pH 7.0), 0.1 M NaCl, 20% glycerol, 10 mM TCEP and loaded onto SP Sepharose Fast Flow resin (GE Life Sciences). The resin was washed with 40 ml of this low-salt buffer, and the protein eluted with 5 ml of activity buffer containing 50 mM tris(hydroxymethyl)-aminomethane (Tris)-HCl (pH 7.0), 0.5 M NaCl, 20% glycerol, 10 mM TCEP. The eluted proteins were quantified by SDS—PAGE. 
     In Vitro Transcription of sgRNAs. 
     Linear DNA fragments containing the T7 promoter followed by the 20-bp sgRNA target sequence were transcribed in vitro using the TranscriptAid T7 High Yield Transcription Kit (ThermoFisher Scientific) according to the manufacturer&#39;s instructions. sgRNA products were purified using the MEGAclear Kit (ThermoFisher Scientific) according to the manufacturer&#39;s instructions and quantified by UV absorbance. 
     Preparation of Cy3-Conjugated dsDNA Substrates. 
     Typically, unlabled sequence strands (e.g. sequences of 80-nt unlabelled strands) were ordered as PAGE-purified oligonucleotides from IDT. A 25-nt Cy3-labelled primer complementary to the 3′ end of each 80-nt substrate was ordered as an HPLC-purified oligonucleotide from IDT. To generate the Cy3-labelled dsDNA substrates, the 80-nt strands (5 μl of a 100 μM solution) were combined with the Cy3-labelled primer (5 μl of a 100 μM solution) in NEBuffer 2 (38.25 μl of a 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9 solution, New England Biolabs) with dNTPs (0.75 μl of a 100 mM solution) and heated to 95° C. for 5 min, followed by a gradual cooling to 45° C. at a rate of 0.1° C. per s. After this annealing period, Klenow exo- (5 U, New England Biolabs) was added and the reaction was incubated at 37° C. for 1 h. The solution was diluted with buffer PB (250 μl, Qiagen) and isopropanol (50 μl) and purified on a QIAprep spin column (Qiagen), eluting with 50 μl of Tris buffer. Deaminase assay on dsDNA. The purified fusion protein (20 μl of 1.9 μM in activity buffer) was combined with 1 equivalent of appropriate sgRNA and incubated at ambient temperature for 5 min. The Cy3-labelled dsDNA substrate was added to final concentration of 125 nM and the resulting solution was incubated at 37° C. for 2 h. The dsDNA was separated from the fusion by the addition of buffer PB (100 Qiagen) and isopropanol (25 μl) and purified on an EconoSpin micro spin column (Epoch Life Science), eluting with 20 μl of CutSmart buffer (New England Biolabs). USER enzyme (1 U, New England Biolabs) was added to the purified, edited dsDNA and incubated at 37° C. for 1 h. The Cy3-labeled strand was fully denatured from its complement by combining 5 μl of the reaction solution with 15 μl of a DMSO-based loading buffer (5 mM Tris, 0.5 mM EDTA, 12.5% glycerol, 0.02% bromophenol blue, 0.02% xylene cyan, 80% DMSO). The full-length C-containing substrate was separated from any cleaved, U-containing edited substrates on a 10% TBE-urea gel (Bio-Rad) and imaged on a GE Amersham Typhoon imager. 
     Preparation of In Vitro-Edited dsDNA for High-Throughput Sequencing. 
     Oligonucleotides were obtained from IDT. Complementary sequences were combined (5 μl of a 100 μM solution) in Tris buffer and annealed by heating to 95° C. for 5 min, followed by a gradual cooling to 45° C. at a rate of 0.1° C. per s to generate 60-bp dsDNA substrates. Purified fusion protein (20 μl of 1.9 μM in activity buffer) was combined with 1 equivalent of appropriate sgRNA and incubated at ambient temperature for 5 min. The 60-mer dsDNA substrate was added to final concentration of 125 nM, and the resulting solution was incubated at 37° C. for 2 h. The dsDNA was separated from the fusion by the addition of buffer PB (100 μl, Qiagen) and isopropanol (25 μl) and purified on a EconoSpin micro spin column (Epoch Life Science), eluting with 20 μl of Tris buffer. The resulting edited DNA (1 μl was used as a template) was amplified by PCR using high-throughput sequencing primer pairs and VeraSeq Ultra (Enzymatics) according to the manufacturer&#39;s instructions with 13 cycles of amplification. PCR reaction products were purified using RapidTips (Diffinity Genomics), and the purified DNA was amplified by PCR with primers containing sequencing adapters, purified, and sequenced on a MiSeq high-throughput DNA sequencer (Illumina) as previously described. 
     Cell Culture. 
     HEK293T (ATCC CRL-3216) and U2OS (ATCC HTB-96) were maintained in Dulbecco&#39;s Modified Eagle&#39;s Medium plus GlutaMax (ThermoFisher) supplemented with 10% (v/v) fetal bovine serum (FBS), at 37° C. with 5% CO2. HCC1954 cells (ATCC CRL-2338) were maintained in RPMI-1640 medium (ThermoFisher Scientific) supplemented as described above. Immortalized cells containing LRRK2) (Taconic Biosciences) were cultured in Dulbecco&#39;s Modified Eagle&#39;s Medium plus GlutaMax (ThermoFisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS) and 200 μg ml-1 Geneticin (ThermoFisher Scientific). 
     Transfections. 
     HEK293T cells were seeded on 48-well collagen-coated BioCoat plates (Corning) and transfected at approximately 85% confluency. Briefly, 750 ng of BE and 250 ng of sgRNA expression plasmids were transfected using 1.5 μl of Lipofectamine 2000 (ThermoFisher Scientific) per well according to the manufacturer&#39;s protocol. HEK293T cells were transfected using appropriate Amaxa Nucleofector II programs according to manufacturer&#39;s instructions (V kits using program Q-001 for HEK293T cells). 
     High-Throughput DNA Sequencing of Genomic DNA Samples. 
     Transfected cells were harvested after 3 days and the genomic DNA was isolated using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter) according to the manufacturer&#39;s instructions. On-target and off-target genomic regions of interest were amplified by PCR with flanking high-throughput sequencing primer pair. PCR amplification was carried out with Phusion high-fidelity DNA polymerase (ThermoFisher) according to the manufacturer&#39;s instructions using 5 ng of genomic DNA as a template. Cycle numbers were determined separately for each primer pair as to ensure the reaction was stopped in the linear range of amplification. PCR products were purified using RapidTips (Diffinity Genomics). Purified DNA was amplified by PCR with primers containing sequencing adaptors. The products were gel purified and quantified using the Quant-iT PicoGreen dsDNA Assay Kit (ThermoFisher) and KAPA Library Quantification Kit-Illumina (KAPA Biosystems). Samples were sequenced on an Illumina MiSeq as previously described (Pattanayak, Nature Biotechnol. 31, 839-843 (2013)). 
     Data Analysis. 
     Sequencing reads were automatically demultiplexed using MiSeq Reporter (Illumina), and individual FASTQ files were analysed with a custom Matlab. Each read was pairwise aligned to the appropriate reference sequence using the Smith-Waterman algorithm. Base calls with a Q-score below 31 were replaced with Ns and were thus excluded in calculating nucleotide frequencies. This treatment yields an expected MiSeq base-calling error rate of approximately 1 in 1,000. Aligned sequences in which the read and reference sequence contained no gaps were stored in an alignment table from which base frequencies could be tabulated for each locus. Indel frequencies were quantified with a custom Matlab script using previously described criteria (Zuris, et al., Nature Biotechnol. 33, 73-80 (2015). Sequencing reads were scanned for exact matches to two 10-bp sequences that flank both sides of a window in which indels might occur. If no exact matches were located, the read was excluded from analysis. If the length of this indel window exactly matched the reference sequence the read was classified as not containing an indel. If the indel window was two or more bases longer or shorter than the reference sequence, then the sequencing read was classified as an insertion or deletion, respectively. 
     PAM Variant Validation in Base Editors 
     Novel CRISPR systems and PAM variants enable base editors (e.g., PV1-PV28) to make precise corrections at a target SNP present in an LRRK2 polynucleotide. Several novel PAM variants have been evaluated and validated. Details of PAM evaluations and base editors are described, for example, in International PCT Application Nos. PCT/2017/045381 (WO2018/027078); PCT/US2016/058344 (WO2017/070632); Kleinstiver, B. P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); and Kleinstiver, B. P., et al., “Broadening the targeting range of  Staphylococcus aureus  CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015), each of which is incorporated herein by reference in its entirety. Also see Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); and Komor, A C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), the entire contents of each of which are hereby incorporated by reference. 
     Gene Editing to Correct Parkinson&#39;s Disease Mutations 
     Pathogenic mutations R1441C and R1441H in LRRK2 are associated with Parkinson&#39;s Disease. As shown in  FIG.  55   , the R1441C mutation, which is associated with a G&gt;A mutation in the antisense strand of the LRRK2 gene, which is corrected using a base editor having adenosine deaminase activity and AGA PAM specificity. The R1441H in LRRK, which is encoded by a G&gt;A mutation in the LRRK2 gene, is corrected using a base editor having adenosine deaminase activity and TGA PAM specificity at position 3 or having TGT specificity at position 5 of the target sequence shown at  FIG.  55   . 
       FIG.  56    is a schematic diagram showing target sequences for correction of the Y1699C, G2019S, and I2020T mutations in LRRK2 associated with Parkinson&#39;s Disease. The Y1699C mutation is associated with a T&gt;C mutation on the antisense strand of the LRRK2 gene, which is corrected using a base editor having cytidine deaminase activity. The G2019S mutation is associated with a G&gt;A mutation on the antisense strand of the LRRK2 gene, which is corrected using a base editor having adenosine deaminase activity. I2020T is encoded by a T&gt;C mutation in the LRRK2 gene, which is corrected using a base editor having cytidine deaminase activity and TGC PAM specificity. 
     As shown in  FIG.  57 A- 57 C , Editors PV 1-14 were used to edit LRRK2 R1441C using a guide RNA having the sequence shown in  FIG.  57 B , but where all thymidines (Ts) in the target sequence are substituted by uridines (Us) (guide RNA1: 5′-AAGCGCAAGCCUGGAGGGAA-3′ (SEQ ID NO: 11)). The percent conversion of A to G is shown at  FIG.  57 A . An exemplary sequence read is shown at  FIG.  57 C . 
     As shown in  FIG.  58 A- 58 C , Editors PV 15-28 were used to edit LRRK2 G2019S using a guide RNA having the sequence shown in  FIG.  58 B , but where all thymidines (Ts) in the target sequence are substituted by uridines (Us) (guide RNA2: 5′-ACUACAGCAUUGCUCAGUAC-3′ (SEQ ID NO: 12)). The percent conversion of A to G at target and off-target sites is shown at  FIG.  58 A . An exemplary sequence read is shown at  FIG.  58 C . Editors (PV15-28) were used to edit G2019S. 
     A description of the editors (PV15-28) used for correction of the LRRK2 mutations follows: 
     
       
         
           
               
               
            
               
                 PV1 (also PV15). pCMV_monoABE8.1_bpNLS + Y147T 
                   
               
               
                 (SEQ ID NO: 21) 
                   
               
               
                   MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD  
               
               
                   
               
               
                 PV2 (also PV16). pCMV_monoABE8.1_bpNLS + Y147R 
               
               
                 (SEQ ID NO: 88) 
                   
               
               
                   MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCRFFRMPRQVFNAQKKAQSSTD  
               
               
                   
               
               
                 PV3 (also PV17). pCMV_monoABE8.1_bpNLS + Q154S 
               
               
                 (SEQ ID NO: 89) 
                   
               
               
                   MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPRSVFNAQKKAQSSTD  
               
               
                   
               
               
                 PV4 (also PV18). pCMV_monoABE8.1_bpNLS + Y123H 
               
               
                 (SEQ ID NO: 90) 
                   
               
               
                   MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD  
               
               
                   
               
               
                 PV5 (also PV19). pCMV_monoABE8.1_bpNLS + V82S 
               
               
                 (SEQ ID NO: 91) 
                   
               
               
                   MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD  
               
               
                   
               
               
                 PV6 (also PV20). pCMV_monoABE8.1_bpNLS + T166R 
               
               
                 (SEQ ID NO: 92) 
                   
               
               
                   MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSRD  
               
               
                   
               
               
                 PV7 (also PV21). pCMV_monoABE8.1_bpNLS + Q154R 
               
               
                 (SEQ ID NO: 93) 
                   
               
               
                   MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPRRVFNAQKKAQSSTD  
               
               
                   
               
               
                 PV8 (also PV22). pCMV_monoABE8.1_bpNLS + Y147R_Q154R_Y123H 
               
               
                 (SEQ ID NO: 94) 
                   
               
               
                   MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD  
               
               
                   
               
               
                 PV9 (also PV23). pCMV_monoABE8.1_bpNLS + Y147R_Q154R_I76Y 
               
               
                 (SEQ ID NO: 95) 
                   
               
               
                   MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD  
               
               
                   
               
               
                 PV10 (also PV24). pCMV_monoABE8.1_bpNLS + Y147R_Q154R_T166R 
               
               
                 (SEQ ID NO: 96) 
                   
               
               
                   MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSRD  
               
               
                   
               
               
                 PV11 (also PV25). pCMV_monoABE8.1_bpNLS + Y147T_Q154R 
               
               
                 (SEQ ID NO: 97) 
                   
               
               
                   MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCTFFRMPRRVFNAQKKAQSSTD  
               
               
                   
               
               
                 PV12 (also PV26). pCMV_monoABE8.1_bpNLS + Y147T_Q154S 
               
               
                 (SEQ ID NO: 98) 
                   
               
               
                   MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCTFFRMPRSVFNAQKKAQSSTD  
               
               
                   
               
               
                 PV13 (also PV27). pCMV_monoABE8.1_bpNLS + H123Y123H_Y147R_Q154R_I76Y 
               
               
                 (SEQ ID NO: 99) 
                   
               
               
                   MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD  
               
               
                   
               
               
                 PV14 (also PV28). pCMV_monoABE8.1_bpNLS + V82S + Q154R 
               
               
                 (SEQ ID NO: 100) 
                   
               
               
                   MSEVEFSHEY WMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR 
                   
               
               
                   
               
               
                 QGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH 
               
               
                   
               
               
                 RVEITEGILADECAALLCYFFRMPRRVFNAQKKAQSSTD  
               
            
           
         
       
     
       FIGS.  59 A- 59 L  provides exemplary sequence reads for the A to G transition at position 7 of the LRRK2 target sequence, which encodes R1441C. The editor is indicated (PV1-14). 
       FIGS.  60 A- 60 W  depicts the sequence reads for the A to G transition at positions 4 and 6 of the LRRK2 target sequence, which encodes G2019S. The editor is indicated (PV15-28). Other pathogenic mutations in LRRK2 associated with Parkinson Disease are corrected using similar strategies ( FIG.  61 A- 61 D ). 
     Example 9. Hitler Syndrome Base Editing for the Correction of the W401X Mutation in the Mouse Alpha-L-Iduronidase (IDUA) Gene 
     Hurler Syndrome is the most severe form of mucopolysaccharidosis type 1 (MPS1). MPS1 is caused by mutations in alpha-L-iduronidase (IDUA) gene. Currently, there are no transgenic mouse models containing the human IDUA gene. However, there is high conservation between the  Mus musculus  IDUA protein amino acid sequence and the  Homo sapiens  IDUA protein amino acid sequence. In humans, a common mutation for MPS1 associated with a severe Hurler Syndrome phenotype is W402X. This mutation is a single base substitution that introduces a stop codon at position 402 (W402X) of the IDUA protein and is associated with an extremely severe clinical phenotype in homozygotes. In mice, the equivalent mutation in the IDUA protein is W401X. ABEs may be used for the correction of the  Mus musculus  IDUA gene by correcting the W401X mutation by efficiently converting A&gt;G at a targeted site. An A&gt;G correction at the SNP changes the stop codon at position 401 (W401X) to tryptophan in the IDUA polypeptide. 
     The W401X mutation was targeted for reversion to wild-type sequence using A•T to G•C DNA base editors (ABEs) that employ Cas9 moieties with validated protospacer adjacent motif (PAM) sequence preferences. To determine which guide RNA (gRNA) and ABE8-Cas9 platform is able to most efficiently and precisely correct a targeted IDUA mutation, a  Mus musculus  IDUA allele bearing the Hurler Syndrome W401X targetable mutation was genomically integrated in HEK293T cells by lentivirus transduction. The HEK293T cells were transfected and plated at 30,000 cells per well of a 48-well plate with 250 ng of gRNA and 750 ng of ABE8 variant base editor expression plasmid using Opti-MEM media and Lipofectamine 2000 as described above. The ABE8 base editor variants contained the NGG PAM sequence (i.e., SpCas9). The cells were lysed and prepped for sequencing 5 days post-transfection (with a media change at day 3 post-transfection) and analyzed for base editing at the desired site by miSeq analysis. 
     The DNA targeted/insert sequence for  Mus musculus  IDUA is shown below and corresponds to nucleic acids 1077-1358 of the representative  Mus musculus  IDUA gene sequence found under NCBI Reference Sequence No. NM_008325.4. 
     
       
         
           
               
            
               
                 (SEQ ID NO: 433) 
               
               
                 CTCCCAGCGCACACTTACTGCTCGATTCCAGGTCAACAATACTCACCCA 
               
               
                   
               
               
                 CCCCACGTGCAGTTGCTGCGAAAGCCAGTACTCACAGTCATGGGGCTCA 
               
               
                   
               
               
                 TGGCCCTGTTGGATGGAGAACAACTCT   A   GGCAGAGGTCTCAAAGGCTGG 
               
               
                   
               
               
                 GGCTGTGTTGGACAGCAATCATACAGTGGGTGTCCTGGCCAGCACCCAT 
               
               
                   
               
               
                 CACCCTGAAGGCTCCGCAGCGGCCTGGAGTACCACAGTCCTCATCTACA 
               
               
                   
               
               
                 CTAGTGATGACACCCACGCACACCCCAACCACAGTAT  
               
            
           
         
       
     
     The above  Mus musculus  DNA target/insert sequence for the W401X mutation contains an “A” nucleobase (shown in bold and underlined), while the  Mus musculus  IDUA gene sequence contains a “G” nucleobase at position 1202. 
     Two guide RNAs were tested for base editing of the  Mus musculus  IDUA W401X mutation. The gRNA encompasses the scaffold sequence and the spacer sequence (target sequence) for disease-associated genes as provided herein or as determined based on the knowledge of the skilled practitioner and as would be understood to the skilled practitioner in the art. (See, e.g., Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, A. C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), and Rees, H. A., et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 December; 19(12):770-788. doi: 10.1038/s41576-018-0059-1). 
     A 21-nucleotide guide RNA (gRNA) was tested targeting the IDUA W401X mutation with ABE8 base editor variants ( FIGS.  62 A and  62 B ). The 21-nucleotide gRNA sequence, which hybridizes to the complement of the above DNA target sequence is shown below: gACTCTAGGCAGAGGTCTCAA AGG  (SEQ ID NO: 434). The NGG PAM sequence (i.e., SpCas9) is underlined above. The lowercase “g” in the gRNA sequence indicates a mismatch in the sequence where a polymerase (e.g. Pol III) must initiate transcription. The guide RNA comprises sequence UUGAGACCUCUGCCUAGAGU (SEQ ID NO: 435). 
     For the above gRNA sequence, the scaffold sequence is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 436) 
               
               
                 GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAA 
               
               
                   
               
               
                 CTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT. 
               
            
           
         
       
     
     The ABE base editors used include ABE8 monomer variants: ABE8.1, ABE8.12, ABE8.13, ABE8.4, ABE8.5, ABE8.6, ABE8.7, ABE8.8, ABE8.9, ABE8.10, ABE8.11, ABE8.12, and ABE8.13. Positive control base editor ABE7.10 and a negative control were also used for comparison. 
     The ABE8 base editor variants had comparable or increased base editing activity compared to the ABE7.10 positive control with about 40% base editing correction of W401X ( FIG.  62 A ). As shown in  FIG.  62 B , the percent of indel formation was comparable to the ABE7.10 positive control at about 0.4-0.6%. 
     A 20-nucleotide guide RNA (gRNA) was also tested targeting the IDUA W401X mutation with ABE8 base editor variants and compared to the 21-nucleotide gRNA described above ( FIG.  63   ). The 20-nucleotide gRNA sequence, which hybridizes to the complement of the above DNA target sequence is shown below: ACTCTAGGCAGAGGTCTCAA  AGG  (SEQ ID NO: 437). The NGG PAM sequence (i.e., SpCas9) is underlined above. 
     For the above gRNA sequence, the scaffold sequence is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 436) 
               
               
                 GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAA 
               
               
                   
               
               
                 CTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT. 
               
            
           
         
       
     
     The ABE base editors used include ABE8 monomer variants: ABE8.1, ABE8.12, ABE8.13, ABE8.4, ABE8.5, ABE8.6, ABE8.7, ABE8.8, ABE8.9, ABE8.10, ABE8.11, ABE8.12, and ABE8.13. Positive control base editor ABE7.10 and a negative control were also used for comparison. 
     The ABE8 base editor variants had comparable base editing activity using either the 20-nucleotide gRNA or the 21-nucleotide gRNA with about 40% base editing correction of W401X ( FIG.  63   ). 
     Example 10. Hurler Syndrome Base Editing for the Correction of the W402X Mutation in the Human Alpha-L-Iduronidase (IDUA) Gene 
     Hurler Syndrome is the most severe form of mucopolysaccharidosis type 1 (MPS1). MPS1 is caused by mutations in alpha-L-iduronidase (IDUA) gene. In humans, a common mutation for MPS1 associated with a severe Hurler Syndrome phenotype is W402X. This mutation is a single base substitution that introduces a stop codon at position 402 (W402X) of the IDUA protein and is associated with an extremely severe clinical phenotype in homozygotes. ABEs may be used for the correction of the human IDUA gene by correcting the W402X mutation by efficiently converting A&gt;G at a targeted site. 
     The W402X mutation was targeted for reversion to wild-type sequence using A•T to G•C DNA base editors (ABEs) that employ Cas9 moieties with validated protospacer adjacent motif (PAM) sequence preferences. As shown in  FIG.  64   , an ABE base editor and guide RNA (gRNA) can be used to target an adenosine (A) nucleobase (boxed) in the  Homo sapiens  IDUA nucleic acid sequence to correct the W402X mutation. An A&gt;G correction at the SNP changes the stop codon at position 402 (W402X) to tryptophan in the IDUA polypeptide. 
     To determine which ABE8-Cas9 platform is able to most efficiently and precisely correct a targeted IDUA mutation, a  Homo sapiens  IDUA allele bearing the Hurler Syndrome W402X targetable mutation was genomically integrated in HEK293T cells by lentivirus transduction. The HEK293T cells were transfected and plated at 30,000 cells per well of a 48-well plate with 250 ng of gRNA and 750 ng of ABE8 variant base editor expression plasmid using Opti-MEM media and Lipofectamine 2000 as described above. The ABE8 base editor variants contained the NGG PAM sequence (i.e., SpCas9). The cells were lysed and prepped for sequencing 5 days post-transfection (with a media change at day 3 post-transfection) and analyzed for base editing at the desired site by miSeq analysis. 
     The DNA targeted/insert sequence in the  Homo sapiens  IDUA polynucleotide sequence is shown below and corresponds to nucleic acids 1076-1358 of the representative  Homo sapiens  IDUA gene sequence found under NCBI Reference Sequence No. NM_000203.5. 
     
       
         
           
               
            
               
                 (SEQ ID NO: 438) 
               
               
                 CCTTCGCGCAGCGCACGCTCACCGCGCGCTTCCAGGTCAACAACACCCG 
               
               
                   
               
               
                 CCCGCCGCACGTGCAGCTGTTGCGCAAGCCGGTGCTCACGGCCATGGGG 
               
               
                   
               
               
                 CTGCTGGCGCTGCTGGATGAGGAGCAGCTCT   A   GGCCGAAGTGTCGCAGG 
               
               
                   
               
               
                 CCGGGACCGTCCTGGACAGCAACCACACGGTGGGCGTCCTGGCCAGCGC 
               
               
                   
               
               
                 CCACCGCCCCCAGGGCCCGGCCGACGCCTGGCGCGCCGCGGTGCTGATC 
               
               
                   
               
               
                 TACGCGAGCGACGACACCCGCGCCCACCCCAACCGCAG  
               
            
           
         
       
     
     The above  Homo sapiens  target/insert sequence contains an “A” nucleobase (shown in bold and underlined), while the  Homo sapiens  IDUA gene sequence contains a “G” nucleobase at position 1205 of the IDUA sequence. 
     A 20-nucleotide guide RNA (gRNA) was tested targeting the W402X mutation with ABE8 base editor variants ( FIG.  65 A ). The gRNA encompasses the scaffold sequence and the spacer sequence (target sequence) for disease-associated genes as provided herein or as determined based on the knowledge of the skilled practitioner and as would be understood to the skilled practitioner in the art. (See, e.g., Komor, A. C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N. M., et al., “Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, A C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017), and Rees, H. A., et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 December; 19(12):770-788. doi: 10.1038/s41576-018-0059-1). 
     The gRNA sequence, which hybridizes to the complement of the above DNA target sequence is: GCTCTAGGCCGAAGTGTCGC AGG (SEQ ID NO: 439). The NGG PAM sequence (i.e., SpCas9) is underlined above. 
     For the above gRNA sequence, the scaffold sequence is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 436) 
               
               
                 GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAA 
               
               
                   
               
               
                 CTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT  
               
            
           
         
       
     
     The ABE base editors used include ABE8 monomer variants: ABE8.1, ABE8.2, ABE8.3, ABE8.4, ABE8.5, ABE8.6, ABE8.7, ABE8.8, ABE8.9, ABE8.10, ABE8.11, ABE8.12, and ABE8.13. Positive control base editor ABE7.10 and a negative control were also used for comparison. 
     The ABE8 base editor variants had comparable or increased base editing activity compared to the ABE7.10 positive control with about 30-40% base editing correction of W402X ( FIG.  65 A ). As shown in  FIG.  65 B , the percent of indel formation was comparable or below the ABE7.10 positive control at about 0.2-0.5%. 
     The efficiency of A to G base editing of the target “A” nucleobase at position 6 in the IDUA nucleic acid sequence as detected by deep sequencing of PCR products is presented in  FIGS.  66 A- 66 O . Table 18 below summarizes the percent of A to G base editing at position 6 in the IDUA nucleic acid target site achieved by ABE8 base editor variants ( FIGS.  66 A- 66 M ) compared with ABE7.10 positive control ( FIG.  66 N ) and negative control ( FIG.  66 O ). 
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 IDUA Target Site Base Editing Percentage 
               
               
                 with Base Editor Variants. 
               
            
           
           
               
               
               
               
            
               
                   
                 Sample 
                 ABE 
                 A to G Base Editing (%) 
               
               
                   
                   
               
               
                   
                 NMG280-1 
                 ABE8.1 
                 31.17% 
               
               
                   
                 NMG280-2 
                 ABE8.1 
                 31.65% 
               
               
                   
                 NMG280-3 
                 ABE8.1 
                 33.61% 
               
               
                   
                 NMG281-1 
                 ABE8.2 
                 41.89% 
               
               
                   
                 NMG281-2 
                 ABE8.2 
                 40.96% 
               
               
                   
                 NMG281-3 
                 ABE8.2 
                 35.02% 
               
               
                   
                 NMG282-1 
                 ABE8.3 
                 34.07% 
               
               
                   
                 NMG282-2 
                 ABE8.3 
                 31.86% 
               
               
                   
                 NMG282-3 
                 ABE8.3 
                 30.78% 
               
               
                   
                 NMG283-1 
                 ABE8.4 
                 32.72% 
               
               
                   
                 NMG283-2 
                 ABE8.4 
                 31.87% 
               
               
                   
                 NMG283-3 
                 ABE8.4 
                 27.74% 
               
               
                   
                 NMG284-1 
                 ABE8.5 
                  8.11% 
               
               
                   
                 NMG284-2 
                 ABE8.5 
                 10.02% 
               
               
                   
                 NMG284-3 
                 ABE8.5 
                  9.5% 
               
               
                   
                 NMG285-1 
                 ABE8.6 
                   39% 
               
               
                   
                 NMG285-2 
                 ABE8.6 
                 31.94% 
               
               
                   
                 NMG285-3 
                 ABE8.6 
                 37.55% 
               
               
                   
                 NMG286-1 
                 ABE8.7 
                 39.84% 
               
               
                   
                 NMG286-2 
                 ABE8.7 
                 32.14% 
               
               
                   
                 NMG286-3 
                 ABE8.7 
                 40.85% 
               
               
                   
                 NMG287-1 
                 ABE8.8 
                 38.92% 
               
               
                   
                 NMG287-2 
                 ABE8.8 
                 38.87% 
               
               
                   
                 NMG287-3 
                 ABE8.8 
                 35.53% 
               
               
                   
                 NMG288-1 
                 ABE8.9 
                 36.73% 
               
               
                   
                 NMG288-2 
                 ABE8.9 
                 38.97% 
               
               
                   
                 NMG288-3 
                 ABE8.9 
                 28.23% 
               
               
                   
                 NMG289-1 
                 ABE8.10 
                  34.1% 
               
               
                   
                 NMG289-2 
                 ABE8.10 
                  33.7% 
               
               
                   
                 NMG289-3 
                 ABE8.10 
                 39.92% 
               
               
                   
                 NMG290-1 
                 ABE8.11 
                 41.41% 
               
               
                   
                 NMG290-2 
                 ABE8.11 
                 36.78% 
               
               
                   
                 NMG290-3 
                 ABE8.11 
                 39.83% 
               
               
                   
                 NMG291-1 
                 ABE8.12 
                 37.87% 
               
               
                   
                 NMG291-2 
                 ABE8.12 
                 45.05% 
               
               
                   
                 NMG291-3 
                 ABE8.12 
                 35.94% 
               
               
                   
                 NMG292-1 
                 ABE8.13 
                 34.44% 
               
               
                   
                 NMG292-2 
                 ABE8.13 
                 33.73% 
               
               
                   
                 NMG292-3 
                 ABE8.13 
                  39.7% 
               
               
                   
                 NP20-1 
                 ABE7.10 
                 33.73% 
               
               
                   
                 NP20-2 
                 ABE7.10 
                 33.93% 
               
               
                   
                 NP20-3 
                 ABE7.10 
                 26.89% 
               
               
                   
                 Negct-1 
                 Negative Control 
                  0.04% 
               
               
                   
                 Negct-2 
                 Negative Control 
                  0.03% 
               
               
                   
                   
               
            
           
         
       
     
     As shown in Table 18 and  FIGS.  66 A- 66 M , an average of about 33.9% A-to-G base editing was achieved at position 6 of the IDUA nucleic acid sequence, the targeted “A” nucleobase site, using ABE8 base editor variants. This was comparable to positive control ABE7.10 ( FIG.  66 N ). 
     Example 11. Cell Culture and Transfection 
     The HEK293T (293T) cell line was obtained from the American Tissue Culture Collection (ATCC). 293T cells were maintained in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37° C. with 5% CO 2 . All cell lines were transfected in 24-well plates with Lipofectamine 2000 (Invitrogen), following the manufacturer&#39;s instructions. The amount of DNA used for lipofection was 1 μg per well. Transfection efficiency was routinely higher than 80% for 293T cells as determined by fluorescent microscopy following delivery of a control GFP expression plasmid. 
     For plasmid transfections, HEK293T cells were plated and transfected with 250 ng of expression plasmid containing a U6 promoter and encoding the gRNA. and with 750 ng of expression plasmid encoding the Cas9/ABE8 variant base editor using Opti-MEM media and Lipofectamine 2000. The ABE8 base editor variants used included the NGG PAM sequence. The cells were maintained 37° C. with 5% CO 2  for 5 days, with a change of medicum at day 3 post transfection. Thereafter, the cells were lysed; genomic DNA was isolated and PCR was performed using standard procedures, typically using 20-100 ng of template DNA. After the addition of adapters (Illumina), the DNA was subjected to deep sequencing. Base editing at the desired site was analyzed by MiSeq analysis. 
     Deep sequencing was performed on PCR amplicons from genomic DNA or RNA harvested from duplicate transfections of 293T cells. After validating the quality of PCR product by gel electrophoresis, the PCR products were isolated by gel extraction, e.g., using the Zymoclean Gel DNA Recovery Kit (Zymo Research). Shotgun libraries were prepared without shearing. The library was quantified by qPCR and sequenced on one MiSeq Nano flowcell for 251 cycles from each end of the fragments using a MiSeq 500-cycle sequencing kit version 2. Fastq files were generated and demultiplexed with the bcl2fastq v2.17.1.14 Conversion Software (Illumina). 
     Example 12: Base Editing Correction of Rett Syndrome Mutations 
     A lenti-HEK line containing a single copy of MECP2 with 6 Rett mutations, including R106W and R255X, was generated and used to screen guide RNA sequences and ABE variants. Plasmids for guide and ABE expression were transfected into the lenti-HEK line, and genomic DNA was collected and analyzed by NextGen sequencing for mutations at the target site. Referring to  FIGS.  67  and  68   , Guide 1 showed overall highest editing, with ABE 8.8, ABE 8.9, and ABE 8.13 working well with both Guides 1 and 2. The guide RNA sequences comprise a spacer CTTTTCACTTTTCCTGCCGGGG (SEQ ID NO: 305) (R255X), AGCTTCCATGTCCAGCCTTC (SEQ ID NO: 306) (R106W), ACCATGAAGTCAAAATCATT (SEQ ID NO: 307) (T158M), or GCTTTCAGCCCCGTTTCTTG (SEQ ID NO: 308) (R270X). 
     Base editor variants having different PAM recognition specificity are tested; the Cas9 substitutions corresponding to PAM alterations are shown in Table 19. *Variant 5 corresponds to 25% editing at R255X. The methods are similar to those for R106W described above, with mutations on ABEs generated by other groups. Referring to  FIGS.  69    and Table 19 below, Mutations contributing to variant 5 showed the highest amount of editing at this particular locus. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 19 
               
               
                   
               
               
                 variant 
                 E1219V 
                 R1335Q 
                 T1337 
                 G1218 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 F 
                 V 
                 T 
                   
               
               
                 2 
                 F 
                 V 
                 R 
               
               
                 3 
                 F 
                 V 
                 Q 
               
               
                 4 
                 F 
                 V 
                 L 
               
               
                 5 
                 F 
                 V 
                 T 
                 R 
               
               
                 6 
                 F 
                 V 
                 R 
                 R 
               
               
                 7 
                 F 
                 V 
                 Q 
                 R 
               
               
                 8 
                 F 
                 V 
                 L 
                 R 
               
               
                 9 
                 L 
                 L 
                 T 
               
               
                 10 
                 L 
                 L 
                 R 
               
               
                 11 
                 L 
                 L 
                 Q 
               
               
                 12 
                 L 
                 L 
                 L 
               
               
                 13 
                 F 
                 I 
                 T 
               
               
                 14 
                 F 
                 I 
                 R 
               
               
                 15 
                 F 
                 I 
                 Q 
               
               
                 16 
                 F 
                 I 
                 L 
               
               
                 17 
                 F 
                 G 
                 C 
               
               
                 18 
                 H 
                 L 
                 N 
               
               
                 19 
                 F 
                 G 
                 C 
                 A 
               
               
                 20 
                 H 
                 L 
                 N 
                 V 
               
               
                 21 
                 L 
                 A 
                 W 
               
               
                 22 
                 L 
                 A 
                 F 
               
               
                 23 
                 L 
                 A 
                 Y 
               
               
                 24 
                 I 
                 A 
                 W 
               
               
                 25 
                 I 
                 A 
                 F 
               
               
                 26 
                 I 
                 A 
                 Y 
               
               
                   
               
            
           
         
       
     
     Example 13: Hurler/IDUA Mutation Correction 
     A lenti-HEK line containing a single copy of MECP2 with Hurler mutations was generated and used to screen guide RNA sequences and variants as described above. Referring to  FIGS.  70 A- 70 C , correction of inherited IDUA loss of function (W402X) mutation was examined using base editor variants. Fibroblasts from two W402X homozygous Hurler patients (GM06214 and GM00798) as well as an unaffected heterozygous parent (GM00799) were obtained from Coriell. Patient-derived and BJ fibroblasts were electroporated with mRNA for ABE 8.8 and a human W402X guide. Genomic DNA was extracted with QuickExract lysis solution and sequenced with NextGen sequencing to assess A to G editing. Iduronidase activity was assessed by a spectrophotometric assay. Lysates were incubated with 4-methylumbelliferone-iduronide in an acidic buffer for 2 hours, then quenched with an alkaline solution. 4-methylumbelliferone cleavage was measured by fluorescence (365 nm excitation, 445 nm emission). High editing was observed in the patient fibroblasts, and this led to an increase in enzymatic activity comparable to that observed in the unaffected heterozygote, GM00799. 
     Example 14: In Vivo Base Editing with ABE 8.8 
     Referring to  FIG.  70   , Viral genomes encoding split intein ABE8.8 and gRNAs for the murine ROSA26 locus were packaged into AAV9 and PHP.eB capsids. 9e11 total vg AAV9 (4.5e11 of each intein split) and 7.5e11 total vg PHP.eB (3.75e11 of each intein split) were injected into the lateral ventricle of C57BL/6 mice. 6 weeks later, brains and spinal cord were harvested and dissected for genomic DNA extraction and sequencing. AAV9 transduction was highest in the hippocampus, which is the closest structure to the lateral ventricle, reaching up to 13% editing. 
     Example 15: ABE8 Variant Base Editing 
     To determine the optimal base editor for reverting a G1961E mutation in ABCA4, forty unique ABE8 variants were compared to ABE7.10 for making an A-to-G base conversion at the disease allele ( FIGS.  72 A- 72 B ) in a lentiviral knock-in model cell line using a sgRNA with a 21-nt spacer sequence that we previously demonstrated as the optimal spacer length on this target site ( FIG.  73   ). All variants provided measurable A-to-G editing at the disease allele and the wobble base. Editing at the wobble base results in a silent mutation and is not deleterious. Six of the best-performing variants were subsequently codon optimized and incorporated into a split AAV system for further validation. Because the size of the DNA sequences that encode the base editor, the sgRNA, and the expression regulatory elements exceed the packaging limit of a single AAV particle, the requisite parts can be divided between two AAV particles and co-delivered. In this delivery methodology, the gene encoding the base editor is split between two viruses, and a split intein is used to reconstitute the full-length protein after co-infection ( FIG.  74   ). The split ABE8 variants lacking a wild type TadA domain (ABE8-m) were packaged into pairs of AAV2 vectors, one of which also encodes a single copy of a sgRNA targeting the wildtype ABCA4 site of interest. In this experiment editing was assessed at the wobble base of the ABCA4 G1961 codon as a surrogate for the disease allele that is not present in the wild type cells. Wild type ARPE-19 cells were co-transduced with the dual AAVs, and base editing rates were assessed on the 21-nt target sequence ( FIGS.  75 A- 75 B ) of interest. ABE variant 7.9, 7.10, 8.5-m, 8.8-m, 8.9-m, and 8.18-m were equally efficient at converting the surrogate site 8A, however, variants 8.8-m, 8.9-m, and 8.18-m also catalyzed undesirable C-to-T conversion at position 5C. A variant of ABE7.10 wherein the wild type TadA domain was removed (ABE7.10-m) had an apparent 50% loss of activity compared to the parent ABE7.10 variant. These results show that variant ABE8.5-m is the most efficient editor at the site of interest that also lacks a wild type TadA domain, which reduces the total size of the base editor by 594 bp of DNA or 198 amino acid residues. 
     
       
         
           
               
            
               
                 Guide RNA sequences target ABCA4 gene at sequence 
               
               
                 (SEQ ID NO: 291) 
               
               
                 GCTGTGTGTCGAAGTTCGCCCTGGAG AGG TG   
               
               
                 or 
               
               
                 (SEQ ID NO: 292) 
               
               
                 GCTGTGTGTCGGAGTTCGCCCTGGAG AGG TG,  
               
               
                 where the PAM sequence is underlined.  
               
               
                   
               
               
                 The guide RNA comprises a sequence 
               
               
                 (SEQ ID NO: 293) 
               
               
                 CACCUCUCCAGGGCGAACUUCGACACACAGC   
               
               
                 or 
               
               
                   
               
               
                 CACCUCUCCAGGGCGAACUCCGACACACAGC (SEQ ID NO: 294). 
               
            
           
         
       
     
     The potential for off-target base editing within the human genome using the 21-nt spacer length sgRNA targeting the ABCA4 G1961E locus was assessed. In silico prediction of potential off target sites within the genome was performed by computationally scanning the human reference genome (GRCh38) for all imperfect matches to the ABCA4 G1961E gRNA protospacer sequence followed by a 3′ sequence matching the SpCas9 NGG PAM. All sequences containing up to 5 mismatches and a single RNA or DNA bulge were evaluated. Potential off-target sites were prioritized for experimental assessment based on (a) low number of mismatches, and (b) overlap with coding exons (as determined by GENCODE transcript annotations) and cancer-associated genes (as reported in the COSMIC cancer gene census). No predicted off-targets in the genome with three or fewer mismatches against the 21-nt spacer sequence were found by in silico analysis. We used a dual AAV system to co-deliver ABE7.10 and the 21-nt spacer sgRNA targeting the ABCA4 G1961E disease allele into wild type ARPE-19 cells and assessed editing by targeted amplicon sequencing of 28 in silico-predicted off target sites. None of the predicted off-target sites were significantly base edited in treated cells compared to untreated cells ( FIGS.  76 A- 76 B ) and no significant indels were found at the off-target sites or at the on-target site ( FIG.  77   ). The only significant editing observed occurred at the ABCA4 G1961 wobble base in the treated cells as would be expected since the sgRNA targeting the ABCA4 G1961E disease allele contains only a single mismatched base pair with the wild type allele present in these cells. These results indicate that the sgRNA does not promote off-target DNA editing at any of the in silico-predicted off-target sites that were assessed. 
     Example 20: Primate Retina Examples 
     Non-human primate eyes were harvested 1-2 hours post-mortem and put in culture between 4-8 hours post-mortem. A 6 mm biopsy punch was used to take punches from the entire neural retina. The retina with the photoreceptor side facing down was placed on top of a nucleopore membrane in a 6-well tissue culture plate. Vector (10 ul at 1.26E+12 vg/ml) was pipetted between the neural retina and the membrane to form a bleb under the retinal tissue. Media was replaced every 3 days and tissues were incubated for 0-22 days. Tissues were collected at different timepoints and fixed in 10% neutral buffered formalin and processed for histology. 
     ‘Primate Retina Integrity’ 
     Sections were immunolabeled with anti-Rhodopsin, anti-GFP, and biotinylated peanut agglutinin antibodies overnight at 4C. After washing in PBS, samples were incubated with secondary antibodies for 1 hour at room temperature. Slides were washed in PBS and mounted with a glycerol-based liquid mountant containing DAPI. 
     Retinal explants from non-human primates were harvested at day (D) 0 and 22. Histological staining comparing DO and D22 untransduced retinal explants showed cell types of the retina to be preserved when cultured up to 22 days. At D22, GFP expression was qualitatively brighter in retinal cultures exposed to Anc80L65.CMV.eGFP compared to a photoreceptor-specific GFP expression vector (Anc80L65.hGRK.eGFP). Transduction of retinal explants with Anc80L65.hGRK1.eGFP demonstrated GFP to be exclusively in the photoreceptor-containing outer nuclear layer (ONL), confirming photoreceptor-specific activity of the hGRK1 promoter. See,  FIG.  78   . 
     ‘Cas9 Expression in NHP’ 
     Sections were immunolabeled with mouse and rabbit monoclonal Cas9 antibodies overnight at 4° C. After washing in PBS, samples were incubated with secondary antibodies for 1 hour at room temperature. Slides were washed in PBS and mounted with a glycerol-based liquid mountant containing DAPI. 
     Dual AAV2 particles encoding the optimized split ABE (ABE7.10, 8.5, 8.9) base editors were tested on non-human primate retinal explants. To test reconstitution of full-length based editors, following coinfection by AAV particles expressing each base editor-split intein halfs, tissues were collected at different timepoints and stained for Cas9 N and C-termini. We observed expression of both Cas9 N (green stain) and C (red stain)-termini as early as day 6 and maintained up to day 17 post infection suggesting a possible editing activity window for the base editors. These results establish that the dual AAV split-intein base editor expresses Cas9 in non-human primate retinal explants. See,  FIG.  79   . 
     Other Embodiments 
     From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. 
     The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. 
     All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Absent any indication otherwise, publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entireties.