Patent Publication Number: US-2023159956-A1

Title: Methods of editing single nucleotide polymorphism using programmable base editor systems

Description:
RELATED APPLICATIONS 
     This application is the U.S. national phase application, pursuant to 35 U.S.C. § 371, of PCT International Application No.: PCT/US2019/031898, filed May 11, 2019, designating the United States and published in English, which claims priority to and benefit of U.S. Provisional Application No. 62/670,588, filed May 11, 2018, U.S. Provisional Application No. 62/780,838, filed Dec. 17, 2018 and U.S. Provisional Application No. 62/817,986, filed Mar. 13, 2019, each of which is incorporated herein by reference in its 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. The ASCII copy, created on Apr. 23, 2021, is named 52885-729_601_SL.txt and is 732,672 bytes in size. 
     BACKGROUND OF THE DISCLOSURE 
     For most known genetic diseases, correction of a point mutation in the target locus, rather than stochastic disruption of the gene, is needed to study or address the underlying cause of the disease. Current genome editing technologies utilizing the clustered regularly interspaced short palindromic repeat (CRISPR) system introduce double-stranded DNA breaks at a target locus as the first step to gene correction. In response to double-stranded DNA breaks, cellular DNA repair processes mostly result in random insertions or deletions (indels) at the site of DNA cleavage through non-homologous end joining. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Therefore, there is a need for an improved form of genome editing that is more efficient and with far fewer undesired products such as stochastic insertions or deletions (indels) or translocations. 
     Alpha-1 Antitrypsin Deficiency (A1AD) is a genetic disease in which pathogenic mutations in the SERPINA1 gene that encodes the alpha-1 antitrypsin (A1AT) protein lead to diminished protein production in individuals having the disease. A1AT is a particularly good inhibitor of neutrophil elastase and protects tissues and organs such as the lung from elastin degradation. Consequently, elastin in the lungs of patients having A1AD is degraded more readily by neutrophil elastase, and over time, the loss in lung elasticity develops into chronic obstructive pulmonary disease (COPD). In healthy individuals, A1AT is produced by hepatocytes within the liver and is secreted into systemic circulation where the protein functions as a protease inhibitor. 
     The most common pathogenic A1AT variant is a Guanine to Adenine (G→A) mutation in the SERPINA1 gene, which results in a glutamate to lysine substitution at amino acid 342 of the A1AT protein. This substitution causes the protein to misfold and polymerize within hepatocytes, and ultimately, the toxic aggregates can lead to liver injury and cirrhosis. While the liver toxicity might potentially be addressed by a gene knockout (CRISPR/ZFN/TALEN) or gene knockdown (siRNA), neither of these approaches addresses the pulmonary pathology. Although pulmonary pathology may be addressed with protein replacement therapy, this therapy fails to address the liver toxicity. Gene therapy also would be inadequate to address the A1AT genetic defect. Because the livers of patients with A1AD are already under a severe disease burden caused by the endogenous A1 AT aggregation, gene therapy that increases A1AT in the liver would be counterproductive. Therefore, there is a need for a method of treating patients with A1AD that addresses both the lung pathology and the liver toxicity which accompany the disease. 
     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. 
     SUMMARY OF THE DISCLOSURE 
     As described herein, compositions and methods for the precise correction of pathogenic amino acids in a protein associated with a disease or disorder using a programmable nucleobase editor are provided. In a particular aspect, the described compositions and methods are useful for the treatment of alpha-1 antitrypsin deficiency (A1AD). In an embodiment, the described compositions and methods for treating A1AD utilize an adenosine (A) base editor (ABE-(NGC variant)) to precisely correct a deleterious, single nucleotide polymorphism (SNP) in the endogenous SERPINA1 gene. In an embodiment, the compositions and methods correct the deleterious mutation, E342K, which affects the activity and function of the encoded alpha-1 antitrypsin (A1AT) protein. This correction simultaneously eliminates the pathogenic protein burden on the liver and restores functional protein to the lungs. 
     In one aspect, a method of editing a SERPINA1 polynucleotide containing a single nucleotide polymorphism (SNP) associated with Alpha1 Anti-Trypsin Deficiency (A1AD) is provided, in which the method involves contacting the SERPINA1 polynucleotide with a base editor in complex with one or more guide polynucleotides, where the base editor contains a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and where one or more of the guide polynucleotides target the base editor to effect an A⋅T to G⋅C alteration of the SNP in the SERPINA1 gene, which is associated with A1AD. In one embodiment, the method involves contacting a cell, e.g., a eukaryotic cell, a mammalian cell, or human cell. In another embodiment, the cell is in vivo or ex vivo. 
     In another aspect, the invention features a cell produced by introducing into the cell, or a progenitor thereof, a base editor, a polynucleotide encoding the base editor, where the base editor contains a polynucleotide programmable DNA binding domain and an adenosine deaminase domain; and one or more guide polynucleotides that target the base editor to effect an A⋅T to G⋅C alteration of the SNP in a gene, e.g., the SERPINA1 gene, associated with A1AD. In one embodiment, the cell produced is a hepatocyte. In another embodiment, the cell or progenitor thereof is an embryonic stem cell, induced pluripotent stem cell, or a hepatocyte. In another embodiment, the hepatocyte expresses an A1AT polypeptide. In another embodiment, the cell is from a subject having A1AD. In yet another embodiment, the cell is a mammalian cell or a human cell. 
     In another aspect, the invention features a method of treating A1AD in a subject containing administering to subject in need thereof a cell as described in the above delineated aspects and embodiments. In one embodiment, the cell is autologous or is allogeneic or xenogeneic to the subject. 
     In another aspect, the invention features aa isolated cell or population of cells propagated or expanded from the cell of any above-delineated aspect. 
     In another aspect, the invention features a method of treating A1AD in a subject, in which the method comprises administering to a subject in need thereof a base editor, or a polynucleotide encoding the base editor, where the base editor contains a polynucleotide programmable DNA binding domain and an adenosine deaminase domain; and one or more guide polynucleotides that target the base editor to effect an A⋅T to G⋅C alteration of the SNP in a gene, e.g., the SERPINA1 gene, associated with A1AD. In one embodiment, the subject is a mammal or a human. In another embodiment, the method involves delivering the base editor, or polynucleotide encoding the base editor, and the one or more guide polynucleotides to a cell of the subject. In yet another embodiment, the cell is a hepatocyte. In another embodiment, the cell is a progenitor of a hepatocyte. In yet another embodiment, the hepatocyte expresses an A1AT polypeptide containing a mutation. 
     In another aspect, the invention features a method of producing a hepatocyte or progenitor thereof, in which the method involves (a) introducing into an induced pluripotent stem cell or hepatocyte progenitor containing an SNP in a gene, e.g., the SERPINA1 gene, associated with A1AD, a base editor, or a polynucleotide encoding the base editor, where the base editor contains a polynucleotide-programmable nucleotide-binding domain and an adenosine deaminase domain; and one or more guide polynucleotides, where the one or more guide polynucleotides target the base editor to effect an A⋅T to G⋅C alteration of the SNP associated with A1AD; and (b) differentiating the induced pluripotent stem cell or hepatocyte progenitor into hepatocyte. In one embodiment, the method involves differentiating the induced pluripotent stem cell into a hepatocyte or progenitor thereof. In another embodiment, the induced pluripotent stem cell contains an E342K mutation. In another embodiment, the hepatocyte progenitor is obtained from a subject having A1AD. In yet another embodiment, the hepatocyte or hepatocyte progenitor is a mammalian cell or human cell. 
     In another aspect, the base editor (BE) used in the described compositions and methods comprises a polypeptide comprising the amino acid sequence: (i): MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTA HAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGA AGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGSS GGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAV LVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCA GAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFF RMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAI GTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARR RYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHE KYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQ TYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNT EITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQ EEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFY PFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQS FIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDN EENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLIN GIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIE EGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQ SFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK AERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSK LVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDV RKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGR DFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFmqP TVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIK LPKYSLFELENGRKRMLASAkfLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTN LGAPrAFKYFDTTIaRKeYrSTKEVLDATLIHQSITGLYETRIDLSQLGGDEGADKRTADGS EFESPKKKRK (SEQ ID NO: 1); and (ii) an adenosine deaminase domain. 
     In another aspect the invention features a guide RNA (gRNA) containing a nucleic acid sequence from among the following: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 2) 
               
               
                 5′-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAU 
               
               
                   
               
               
                 CAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3′; 
               
               
                   
               
               
                 (SEQ ID NO: 3) 
               
               
                 5′- 
               
               
                 ACCAUCGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGUUA 
               
               
                   
               
               
                 AAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG 
               
               
                   
               
               
                 CUUUU-3′; 
               
               
                   
               
               
                 (SEQ ID NO: 4) 
               
               
                 5′-CCAUCGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGU 
               
               
                   
               
               
                 UAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG 
               
               
                   
               
               
                 UGCUUUU-3′; 
               
               
                   
               
               
                 (SEQ ID NO: 5) 
               
               
                 5′-CAUCGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGUU 
               
               
                   
               
               
                 AAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU 
               
               
                   
               
               
                 GCUUUU-3′; 
               
               
                   
               
               
                 (SEQ ID NO: 6) 
               
               
                 5′-AUCGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGUUA 
               
               
                   
               
               
                 AAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG 
               
               
                   
               
               
                 CUUUU-3′; 
               
               
                   
               
               
                 (SEQ ID NO: 7) 
               
               
                 5′-UCGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGUUAA 
               
               
                   
               
               
                 AAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC 
               
               
                   
               
               
                 UUUU-3′; 
               
               
                 and 
               
               
                   
               
               
                 (SEQ ID NO: 8) 
               
               
                 5′-CGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAA 
               
               
                   
               
               
                 AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU 
               
               
                   
               
               
                 UUU-3′. 
               
            
           
         
       
     
     In another aspect, the invention features a protein nucleic acid complex containing the base editor and a guide RNA of any of the foregoing aspects or embodiments delineated herein. 
     In various embodiments of the above aspects or any other aspect of the invention delineated herein, the A⋅T to G⋅C alteration at the SNP associated with A1AD changes a lysine to a glutamic acid in the A1AT polypeptide. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the SNP associated with A1AD results in expression of an A1AT polypeptide having a lysine at amino acid position 342. In another embodiment, the base editor correction replaces the lysine at position 342 of an A1AT polypeptide associated with A1AD with a glutamic acid. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the polynucleotide programmable DNA binding domain is a modified  Streptococcus pyogenes  Cas9 (SpCas9), or variants thereof. 
     In various embodiments of the above aspects or any other aspect of the invention delineated herein, the polynucleotide programmable DNA binding domain contains a modified SpCas9 having an altered protospacer-adjacent motif (PAM) specificity. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the modified SpCas9 has specificity for the nucleic acid sequence 5′-AGC-3′. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the modified SpCas9 comprises the amino acid substitution D1332A and one or more of D1135M, S1136Q, G1218K, E1219F, D1332A, R1335E, and T1337R, or corresponding amino acid substitutions thereof. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the polynucleotide programmable DNA binding domain contains a variant of SpCas9 having an altered protospacer-adjacent motif (PAM) specificity. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the variant of SpCas9 has specificity for the nucleic acid sequence 5′-NGC-3′. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the modified SpCas9 contains amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R, or corresponding amino acid substitutions thereof. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the polynucleotide programmable DNA binding domain is a nuclease inactive or nickase variant. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the nickase variant contains an amino acid substitution D10A or a corresponding amino acid substitution thereof. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the adenosine deaminase domain is capable of deaminating adenosine in deoxyribonucleic acid (DNA). In various embodiments of the above aspects or any other aspect of the invention delineated herein, the adenosine deaminase is a modified adenosine deaminase that does not occur in nature. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the adenosine deaminase is a TadA deaminase (e.g., TadA*7.10). In various embodiments of the above aspects or any other aspect of the invention delineated herein, the one or more guide RNAs contains a CRISPR RNA (crRNA) and a trans-encoded small RNA (tracrRNA), where the crRNA contains a nucleic acid sequence complementary to a SERPINA1 nucleic acid sequence containing the SNP associated with A1AD. In various embodiments of the above aspects, the base editor is in complex with a single guide RNA (sgRNA) containing a nucleic acid sequence complementary to an SERPINA1 nucleic acid sequence containing the SNP associated with A1AD. 
     In yet another aspect, provided herein is a base editor system for correcting a pathogenic single nucleotide polymorphism (SNP) in a gene, wherein the base editor system comprises (a) a base editor comprising: (i) a polynucleotide-programmable DNA-binding domain, and (ii) a deaminase domain capable of deaminating the pathogenic SNP or its complement nucleobase; and (b) a guide polynucleotide in conjunction with the polynucleotide-programmable DNA-binding domain, wherein the guide polynucleotide targets the base editor to a target polynucleotide sequence at least a portion of which is located in the gene or its reverse complement; wherein deaminating the pathogenic SNP or its complement nucleobase results in a conversion of the pathogenic SNP to its wild-type allele, thereby correcting a pathogenic mutation, such as those listed in Tables 3A and 3B herein. 
     In another aspect, provided herein is a method for correcting a pathogenic single nucleotide polymorphism (SNP) in a gene, in which the method comprises: contacting a target nucleotide sequence, at least a portion of which is located in the gene or its reverse complement, with a base editor comprising: (i) a polynucleotide-programmable DNA-binding domain in conjunction with a guide polynucleotide that targets the base editor to the target polynucleotide sequence, at least a portion of which is located in the gene or its reverse complement, and (ii) a deaminase domain capable of deaminating the pathogenic SNP or its complement nucleobase; and editing the pathogenic SNP by deaminating the pathogenic SNP or its complement nucleobase upon targeting of the base editor to the target nucleotide sequence, wherein deaminating the pathogenic SNP or its complement nucleobase results in a conversion of the pathogenic SNP to its wild-type allele, thereby correcting a pathogenic mutation, such as listed in Table 3A or Table 3B herein. 
     In another aspect, provided herein is a method of treating a genetic disorder in a subject by correcting a pathogenic single nucleotide polymorphism (SNP) in a gene, in which the method comprises administering a base editor, or a polynucleotide encoding the base editor, to a subject in need thereof, wherein the base editor comprises: (i) a polynucleotide-programmable DNA-binding domain, and (ii) a deaminase domain capable of deaminating the pathogenic SNP or its complement nucleobase; and administering a guide polynucleotide to the subject, wherein the guide polynucleotide targets the base editor to a target nucleotide sequence, at least a portion of which is located in the gene or its reverse complement; and editing the pathogenic SNP by deaminating the pathogenic SNP or its complement nucleobase upon targeting of the base editor to the target nucleotide sequence, wherein deaminating the pathogenic SNP or its complement nucleobase results in a conversion of the pathogenic SNP to its wild-type allele, thereby correcting a pathogenic mutation, such as listed in Table 3A or 3B, and treating the genetic disorder. 
     Provided herein is a method of producing a cell, tissue, or organ for treating a genetic disorder in a subject in need thereof by correcting a pathogenic single nucleotide polymorphism (SNP) in a gene of the cell, tissue, or organ, in which the method comprises: contacting the cell, tissue, or organ with a base editor, wherein the base editor comprises: (i) a polynucleotide-programmable DNA-binding domain, and (ii) a deaminase domain capable of deaminating the pathogenic SNP or its complement nucleobase; and contacting the cell, tissue, or organ with a guide polynucleotide, wherein the guide polynucleotide targets the base editor to a target nucleotide sequence at least a portion of which is located in the gene or its reverse complement; and editing the pathogenic SNP by deaminating the pathogenic SNP or its complement nucleobase upon targeting of the base editor to the target nucleotide sequence, wherein deaminating the pathogenic SNP or its complement nucleobase results in a conversion of the pathogenic SNP to its wild-type allele, thereby correcting a pathogenic mutation, such as listed in Table 3A or 3B, and producing the cell, tissue, or organ for treating the genetic disorder. In some embodiments, the method further comprises administering the cell, tissue, or organ to the subject. In some embodiments, the cell, tissue, or organ is autologous to subject. In some embodiments, the cell, tissue, or organ is allogeneic to the subject. In some embodiments, the cell, tissue, or organ is xenogeneic to the subject 
     In some embodiments, the 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*. 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. 
     In some embodiments, the pathogenic SNP is associated with primary hypoxaluria; optionally, the pathogenic SNP is in an AGXT gene; and optionally, the pathogenic mutation comprises G170R. In some embodiments, the pathogenic SNP is associated with autosomal recessive hypercholesterolemia; optionally; optionally, the pathogenic SNP is in an ARH gene; optionally, the pathogenic mutation comprises Q136*. In some embodiments, the pathogenic SNP is associated with metachromatic leukodystrophy; optionally, the pathogenic SNP is in an ARSA gene; optionally, the pathogenic mutation comprises P426L, c. 459+1G&gt;A, or a combination thereof. In some embodiments, the pathogenic SNP is associated with Marteauz-Lamy Syndrome (MSPVI); optionally, the pathogenic SNP is in an ARSB gene; optionally, the pathogenic mutation comprises Y210C. In some embodiments, the pathogenic SNP is associated with Citrullinemia Type I; optionally, the pathogenic SNP is in an ASS gene; optionally, the pathogenic mutation comprises G390R. In some embodiments, the pathogenic SNP is associated with Darier disease; optionally, the pathogenic SNP is in an ATP2A2 gene; optionally, the pathogenic mutation comprises N767S. 
     In some embodiments, the pathogenic SNP is associated with classic homocysteinuria; optionally, the pathogenic SNP is in a CBS gene; optionally, the pathogenic mutation comprises G307S, T191M, or a combination thereof. In some embodiments, the pathogenic SNP is associated with cystic fibrosis; optionally, the pathogenic SNP is in a CFTR gene; optionally, the pathogenic mutation comprises G551D, W1282*, R553*, R117H, or a combination thereof. In some embodiments, the pathogenic SNP is associated with choroideremia; optionally, the pathogenic SNP is in a CHM gene; optionally, the pathogenic mutation comprises R293*, R270*, A117A, or a combination thereof. In some embodiments, the pathogenic SNP is associated with Neuronal ceroid lipofuscinosis (NCL); optionally, the pathogenic SNP is in a CLN2 gene; optionally, the pathogenic mutation comprises R208*. In some embodiments, the pathogenic SNP is associated with autosomal dominant deafness; optionally, the pathogenic SNP is in a COCH gene; optionally, the pathogenic mutation comprises G88E. In some embodiments, the pathogenic SNP is associated with carnitine palmitoyltransferase II deficiency; optionally, the pathogenic SNP is in a CPT2 gene; optionally, the pathogenic mutation comprises S113L. 
     In some embodiments, the pathogenic SNP is associated with cystinosis; optionally, the pathogenic SNP is in a CTNS gene; optionally, the pathogenic mutation comprises W138*. In some embodiments, the pathogenic SNP is associated with autosomal recessive deafness; optionally, the pathogenic SNP is in a CX30 gene; optionally, the pathogenic mutation comprises TSM. In some embodiments, the pathogenic SNP is associated with autosomal recessive deafness; optionally, the pathogenic SNP is in an DFNB59 gene; and optionally, the pathogenic mutation comprises R183W. In some embodiments, the pathogenic SNP is associated with isolated agammaglobulinemia; optionally, the pathogenic SNP is in an E47 gene; and optionally, the pathogenic mutation comprises E555K. In some embodiments, the pathogenic SNP is associated with congenital factor XI deficiency; optionally, the pathogenic SNP is in an F11 gene; and optionally, the pathogenic mutation comprises E117*, F283L, or a combination thereof. In some embodiments, the pathogenic SNP is associated with congenital factor V deficiency; optionally, the pathogenic SNP is in an F5 gene; and optionally, the pathogenic mutation comprises R506Q, R534Q, or a combination thereof. In some embodiments, the pathogenic SNP is associated with congenital factor VII deficiency; optionally, the pathogenic SNP is in an F7 gene; and optionally, the pathogenic mutation comprises A294V, C310F, R304Q, Q100R, or a combination thereof. 
     In some embodiments, the pathogenic SNP is associated with hemophilia A; optionally, the pathogenic SNP is in an F8 gene; and optionally, the pathogenic mutation comprises R2169H, R1985Q, R2178C, R550C, or a combination thereof. In some embodiments, the pathogenic SNP is associated with hemophilia B; optionally, the pathogenic SNP is in an F9 gene; and optionally, the pathogenic mutation comprises T342M, R294Q, R43Q, R191H, G106S, A279T, R75*, R294*, R379Q, or a combination thereof. In some embodiments, the pathogenic SNP is associated with tyrosinemia type 1; optionally, the pathogenic SNP is in a FAH gene; and optionally, the pathogenic mutation comprises P261L. In some embodiments, the pathogenic SNP is associated with autosomal dominant hypophosphatemic rickets; optionally, the pathogenic SNP is in an FGF23 gene; and optionally, the pathogenic mutation comprises R176Q. 
     In some embodiments, the pathogenic SNP is associated with von Gierke disease; optionally, the pathogenic SNP is in a G6PC gene; and optionally, the pathogenic mutation comprises Q347*. In some embodiments, the pathogenic SNP is associated with Mediterranean G6PD deficiency; optionally, the pathogenic SNP is in a G6PD gene; and optionally, the pathogenic mutation comprises S188D. In some embodiments, the pathogenic SNP is associated with Morquio Syndrome (MPSIVA); optionally, the pathogenic SNP is in a GALNS gene; and optionally, the pathogenic mutation comprises R386C. In some embodiments, the pathogenic SNP is associated with classic galactosemia; optionally, the pathogenic SNP is in an GALT gene; and optionally, the pathogenic mutation comprises Q188R. 
     In some embodiments, the pathogenic SNP is associated with Gaucher disease; optionally, the pathogenic SNP is in an GBA gene; and optionally, the pathogenic mutation comprises N370S, L444P, or a combination thereof. In some embodiments, the pathogenic SNP is associated with glutaryl-CoA dehydrogenase deficiency; optionally, the pathogenic SNP is in a GCDH gene; and optionally, the pathogenic mutation comprises R138G, M263V, R402W, or a combination thereof. In some embodiments, the pathogenic SNP is associated with glycine encephalopathy; optionally, the pathogenic SNP is in a GLDC gene; and optionally, the pathogenic mutation comprises A389V, G771R, T269M, or a combination thereof. In some embodiments, the pathogenic SNP is associated with cone-rod dystrophy; optionally, the pathogenic SNP is in a GUCY2D gene; and optionally, the pathogenic mutation comprises R838C. In some embodiments, the pathogenic SNP is associated with Sly Syndrome (MPSVII); optionally, the pathogenic SNP is in a GUSB gene; and optionally, the pathogenic mutation comprises L175F. 
     In some embodiments, the pathogenic SNP is associated with sickle cell disease; optionally, the pathogenic SNP is in a HBB gene; and optionally, the pathogenic mutation comprises E26K; E7K; c.-138C&gt;T; IVS2, 654 C&gt;T; or a combination thereof. In some embodiments, the pathogenic SNP is associated with intermittent porphyria; optionally, the pathogenic SNP is in a HMBS gene; and optionally, the pathogenic mutation comprises R173W. In some embodiments, the pathogenic SNP is associated with Lesch-Nyhan syndrome; optionally, the pathogenic SNP is in a HPRT1 gene; and optionally, the pathogenic mutation comprises R51*, R170*, or a combination thereof. In some embodiments, the pathogenic SNP is associated with Hunter syndrome; optionally, the pathogenic SNP is in an IDS gene; and optionally, the pathogenic mutation comprises R88C, G374G, or a combination thereof. In some embodiments, the pathogenic SNP is associated with Hurler syndrome (MPS1); optionally, the pathogenic SNP is in an IDUA gene; and optionally, the pathogenic mutation comprises Q70*. 
     In some embodiments, the pathogenic SNP is associated with retinitis pigmentosa; optionally, the pathogenic SNP is in an IMPDH1 gene; and optionally, the pathogenic mutation comprises D226N. In some embodiments, the pathogenic SNP is associated with Andersen-Tawil syndrome; optionally, the pathogenic SNP is in a KCNJ2 gene; and optionally, the pathogenic mutation comprises R218W. In some embodiments, the pathogenic SNP is associated with Meesmann epithelial corneal dystrophy; optionally, the pathogenic SNP is in a KRT12 gene; and optionally, the pathogenic mutation comprises L132P. In some embodiments, the pathogenic SNP is associated with Parkinson&#39;s disease; optionally, the pathogenic SNP is in a LRRK2 gene; and optionally, the pathogenic mutation comprises G2109S. In some embodiments, the pathogenic SNP is associated with Rett syndrome; optionally, the pathogenic SNP is in a MECP2 gene; and optionally, the pathogenic mutation comprises R106W, R133C, R306C, R168*, R255*, or a combination thereof. In some embodiments, the pathogenic SNP is associated with Sanfilippo syndrome B (MPSIIIB); optionally, the pathogenic SNP is in a NAGLU gene; and optionally, the pathogenic mutation comprises R297*, Y140C, or a combination thereof. 
     In some embodiments, the pathogenic SNP is associated with CADASIL syndrome; optionally, the pathogenic SNP is in a NOTCH3 gene; and optionally, the pathogenic mutation comprises R90C, R141C, or a combination thereof. In some embodiments, the pathogenic SNP is associated with blue-cone monochromatism; optionally, the pathogenic SNP is in an OPN1LW gene; and optionally, the pathogenic mutation comprises C203R. In some embodiments, the pathogenic SNP is associated with phenylketonuria; optionally, the pathogenic SNP is in a PAH gene; and optionally, the pathogenic mutation comprises R408W, I65T, R261Q, IVS10-11G&gt;A, or a combination thereof. In some embodiments, the pathogenic SNP is associated with Usher syndrome type 1F; optionally, the pathogenic SNP is in a PCDH15 gene; and optionally, the pathogenic mutation comprises R245*. In some embodiments, the pathogenic SNP is associated with retinitis pigmentosa; optionally, the pathogenic SNP is in a PDE6A gene; and optionally, the pathogenic mutation comprises V685M, D670G, or a combination thereof. 
     In some embodiments, the pathogenic SNP is associated with Pendred syndrome; optionally, the pathogenic SNP is in a PDS gene; and optionally, the pathogenic mutation comprises L236P; c.1001+1G&gt;A; IVS8, +1 G&gt;A, or a combination thereof. In some embodiments, the pathogenic SNP is associated with variegate porphyria; optionally, the pathogenic SNP is in a PPDX gene; and optionally, the pathogenic mutation comprises R59W. In some embodiments, the pathogenic SNP is associated with neuronal ceroid lipofuscinosis 1; optionally, the pathogenic SNP is in a PPT1 gene; and optionally, the pathogenic mutation comprises R151*. In some embodiments, the pathogenic SNP is associated with Creutzfeldt-Jakob disease (CJD); optionally, the pathogenic SNP is in a PRNP gene; and optionally, the pathogenic mutation comprises M129V, P102L, D178N, or a combination thereof. In some embodiments, the pathogenic SNP is associated with retinitis pigmentosa; optionally, the pathogenic SNP is in a PRPF3 gene; and optionally, the pathogenic mutation comprises T494M. In some embodiments, the pathogenic SNP is associated with retinitis pigmentosa; optionally, the pathogenic SNP is in a PRPF8 gene; and optionally, the pathogenic mutation comprises H2309R. 
     In some embodiments, the pathogenic SNP is associated with hereditary chronic pancreatitis; optionally, the pathogenic SNP is in a PRSS1 gene; and optionally, the pathogenic mutation comprises R122H. In some embodiments, the pathogenic SNP is associated with retinitis pigmentosa; optionally, the pathogenic SNP is in a RHO gene; and optionally, the pathogenic mutation comprises P347L, D190N, or a combination thereof. In some embodiments, the pathogenic SNP is associated with retinitis pigmentosa; optionally, the pathogenic SNP is in a RP1 gene; and optionally, the pathogenic mutation comprises R667*. In some embodiments, the pathogenic SNP is associated with Leber congenital amaurosis 2; optionally, the pathogenic SNP is in a RPE65 gene; and optionally, the pathogenic mutation comprises R44*; IVS1, G-A, +5; or a combination thereof. In some embodiments, the pathogenic SNP is associated with Blackfan-Diamond anemia; optionally, the pathogenic SNP is in a RPS19 gene; and optionally, the pathogenic mutation comprises R62Q. 
     In some embodiments, the pathogenic SNP is associated with X-linked retinoschisis; optionally, the pathogenic SNP is in a retinoschisin (RS1) gene; and optionally, the pathogenic mutation comprises R102W, R141C, or a combination thereof. In some embodiments, the pathogenic SNP is associated with A1AD; optionally, the pathogenic SNP is in a SERPINA1 gene; and optionally, the pathogenic mutation comprises E342K, R48C (R79C), or a combination thereof. In some embodiments, the pathogenic SNP is associated with Sanfilippo syndrome A (MPSIIIA); optionally, the pathogenic SNP is in a SGSH gene; and optionally, the pathogenic mutation comprises R74C. In some embodiments, the pathogenic SNP is associated with Neimann-Pick disease type A; optionally, the pathogenic SNP is in a SMPD1 gene; and optionally, the pathogenic mutation comprises L302P. 
     In some embodiments, the pathogenic SNP is associated with autosomal dominant Parkinson&#39;s disease; optionally, the pathogenic SNP is in a SNCA gene; and optionally, the pathogenic mutation comprises A53T. In some embodiments, the pathogenic SNP is associated with familial amyotrophic lateral sclerosis (ALS); optionally, the pathogenic SNP is in a superoxide dismutase 1 (SOD1) gene; and optionally, the pathogenic mutation comprises A4V, H46R, G37R, or a combination thereof. In some embodiments, the pathogenic SNP is associated with autosomal dominant deafness; optionally, the pathogenic SNP is in a TECTA gene; and optionally, the pathogenic mutation comprises Y1870C. In some embodiments, the pathogenic SNP is associated with autosomal recessive deafness; optionally, the pathogenic SNP is in a TMC1 gene; and optionally, the pathogenic mutation comprises Y182C. In some embodiments, the pathogenic SNP is associated with ATTR amyloidosis; optionally, the pathogenic SNP is in a TTR gene; and optionally, the pathogenic mutation comprises V50M/V30M. In some embodiments, the pathogenic SNP is associated with retinitis pigmentosa/Usher syndrome type 1C; optionally, the pathogenic SNP is in an USH1C gene; and optionally, the pathogenic mutation comprises V72V. 
     In some embodiments, the pathogenic SNP is associated with retinitis pigmentosa; optionally, the pathogenic SNP is in an USH2a gene; and optionally, the pathogenic mutation comprises C759F. In some embodiments, the pathogenic SNP is associated with myotubular myopathy; optionally, the pathogenic SNP is in a MTM1 gene; and optionally, the pathogenic mutation comprises c.1261-10A&gt;G. 
     In some embodiments, any of the base editor system or the methods provided herein can further comprise a second guide polynucleotide for editing of an additional nucleobase. In some embodiments, the additional nucleobase is not located in the gene. In some embodiments, the additional nucleobase is located in the gene. In some embodiments, additional nucleobase is located in a protein coding region. In some embodiments, the additional nucleobase is located in a protein non-coding region. In some embodiments, the protein non-coding region is a gene regulatory element. In some embodiments, the deaminase domain is a cytidine deaminase domain or an adenosine deaminase domain. In some embodiments, the deaminase domain is a cytidine deaminase domain. In some embodiments, the deaminase domain is an adenosine deaminase domain. In some embodiments, the adenosine deaminase domain is capable of deaminating adenosine in deoxyribonucleic acid (DNA). In some embodiments, the guide polynucleotide comprises ribonucleic acid (RNA), or deoxyribonucleic acid (DNA). In some embodiments, the guide polynucleotide comprises a CRISPR RNA (crRNA) sequence, a trans-activating CRISPR RNA (tracrRNA) sequence, or a combination thereof. 
     In some embodiments, any of the base editor system or the methods provided herein can further comprise a second guide polynucleotide. In some embodiments, the second guide polynucleotide comprises ribonucleic acid (RNA), or deoxyribonucleic acid (DNA). In some embodiments, the second guide polynucleotide comprises a CRISPR RNA (crRNA) sequence, a trans-activating CRISPR RNA (tracrRNA) sequence, or a combination thereof. In some embodiments, the second guide polynucleotide targets the base editor to a second target nucleotide sequence. 
     In some embodiments, in any of the base editor system or the methods provided herein, the polynucleotide-programmable DNA-binding domain comprises a Cas9 domain, a Cpf1 domain, a CasX domain, a CasY domain, a Cas12b/C2c1 domain or a Cas12c/C2c3 domain. In some embodiments, the polynucleotide-programmable DNA-binding domain is nuclease dead. In some embodiments, the polynucleotide-programmable DNA-binding domain is a nickase. In some embodiments, the polynucleotide-programmable DNA-binding domain comprises a Cas9 domain. In some embodiments, the Cas9 domain comprises a nuclease dead Cas9 (dCas9), a Cas9 nickase (nCas9), or a nuclease active Cas9. In some embodiments, the Cas9 domain comprises a Cas9 nickase. In some embodiments, the polynucleotide-programmable DNA-binding domain is an engineered or a modified polynucleotide-programmable DNA-binding domain. 
     In some embodiments, any of the base editor system or the methods provided herein can further comprise a second base editor. In some embodiments, the second base editor comprises a different deaminase domain than the base editor. 
     In some embodiments, in any of the methods provided herein, the editing results in less than 20% indel formation. In some embodiments, the editing results in less than 15% indel formation. In some embodiments, the editing results in less than 10% indel formation. In some embodiments, the editing results in less than 5% indel formation. In some embodiments, the editing results in less than 4% indel formation. In some embodiments, the editing results in less than 3% indel formation. In some embodiments, the editing results in less than 2% indel formation. In some embodiments, the editing results in less than 1% indel formation. In some embodiments, the editing results in less than 0.5% indel formation. In some embodiments, the editing results in less than 0.1% indel formation. In some embodiments, the editing does not result in translocations. 
     In one aspect, the invention provides a method of editing a G6PC polynucleotide comprising a single nucleotide polymorphism (SNP) associated with glycogen storage disorder Type 1a (GSD1a), the method comprising contacting the G6PC polynucleotide with a base editor in complex with one or more guide polynucleotides, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and wherein one or more of the guide polynucleotides target the base editor to effect an A⋅T to G⋅C alteration of the SNP associated with GSD1a. In one embodiment, the A⋅T to G⋅C alteration at the SNP associated with glycogen storage disorder Type 1a (GSD1a) changes a glutamine (Q) to a non-glutamine (X) amino acid. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the A⋅T to G⋅C alteration at the SNP associated with glycogen storage disorder Type 1a (GSD1a) changes an arginine (R) to a non-arginine (X) in the G6PC polypeptide. 
     In various embodiments of the above aspects or any other aspect of the invention delineated herein, the SNP associated with GSD1a results in expression of an G6PC polypeptide having a non-glutamine (X) amino acid at position 347 or a non-arginine (X) amino acid at position 83. In one embodiment, the base editor correction replaces the glutamine at position 347 with a non-glutamine amino acid (X). In another embodiment, the base editor correction replaces the arginine at position 83 with a non-arginine amino acid (X). 
     In various embodiments of the above aspects or any other aspect of the invention delineated herein, the polynucleotide programmable DNA binding domain is a modified  Streptococcus pyogenes  Cas9 (SpCas9), or variants thereof. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the polynucleotide programmable DNA binding domain comprises a modified SpCas9 having an altered protospacer-adjacent motif (PAM) specificity. In one embodiment, the modified SpCas9 has specificity for the nucleic acid sequences 5′-NGA-3′ or 5′-NGG-3′. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the adenosine deaminase is ABE7.10. 
     In one aspect, a cell is produced by introducing into the cell, or a progenitor thereof: a base editor, a polynucleotide encoding the base editor, to the cell, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain; and one or more guide polynucleotides that target the base editor to effect an AT to GC alteration of the SNP associated with glycogen storage disorder Type 1a (GSD1a). In various embodiments of the above aspects or any other aspect of the invention delineated herein, the cell is a hepatocyte, a hepatocyte precursor, or an iPSc-derived hepatocyte. 
     In various embodiments of the above aspects or any other aspect of the invention delineated herein, the cell is from a subject having GSD1a. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the cell harbors a Q347X mutation. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the A⋅T to G⋅C alteration at the SNP associated with GSD1a changes a glutamine to a non-glutamine (X) amino acid. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the A⋅T to G⋅C alteration at the SNP associated with GSD1a changes an arginine to a non-arginine (X) amino acid in the G6PC polypeptide. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the SNP associated with GSD1a results in expression of an G6PC polypeptide having a non-glutamine (X) amino acid at position 347 or a non-arginine (X) amino acid at position 83. 
     In one aspect, the invention provides a method of treating glycogen storage disorder Type 1a (GSD1a) or von Gierke Disease in a subject in need thereof, the method comprising administering to the subject the cell of various embodiments of the above aspects or any other aspect of the invention delineated herein. 
     In another aspect, the invention provides a method of producing a hepatocyte, or progenitor thereof, the method comprising: (a) introducing into an induced pluripotent stem cell or hepatocyte progenitor comprising an SNP associated with GSD1a, a base editor, or a polynucleotide encoding the base editor, wherein the base editor comprises a polynucleotide-programmable nucleotide-binding domain and an adenosine deaminase domain; and one or more guide polynucleotides, wherein the one or more guide polynucleotides target the base editor to effect an A⋅T to G⋅C alteration of the SNP associated with GSD1a; and (b) differentiating the induced pluripotent stem cell or hepatocyte progenitor into hepatocyte. In a further aspect, the method includes differentiating the induced pluripotent stem cell into a hepatocyte or progenitor thereof. In various embodiments, the induced pluripotent stem cell of step (a) comprises a Q347X mutation. 
     In various embodiments of the above aspects or any other aspect of the invention delineated herein, the hepatocyte progenitor is obtained from a subject having GSD1a. In various embodiments, the hepatocyte or hepatocyte progenitor is a mammalian cell or human cell. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the A⋅T to G⋅C alteration at the SNP associated with GSD1a changes a glutamine to a non-glutamine (X) amino acid or changes an arginine to a non-arginine (X) amino acid in the G6PC polypeptide. In various embodiments, the SNP associated with GSD1a results in expression of an G6PC polypeptide having a non-glutamine (X) amino acid at position 347. In various embodiments, the SNP associated with GSD1a results in expression of an G6PC polypeptide having a non-arginine (X) amino acid at position 83. In various embodiments, the SNP associated with GSD1a substitutes a glutamine with a non-glutamine (X) amino acid. In various embodiments, the SNP associated with GSD1a substitutes an arginine with a non-arginine (X) amino acid. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the cell is selected for the A⋅T to G⋅C alteration of the SNP associated with GSD1a. 
     In one aspect, the invention provides a method of editing a IDUA polynucleotide comprising a single nucleotide polymorphism (SNP) associated with mucopolysaccharidosis type 1 (MPS1), the method comprising contacting the IDUA polynucleotide with a base editor in complex with one or more guide polynucleotides, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and wherein one or more of the guide polynucleotides target the base editor to effect an A⋅T to G⋅C alteration of the SNP associated with MPS1. In one embodiment, the polynucleotide programmable DNA binding domain is a modified  Streptococcus pyogenes  Cas9 (SpCas9), or variants thereof. In a further embodiment, the polynucleotide programmable DNA binding domain comprises a modified SpCas9 having an altered protospacer-adjacent motif (PAM) specificity. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the modified SpCas9 has specificity for the nucleic acid sequence 5′-NGG-3′. In various embodiments of the above aspects or any other aspect of the invention delineated herein, the adenosine deaminase is ABE7.10. In various embodiments, the guide polynucleotide comprises the human nucleic acid sequence ACTCTaGGCAGAGGTCTCAA AGG (SEQ ID NO: 9). In various embodiments, the guide polynucleotide comprises the mouse nucleic acid sequence GCTCTaGGCCGAAGTGTCGC AGG (SEQ ID NO: 10). 
     In one aspect, a cell is produced by introducing into the cell, or a progenitor thereof: a base editor, a polynucleotide encoding the base editor, to the cell, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain; and one or more guide polynucleotides that target the base editor to effect an AT to GC alteration of the SNP associated with mucopolysaccharidosis type 1 (MPS1). In various embodiments, the cell is a stem cell, a stem cell precursor, or an induced pluripotent stem cell (iPSC). In various embodiments of the above aspects or any other aspect of the invention delineated herein, the cell is from a subject having MPS1. 
     In another aspect, the invention provides a method of treating MPS1 in a subject in need thereof, the method comprising administering to the subject a cell of the above aspects or any other aspect of the invention delineated herein. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       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 the accompanying drawings of which: 
         FIG.  1    is schematic diagram comparing a healthy subject and a patient with antitrypsin deficiency (A1AD). In the healthy subject, alpha-1 antitrypsin (A1AT) protects lung from protease damage, and the liver releases alpha-1 antitrypsin into the blood. In a patient having A1AD, the deficiency of normally functioning A1AT protein leads to lung tissue damage. In addition, an accumulation of abnormal A1AT in hepatocytes leads to cirrhosis of the liver. 
         FIG.  2    shows typical ranges of serum alpha-1 antitrypsin (A1AT) levels for different genotypes (normal (MM); heterozygous carriers of alpha-1 antitrypsin deficiency (MZ, SZ); and homozygous deficiency (SS, ZZ)). Serum alpha-1 antitrypsin (AAT) concentration is expressed in μM in the left “y” axis, which is common in the literature. The right “y” axis shows an approximate conversion of serum AAT concentration into mg/dL units, as commonly reported by clinical laboratories and by different measurement technologies (nephelometry or radial immunodiffusion) 
         FIGS.  3 A- 3 C  present a base editing target sequence, and graphs related to precise corrections of pathogenic mutations in the SERPINA1 gene which encodes the A1AT protein.  FIG.  3 A  shows a precise correction base editing strategy for a mutation in the SERPINA1 gene which encodes A1AT. A7 (“Target A”) can be edited to restore wild-type (WT) phenotype. In some cases, “A” nucleobases A5/A7 can be edited to introduce amino acid D341G into the A1AT protein. In some cases, A7/A8 can be edited to introduce amino acid E342G into the A1AT protein.  FIG.  3 A  discloses SEQ ID NOS 309-310, respectively, in order of appearance.  FIG.  3 B  provides a nucleic acid sequence showing the position of target A nucleobases within the SERPINA1 gene and the encoded amino acids, as well as a graph showing levels of A1AT (ng/ml) secreted from HEK293T cells that express wild-type (WT), or A1AT variants containing E342K, D341G, or E342G.  FIG.  3 B  discloses SEQ ID NOS 309-310, respectively, in order of appearance.  FIG.  3 C  is a graph showing elastase activity in wild-type (WT) A1AT protein versus that in A1AT variants containing E342K or D341G. 
         FIG.  4    is a schematic diagram showing a strategy to evolve a DNA deoxyadenosine deaminase starting from TadA tRNA deaminase. Shown are a library of  E. coli  harboring a plasmid library of mutant ecTadA (TadA*) genes fused to dCas9 and a selection plasmid requiring targeted A⋅T to G⋅C mutations to repair antibiotic resistance genes. Mutations from surviving TadA* variants were imported into an ABE architecture for base editing in human cells. 
         FIG.  5    provides a nucleic acid sequence showing the position of target “A” nucleobases within the SERPINA1 gene and the encoded amino acids, as well as a graph showing percent editing at positions A5 or A7 in the SERPINA1 gene as a function of guide RNA length.  FIG.  5    discloses SEQ ID NOS 309-310, respectively, in order of appearance. 
         FIGS.  6 A and  6 B  depict a library of SpCas9 mutants that were generated to enrich for mutations within the PAM-interacting (PI) domain of Cas9. This library can be screened for SpCas9s having altered PAM specificities. 
         FIG.  7    is a schematic diagram showing a strategy for the correction of the Q347X mutation using a base editor to convert A&gt;G at the targeted site (highlighted) using NGG and NGA PAM recognition sequences. A precise correction would yield the coversion TAG&gt;CAG (stop codon&gt;Glutamine).  FIG.  7    discloses SEQ ID NOS 311-312, respectively, in order of appearance. 
         FIGS.  8 A and  8 B  provide a transfection schedule based on the maturation cycle for GSD1a iPSc-derived hepatocytes.  FIG.  8 A  provides a timeline of the transfection schedule showing representative time points for plating, transfection, and cell harvest.  FIG.  8 B  shows images of maturing GSD1a iPSc-derived hepatocytes on Day 5 and Day 7. 
         FIGS.  9 A and  9 B  provide data showing base editing precise correction of G6PC Q347X for GSD1a.  FIG.  9 A  provides nucleic acid sequences showing the positions of on target and bystander “A” nucleobases within the G6PC gene and corresponding NGG and NGA PAM sequences, as well as a graph showing the percentage of base editing efficiency of G6PC Q347X in HEK293T cells for ABE-On target, ABE-Bystander, Indels, and Nuclease-Indels using either NGA PAM or NGG PAM.  FIG.  9 A  discloses SEQ ID NOS 313-314, respectively, in order of appearance.  FIG.  9 B  provides a graph showing the base editing efficiency in G6PC Q347X in patient iPSc-derived hepatocytes for ABE-On target, ABE-Bystander, Indels, and Nuclease-Indels using either NGA PAM or NGG PAM. The solid line denotes mean of experiments. 
         FIG.  10    provides a nucleic acid sequence showing the positions of on target and bystander “A” nucleobases within the G6PC gene and corresponding GGA PAM sequence, as well as a graph showing the percent of A&gt;G base editing of G6PC Q347X in patient iPSc-derived hepatocyes for ABE-On target, ABE-Bystander, and Indel using mRNA variants.  FIG.  10    discloses SEQ ID NOS 314-315, respectively, in order of appearance. 
         FIG.  11    provides a graph showing the percent of base editing efficiency in the mouse and human IDUA gene using an ABE7.10 base editor. 
     
    
    
     DETAILED DESCRIPTION OF THE DISCLOSURE 
     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. 
     All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. 
     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. 
     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 as 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 referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. 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, preferably within 5-fold, and more preferably 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. 
     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 “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. 
     By “agent” is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof. 
     By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. 
     By “alteration” is meant a change (increase or decrease) in the 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 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels. 
     By “analog” is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog&#39;s function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog&#39;s protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid. 
     “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. Alternatively, or concurrently, administration can be by an oral route. 
     By “alpha-1 antitrypsin (A1AT) protein” is meant a polypeptide or fragment thereof having at least about 95% amino acid sequence identity to the amino acid sequence of UniProt Accession No. P01009. In particular embodiments, an A1AT protein comprises one or more alterations relative to the following reference sequence. In one particular embodiment, an A1AT protein associated with A1AD comprises an E342K mutation. An exemplary A1AT amino acid sequence (&gt;sp1P010091A1AT_HUMAN Alpha-1-antitrypsin OS= Homo sapiens  OX=9606 GN=SERPINA1 PE=1 SV=3) is provided below: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 11) 
               
               
                 MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNKI 
               
               
                   
               
               
                 TPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEI 
               
               
                   
               
               
                 LEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKL 
               
               
                   
               
               
                 VDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKEL 
               
               
                   
               
               
                 DRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGM 
               
               
                   
               
               
                 FNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFL 
               
               
                   
               
               
                 ENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAP 
               
               
                   
               
               
                 LKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIE 
               
               
                   
               
               
                 QNTKSPLFMGKVVNPTQK. 
               
            
           
         
       
     
     By “base editor (BE),” or “nucleobase editor (NBE)” is meant an agent that binds a polynucleotide and has nucleobase modifying activity. In various embodiments, the base editor comprises a nucleobase modifying polypeptide (e.g., a deaminase) and a polynucleotide 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 a base within a DNA molecule. In some embodiments, the base editor is capable of deaminating a cytosine (C) or an adenosine (A) within DNA. In some embodiments, the base editor is a cytidine base editor (CBE). 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 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 (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); 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, the cytidine base editor CBE 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: 12) 
                   
               
            
           
           
               
               
               
            
               
                 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 
               
               
                   
               
               
                 7440 
                 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 
               
            
           
         
       
     
     In some embodiments, the BE4 nucleic acid sequence is selected from one of the following: 
     
       
         
           
               
               
            
               
                 Original BE4 
                   
               
               
                 (SEQ ID NO: 13) 
                   
               
               
                 ATGagctcagagactggcccagtggctgtggaccccacattgagacggcggatcgagccccatgagtttgaggtattcttcgatccgaga 
                   
               
               
                   
               
               
                 gagctccgcaaggagacctgcctgctttacgaaattaattgggggggccggcactccatttggcgacatacatcacagaacactaacaag 
               
               
                   
               
               
                 cacgtcgaagtcaacttcatcgagaagttcacgacagaaagatatttctgtccgaacacaaggtgcagcattacctggtttctcagctgg 
               
               
                   
               
               
                 agcccatgcggcgaatgtagtagggccatcactgaattcctgtcaaggtatccccacgtcactctgtttatttacatcgcaaggctgtac 
               
               
                   
               
               
                 caccacgctgacccccgcaatcgacaaggcctgcgggatttgatctcttcaggtgtgactatccaaattatgactgagcaggagtcagga 
               
               
                   
               
               
                 tactgctggagaaactttgtgaattatagcccgagtaatgaagcccactggcctaggtatccccatctgtgggtacgactgtacgttctt 
               
               
                   
               
               
                 gaactgtactgcatcatactgggcctgcctccttgtctcaacattctgagaaggaagcagccacagctgacattctttaccatcgctctt 
               
               
                   
               
               
                 cagtcttgtcattaccagcgactgcccccacacattctctgggccaccgggttgaaatctggtggttcttctggtggttctagcggcagc 
               
               
                   
               
               
                 gagactcccgggacctcagagtccgccacacccgaaagttctggtggttcttctggtggttctgataaaaagtattctattggtttagcc 
               
               
                   
               
               
                 atcggcactaattccgttggatgggctgtcataaccgatgaatacaaagtaccttcaaagaaatttaaggtgttggggaacacagaccgt 
               
               
                   
               
               
                 cattcgattaaaaagaatcttatcggtgccctcctattcgatagtggcgaaacggcagaggcgactcgcctgaaacgaaccgctcggaga 
               
               
                   
               
               
                 aggtatacacgtcgcaagaaccgaatatgttacttacaagaaatttttagcaatgagatggccaaagttgacgattctttctttcaccgt 
               
               
                   
               
               
                 ttggaagagtccttccttgtcgaagaggacaagaaacatgaacggcaccccatctttggaaacatagtagatgaggtggcatatcatgaa 
               
               
                   
               
               
                 aagtacccaacgatttatcacctcagaaaaaagctagttgactcaactgataaagcggacctgaggttaatctacttggctcttgcccat 
               
               
                   
               
               
                 atgataaagttccgtgggcactttctcattgagggtgatctaaatccggacaactcggatgtcgacaaactgttcatccagttagtacaa 
               
               
                   
               
               
                 acctataatcagttgtttgaagagaaccctataaatgcaagtggcgtggatgcgaaggctattcttagcgcccgcctctctaaatcccga 
               
               
                   
               
               
                 cggctagaaaacctgatcgcacaattacccggagagaagaaaaatgggttgttcggtaaccttatagcgctctcactaggcctgacacca 
               
               
                   
               
               
                 aattttaagtcgaacttcgacttagctgaagatgccaaattgcagcttagtaaggacacgtacgatgacgatctcgacaatctactggca 
               
               
                   
               
               
                 caaattggagatcagtatgcggacttatttttggctgccaaaaaccttagcgatgcaatcctcctatctgacatactgagagttaatact 
               
               
                   
               
               
                 gagattaccaaggcgccgttatccgcttcaatgatcaaaaggtacgatgaacatcaccaagacttgacacttctcaaggccctagtccgt 
               
               
                   
               
               
                 cagcaactgcctgagaaatataaggaaatattctttgatcagtcgaaaaacgggtacgcaggttatattgacggcggagcgagtcaagag 
               
               
                   
               
               
                 gaattctacaagtttatcaaacccatattagagaagatggatgggacggaagagttgcttgtaaaactcaatcgcgaagatctactgcga 
               
               
                   
               
               
                 aagcagcggactttcgacaacggtagcattccacatcaaatccacttaggcgaattgcatgctatacttagaaggcaggaggatttttat 
               
               
                   
               
               
                 ccgttcctcaaagacaatcgtgaaaagattgagaaaatcctaacctttcgcataccttactatgtgggacccctggcccgagggaactct 
               
               
                   
               
               
                 cggttcgcatggatgacaagaaagtccgaagaaacgattactccatggaattttgaggaagttgtcgataaaggtgcgtcagctcaatcg 
               
               
                   
               
               
                 ttcatcgagaggatgaccaactttgacaagaatttaccgaacgaaaaagtattgcctaagcacagtttactttacgagtatttcacagtg 
               
               
                   
               
               
                 tacaatgaactcacgaaagttaagtatgtcactgagggcatgcgtaaacccgcctttctaagcggagaacagaagaaagcaatagtagat 
               
               
                   
               
               
                 ctgttattcaagaccaaccgcaaagtgacagttaagcaattgaaagaggactactttaagaaaattgaatgcttcgattctgtcgagatc 
               
               
                   
               
               
                 tccggggtagaagatcgatttaatgcgtcacttggtacgtatcatgacctcctaaagataattaaagataaggacttcctggataacgaa 
               
               
                   
               
               
                 gagaatgaagatatcttagaagatatagtgttgactcttaccctctttgaagatcgggaaatgattgaggaaagactaaaaacatacgct 
               
               
                   
               
               
                 cacctgttcgacgataaggttatgaaacagttaaagaggcgtcgctatacgggctggggacgattgtcgcggaaacttatcaacgggata 
               
               
                   
               
               
                 agagacaagcaaagtggtaaaactattctcgattttctaaagagcgacggcttcgccaataggaactttatgcagctgatccatgatgac 
               
               
                   
               
               
                 tctttaaccttcaaagaggatatacaaaaggcacaggtttccggacaaggggactcattgcacgaacatattgcgaatcttgctggttcg 
               
               
                   
               
               
                 ccagccatcaaaaagggcatactccagacagtcaaagtagtggatgagctagttaaggtcatgggacgtcacaaaccggaaaacattgta 
               
               
                   
               
               
                 atcgagatggcacgcgaaaatcaaacgactcagaaggggcaaaaaaacagtcgagagcggatgaagagaatagaagagggtattaaagaa 
               
               
                   
               
               
                 ctgggcagccagatcttaaaggagcatcctgtggaaaatacccaattgcagaacgagaaactttacctctattacctacaaaatggaagg 
               
               
                   
               
               
                 gacatgtatgttgatcaggaactggacataaaccgtttatctgattacgacgtcgatcacattgtaccccaatcctttttgaaggacgat 
               
               
                   
               
               
                 tcaatcgacaataaagtgcttacacgctcggataagaaccgagggaaaagtgacaatgttccaagcgaggaagtcgtaaagaaaatgaag 
               
               
                   
               
               
                 aactattggcggcagctcctaaatgcgaaactgataacgcaaagaaagttcgataacttaactaaagctgagaggggtggcttgtctgaa 
               
               
                   
               
               
                 cttgacaaggccggatttattaaacgtcagctcgtggaaacccgccaaatcacaaagcatgttgcacagatactagattcccgaatgaat 
               
               
                   
               
               
                 acgaaatacgacgagaacgataagctgattcgggaagtcaaagtaatcactttaaagtcaaaattggtgtcggacttcagaaaggatttt 
               
               
                   
               
               
                 caattctataaagttagggagataaataactaccaccatgcgcacgacgcttatcttaatgccgtcgtagggaccgcactcattaagaaa 
               
               
                   
               
               
                 tacccgaagctagaaagtgagtttgtgtatggtgattacaaagtttatgacgtccgtaagatgatcgcgaaaagcgaacaggagataggc 
               
               
                   
               
               
                 aaggctacagccaaatacttcttttattctaacattatgaatttctttaagacggaaatcactctggcaaacggagagatacgcaaacga 
               
               
                   
               
               
                 cctttaattgaaaccaatggggagacaggtgaaatcgtatgggataagggccgggacttcgcgacggtgagaaaagttttgtccatgccc 
               
               
                   
               
               
                 caagtcaacatagtaaagaaaactgaggtgcagaccggagggttttcaaaggaatcgattcttccaaaaaggaatagtgataagctcatc 
               
               
                   
               
               
                 gctcgtaaaaaggactgggacccgaaaaagtacggtggcttcgatagccctacagttgcctattctgtcctagtagtggcaaaagttgag 
               
               
                   
               
               
                 aagggaaaatccaagaaactgaagtcagtcaaagaattattggggataacgattatggagcgctcgtcttttgaaaagaaccccatcgac 
               
               
                   
               
               
                 ttccttgaggcgaaaggttacaaggaagtaaaaaaggatctcataattaaactaccaaagtatagtctgtttgagttagaaaatggccga 
               
               
                   
               
               
                 aaacggatgttggctagcgccggagagcttcaaaaggggaacgaactcgcactaccgtctaaatacgtgaatttcctgtatttagcgtcc 
               
               
                   
               
               
                 cattacgagaagttgaaaggttcacctgaagataacgaacagaagcaactttttgttgagcagcacaaacattatctcgacgaaatcata 
               
               
                   
               
               
                 gagcaaatttcggaattcagtaagagagtcatcctagctgatgccaatctggacaaagtattaagcgcatacaacaagcacagggataaa 
               
               
                   
               
               
                 cccatacgtgagcaggcggaaaatattatccatttgtttactcttaccaacctcggcgctccagccgcattcaagtattttgacacaacg 
               
               
                   
               
               
                 atagatcgcaaacgatacacttctaccaaggaggtgctagacgcgacactgattcaccaatccatcacgggattatatgaaactcggata 
               
               
                   
               
               
                 gatttgtcacagcttgggggtgactctggtggttctggaggatctggtggttctactaatctgtcagatattattgaaaaggagaccggt 
               
               
                   
               
               
                 aagcaactggttatccaggaatccatcctcatgctcccagaggaggtggaagaagtcattgggaacaagccggaaagcgatatactcgtg 
               
               
                   
               
               
                 cacaccgcctacgacgagagcaccgacgagaatgtcatgcttctgactagcgacgcccctgaatacaagccttgggctctggtcatacag 
               
               
                   
               
               
                 gatagcaacggtgagaacaagattaagatgctctctggtggttctggaggatctggtggttctactaatctgtcagatattattgaaaag 
               
               
                   
               
               
                 gagaccggtaagcaactggttatccaggaatccatcctcatgctcccagaggaggtggaagaagtcattgggaacaagccggaaagcgat 
               
               
                   
               
               
                 atactcgtgcacaccgcctacgacgagagcaccgacgagaatgtcatgcttctgactagcgacgcccctgaatacaagccttgggctctg 
               
               
                   
               
               
                 gtcatacaggatagcaacggtgagaacaagattaagatgctctctggtggttctAAAAGGACGGCGGACGGATCAGAGTTCGAGAGTCCG 
               
               
                   
               
               
                 AAAAAAAAACGAAAGGTCGAAtaa 
               
               
                   
               
               
                 BE4 Codon Optimization 1 
               
               
                 (SEQ ID NO: 14) 
                   
               
               
                 ATGTCATCCGAAACCGGGCCAGTGGCCGTAGACCCAACACTCAGGAGGCGGATAGAACCCCATGAGTTTGAAGTGTTCTTCGACCCCAGA 
                   
               
               
                   
               
               
                 GAGCTGCGCAAAGAGACTTGCCTCCTGTATGAAATAAATTGGGGGGGTCGCCATTCAATTTGGAGGCACACTAGCCAGAATACTAACAAA 
               
               
                   
               
               
                 CACGTGGAGGTAAATTTTATCGAGAAGTTTACCACCGAAAGATACTTTTGCCCCAATACACGGTGTTCAATTACCTGGTTTCTGTCATGG 
               
               
                   
               
               
                 AGTCCATGTGGAGAATGTAGTAGAGCGATAACTGAGTTCCTGTCTCGATATCCTCACGTCACGTTGTTTATATACATCGCTCGGCTTTAT 
               
               
                   
               
               
                 CACCATGCGGACCCGCGGAACAGGCAAGGTCTTCGGGACCTCATATCCTCTGGGGTGACCATCCAGATAATGACGGAGCAAGAGAGCGGA 
               
               
                   
               
               
                 TACTGCTGGCGAAACTTTGTTAACTACAGCCCAAGCAATGAGGCACACTGGCCTAGATATCCGCATCTCTGGGTTCGACTGTATGTCCTT 
               
               
                   
               
               
                 GAACTGTACTGCATAATTCTGGGACTTCCGCCATGCTTGAACATTCTGCGGCGGAAACAACCACAGCTGACCTTTTTCACGATTGCTCTC 
               
               
                   
               
               
                 CAAAGTTGTCACTACCAGCGATTGCCACCCCACATCTTGTGGGCTACTGGACTCAAGTCTGGAGGAAGTTCAGGCGGAAGCAGCGGGTCT 
               
               
                   
               
               
                 GAAACGCCCGGAACCTCAGAGAGCGCAACGCCCGAAAGCTCTGGAGGGTCAAGTGGTGGTAGTGATAAGAAATACTCCATCGGCCTCGCC 
               
               
                   
               
               
                 ATCGGTACGAATTCTGTCGGTTGGGCCGTTATCACCGATGAGTACAAGGTCCCTTCTAAGAAATTCAAGGTTTTGGGCAATACAGACCGC 
               
               
                   
               
               
                 CATTCTATAAAAAAAAACCTGATCGGCGCCCTTTTGTTTGACAGTGGTGAGACTGCTGAAGCGACTCGCCTGAAGCGAACTGCCAGGAGG 
               
               
                   
               
               
                 CGGTATACGAGGCGAAAAAACCGAATTTGTTACCTCCAGGAGATTTTCTCAAATGAAATGGCCAAGGTAGATGATAGTTTTTTTCACCGC 
               
               
                   
               
               
                 TTGGAAGAAAGTTTTCTCGTTGAGGAGGACAAAAAGCACGAGAGGCACCCAATCTTTGGCAACATAGTCGATGAGGTCGCATACCATGAG 
               
               
                   
               
               
                 AAATATCCTACGATCTATCATCTCCGCAAGAAGCTGGTCGATAGCACGGATAAAGCTGACCTCCGGCTGATCTACCTTGCTCTTGCTCAC 
               
               
                   
               
               
                 ATGATTAAATTCAGGGGCCATTTCCTGATAGAAGGAGACCTCAATCCCGACAATTCTGATGTCGACAAACTGTTTATTCAGCTCGTTCAG 
               
               
                   
               
               
                 ACCTATAATCAACTCTTTGAGGAGAACCCCATCAATGCTTCAGGGGTGGACGCAAAGGCCATTTTGTCCGCGCGCTTGAGTAAATCACGA 
               
               
                   
               
               
                 CGCCTCGAGAATTTGATAGCTCAACTGCCGGGTGAGAAGAAAAACGGGTTGTTTGGGAATCTCATAGCGTTGAGTTTGGGACTTACGCCA 
               
               
                   
               
               
                 AACTTTAAGTCTAACTTTGATTTGGCCGAAGATGCCAAATTGCAGCTGTCCAAAGATACCTATGATGACGACTTGGATAACCTTCTTGCG 
               
               
                   
               
               
                 CAGATTGGTGACCAATACGCGGATCTGTTTCTTGCCGCAAAAAATCTGTCCGACGCCATACTCTTGTCCGATATACTGCGCGTCAATACT 
               
               
                   
               
               
                 GAGATAACTAAGGCTCCCCTCAGCGCGTCCATGATTAAAAGATACGATGAGCACCACCAAGATCTCACTCTGTTGAAAGCCCTGGTTCGC 
               
               
                   
               
               
                 CAGCAGCTTCCAGAGAAGTATAAGGAGATATTTTTCGACCAATCTAAAAACGGCTATGCGGGTTACATTGACGGTGGCGCCTCTCAAGAA 
               
               
                   
               
               
                 GAATTCTACAAGTTTATAAAGCCGATACTTGAGAAAATGGACGGTACAGAGGAATTGTTGGTTAAGCTCAATCGCGAGGACTTGTTGAGA 
               
               
                   
               
               
                 AAGCAGCGCACATTTGACAATGGTAGTATTCCACACCAGATTCATCTGGGCGAGTTGCATGCCATTCTTAGAAGACAAGAAGATTTTTAT 
               
               
                   
               
               
                 CCGTTTCTGAAAGATAACAGAGAAAAGATTGAAAAGATACTTACCTTTCGCATACCGTATTATGTAGGTCCCCTGGCTAGAGGGAACAGT 
               
               
                   
               
               
                 CGCTTCGCTTGGATGACTCGAAAATCAGAAGAAACAATAACCCCCTGGAATTTTGAAGAAGTGGTAGATAAAGGTGCGAGTGCCCAATCT 
               
               
                   
               
               
                 TTTATTGAGCGGATGACAAATTTTGACAAGAATCTGCCTAACGAAAAGGTGCTTCCCAAGCATTCCCTTTTGTATGAATACTTTACAGTA 
               
               
                   
               
               
                 TATAATGAACTGACTAAAGTGAAGTACGTTACCGAGGGGATGCGAAAGCCAGCTTTTCTCAGTGGCGAGCAGAAAAAAGCAATAGTTGAC 
               
               
                   
               
               
                 CTGCTGTTCAAGACGAATAGGAAGGTTACCGTCAAACAGCTCAAAGAAGATTACTTTAAAAAGATCGAATGTTTTGATTCAGTTGAGATA 
               
               
                   
               
               
                 AGCGGAGTAGAGGATAGATTTAACGCAAGTCTTGGAACTTATCATGACCTTTTGAAGATCATCAAGGATAAAGATTTTTTGGACAACGAG 
               
               
                   
               
               
                 GAGAATGAAGATATCCTGGAAGATATAGTACTTACCTTGACGCTTTTTGAAGATCGAGAGATGATCGAGGAGCGACTTAAGACGTACGCA 
               
               
                   
               
               
                 CATCTCTTTGACGATAAGGTTATGAAACAATTGAAACGCCGGCGGTATACTGGCTGGGGCAGGCTTTCTCGAAAGCTGATTAATGGTATC 
               
               
                   
               
               
                 CGCGATAAGCAGTCTGGAAAGACAATCCTTGACTTTCTGAAAAGTGATGGATTTGCAAATAGAAACTTTATGCAGCTTATACATGATGAC 
               
               
                   
               
               
                 TCTTTGACGTTCAAGGAAGACATCCAGAAGGCACAGGTATCCGGCCAAGGGGATAGCCTCCATGAACACATAGCCAACCTGGCCGGCTCA 
               
               
                   
               
               
                 CCAGCTATTAAAAAGGGAATATTGCAAACCGTTAAGGTTGTTGACGAACTCGTTAAGGTTATGGGCCGACACAAACCAGAGAATATCGTG 
               
               
                   
               
               
                 ATTGAGATGGCTAGGGAGAATCAGACCACTCAAAAAGGTCAGAAAAATTCTCGCGAAAGGATGAAGCGAATTGAAGAGGGAATCAAAGAA 
               
               
                   
               
               
                 CTTGGCTCTCAAATTTTGAAAGAGCACCCGGTAGAAAACACTCAGCTGCAGAATGAAAAGCTGTATCTGTATTATCTGCAGAATGGTCGA 
               
               
                   
               
               
                 GATATGTACGTTGATCAGGAGCTGGATATCAATAGGCTCAGTGACTACGATGTCGACCACATCGTTCCTCAATCTTTCCTGAAAGATGAC 
               
               
                   
               
               
                 GCTATCGACAACAAAGTGTTGACGCGATCAGATAAGAACCGGGGAAAATCCGACAATGTACCCTCAGAAGAAGTTGTCAAGAAGATGAAA 
               
               
                   
               
               
                 AACTATTGGAGACAATTGCTGAACGCCAAGCTCATAACACAACGCAAGTTCGATAACTTGACGAAAGCCGAAAGAGGTGGGTTGTCAGAA 
               
               
                   
               
               
                 TTGGACAAAGCTGGCTTTATTAAGCGCCAATTGGTGGAGACCCGGCAGATTACGAAACACGTAGCACAAATTTTGGATTCACGAATGAAT 
               
               
                   
               
               
                 ACCAAATACGACGAAAACGACAAATTGATACGCGAGGTGAAAGTGATTACGCTTAAGAGTAAGTTGGTTTCCGATTTCAGGAAGGATTTT 
               
               
                   
               
               
                 CAGTTTTACAAAGTAAGAGAAATAAACAACTACCACCACGCCCATGATGCTTACCTCAACGCGGTAGTTGGCACAGCTCTTATCAAAAAA 
               
               
                   
               
               
                 TATCCAAAGCTGGAAAGCGAGTTCGTTTACGGTGACTATAAAGTATACGACGTTCGGAAGATGATAGCCAAATCAGAGCAGGAAATTGGG 
               
               
                   
               
               
                 AAGGCAACCGCAAAATACTTCTTCTATTCAAACATCATGAACTTCTTTAAGACGGAGATTACGCTCGCGAACGGCGAAATACGCAAGAGG 
               
               
                   
               
               
                 CCCCTCATAGAGACTAACGGCGAAACCGGGGAGATCGTATGGGACAAAGGACGGGACTTTGCGACCGTTAGAAAAGTACTTTCAATGCCA 
               
               
                   
               
               
                 CAAGTGAATATTGTTAAAAAGACAGAAGTACAAACAGGGGGGTTCAGTAAGGAATCCATTTTGCCCAAGCGGAACAGTGATAAATTGATA 
               
               
                   
               
               
                 GCAAGGAAAAAAGATTGGGACCCTAAGAAGTACGGTGGTTTCGACTCTCCTACCGTTGCATATTCAGTCCTTGTAGTTGCGAAAGTGGAA 
               
               
                   
               
               
                 AAGGGGAAAAGTAAGAAGCTTAAGAGTGTTAAAGAGCTTCTGGGCATAACCATAATGGAACGGTCTAGCTTCGAGAAAAATCCAATTGAC 
               
               
                   
               
               
                 TTTCTCGAGGCTAAAGGTTACAAGGAGGTAAAAAAGGACCTGATAATTAAACTCCCAAAGTACAGTCTCTTCGAGTTGGAGAATGGGAGG 
               
               
                   
               
               
                 AAGAGAATGTTGGCATCTGCAGGGGAGCTCCAAAAGGGGAACGAGCTGGCTCTGCCTTCAAAATACGTGAACTTTCTGTACCTGGCCAGC 
               
               
                   
               
               
                 CACTACGAGAAACTCAAGGGTTCTCCTGAGGATAACGAGCAGAAACAGCTGTTTGTAGAGCAGCACAAGCATTACCTGGACGAGATAATT 
               
               
                   
               
               
                 GAGCAAATTAGTGAGTTCTCAAAAAGAGTAATCCTTGCAGACGCGAATCTGGATAAAGTTCTTTCCGCCTATAATAAGCACCGGGACAAG 
               
               
                   
               
               
                 CCTATACGAGAACAAGCCGAGAACATCATTCACCTCTTTACCCTTACTAATCTGGGCGCGCCGGCCGCCTTCAAATACTTCGACACCACG 
               
               
                   
               
               
                 ATAGACAGGAAAAGGTATACGAGTACCAAAGAAGTACTTGACGCCACTCTCATCCACCAGTCTATAACAGGGTTGTACGAAACGAGGATA 
               
               
                   
               
               
                 GATTTGTCCCAGCTCGGCGGCGACTCAGGAGGGTCAGGCGGCTCCGGTGGATCAACGAATCTTTCCGACATAATCGAGAAAGAAACCGGC 
               
               
                   
               
               
                 AAACAGTTGGTGATCCAAGAATCAATCCTGATGCTGCCTGAAGAAGTAGAAGAGGTGATTGGCAACAAACCTGAGTCTGACATTCTTGTC 
               
               
                   
               
               
                 CACACCGCGTATGACGAGAGCACGGACGAGAACGTTATGCTTCTCACTAGCGACGCCCCTGAGTATAAACCATGGGCGCTGGTCATCCAA 
               
               
                   
               
               
                 GATTCCAATGGGGAAAACAAGATTAAGATGCTTAGTGGTGGGTCTGGAGGGAGCGGTGGGTCCACGAACCTCAGCGACATTATTGAAAAA 
               
               
                   
               
               
                 GAGACTGGTAAACAACTTGTAATACAAGAGTCTATTCTGATGTTGCCTGAAGAGGTGGAGGAGGTGATTGGGAACAAACCGGAGTCTGAT 
               
               
                   
               
               
                 ATACTTGTTCATACCGCCTATGACGAATCTACTGATGAGAATGTGATGCTTTTaACGTCAGACGCTCCCGAGTACAAACCCTGGGCTCTG 
               
               
                   
               
               
                 GTGATTCAGGACAGCAATGGTGAGAATAAGATTAAAATGTTGAGTGGGGGCTCAAAGCGCACGGCTGACGGTAGCGAATTTGAGAGCCCC 
               
               
                   
               
               
                 AAAAAAAAACGAAAGGTCGAAtaa 
               
               
                   
               
               
                 BE4 Codon Optimization 2 
               
               
                 (SEQ ID NO: 15) 
                   
               
               
                 ATGAGCAGCGAGACAGGCCCTGTGGCTGTGGATCCTACACTGCGGAGAAGAATCGAGCCCCACGAGTTCGAGGTGTTCTTCGACCCCAGA 
                   
               
               
                   
               
               
                 GAGCTGCGGAAAGAGACATGCCTGCTGTACGAGATCAACTGGGGCGGCAGACACTCTATCTGGCGGCACACAAGCCAGAACACCAACAAG 
               
               
                   
               
               
                 CACGTGGAAGTGAACTTTATCGAGAAGTTTACGACCGAGCGGTACTTCTGCCCCAACACCAGATGCAGCATCACCTGGTTTCTGAGCTGG 
               
               
                   
               
               
                 TCCCCTTGCGGCGAGTGCAGCAGAGCCATCACCGAGTTTCTGTCCAGATATCCCCACGTGACCCTGTTCATCTATATCGCCCGGCTGTAC 
               
               
                   
               
               
                 CACCACGCCGATCCTAGAAATAGACAGGGACTGCGCGACCTGATCAGCAGCGGAGTGACCATCCAGATCATGACCGAGCAAGAGAGCGGC 
               
               
                   
               
               
                 TACTGCTGGCGGAACTTCGTGAACTACAGCCCCAGCAACGAAGCCCACTGGCCTAGATATCCTCACCTGTGGGTCCGACTGTACGTGCTG 
               
               
                   
               
               
                 GAACTGTACTGCATCATCCTGGGCCTGCCTCCATGCCTGAACATCCTGAGAAGAAAGCAGCCTCAGCTGACCTTCTTCACAATCGCCCTG 
               
               
                   
               
               
                 CAGAGCTGCCACTACCAGAGACTGCCTCCACACATCCTGTGGGCCACCGGACTTAAGAGCGGAGGATCTAGCGGCGGCTCTAGCGGATCT 
               
               
                   
               
               
                 GAGACACCTGGCACAAGCGAGTCTGCCACACCTGAGAGTAGCGGCGGATCTTCTGGCGGCTCCGACAAGAAGTACTCTATCGGACTGGCC 
               
               
                   
               
               
                 ATCGGCACCAACTCTGTTGGATGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGG 
               
               
                   
               
               
                 CACAGCATCAAGAAGAATCTGATCGGCGCCCTGCTGTTCGACTCTGGCGAAACAGCCGAAGCCACCAGACTGAAGAGAACCGCCAGGCGG 
               
               
                   
               
               
                 AGATACACCCGGCGGAAGAACCGGATCTGCTACCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGA 
               
               
                   
               
               
                 CTGGAAGAGTCCTTCCTGGTGGAAGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGATGAGGTGGCCTACCACGAG 
               
               
                   
               
               
                 AAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGAGACTGATCTACCTGGCTCTGGCCCAC 
               
               
                   
               
               
                 ATGATCAAGTTCCGGGGCCACTTTCTGATCGAGGGCGATCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAG 
               
               
                   
               
               
                 ACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCTCTGGCGTGGACGCCAAGGCTATCCTGTCTGCCAGACTGAGCAAGAGCAGA 
               
               
                   
               
               
                 AGGCTGGAAAACCTGATCGCCCAGCTGCCTGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGACTGACCCCT 
               
               
                   
               
               
                 AACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAATCTGCTGGCC 
               
               
                   
               
               
                 CAGATCGGCGATCAGTACGCCGACTTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGATATCCTGAGAGTGAACACC 
               
               
                   
               
               
                 GAGATCACAAAGGCCCCTCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGATCTGACCCTGCTGAAGGCCCTCGTTAGA 
               
               
                   
               
               
                 CAGCAGCTGCCAGAGAAGTACAAAGAGATTTTCTTCGATCAGTCCAAGAACGGCTACGCCGGCTACATTGATGGCGGAGCCAGCCAAGAG 
               
               
                   
               
               
                 GAATTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTGGTCAAGCTGAACAGAGAGGACCTGCTGCGG 
               
               
                   
               
               
                 AAGCAGCGGACCTTCGACAATGGCTCTATCCCTCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGAGACAAGAGGACTTTTAC 
               
               
                   
               
               
                 CCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCAGGATCCCCTACTACGTGGGACCACTGGCCAGAGGCAATAGC 
               
               
                   
               
               
                 AGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACACCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCCAGCGCTCAGTCC 
               
               
                   
               
               
                 TTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCTAACGAGAAGGTGCTGCCCAAGCACTCCCTGCTGTATGAGTACTTCACCGTG 
               
               
                   
               
               
                 TACAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTTCTGAGCGGCGAGCAGAAAAAGGCCATTGTGGAT 
               
               
                   
               
               
                 CTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACAGCGTGGAAATC 
               
               
                   
               
               
                 AGCGGCGTGGAAGATCGGTTCAATGCCAGCCTGGGCACATACCACGACCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAACGAA 
               
               
                   
               
               
                 GAGAACGAGGACATTCTCGAGGACATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACATACGCC 
               
               
                   
               
               
                 CACCTGTTCGACGACAAAGTGATGAAGCAACTGAAGCGGAGGCGGTACACAGGCTGGGGCAGACTGTCTCGGAAGCTGATCAACGGCATC 
               
               
                   
               
               
                 CGGGATAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGAC 
               
               
                   
               
               
                 AGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAAGGCGATTCTCTGCACGAGCACATTGCCAACCTGGCCGGATCT 
               
               
                   
               
               
                 CCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTTGTGAAAGTGATGGGCAGACACAAGCCCGAGAACATCGTG 
               
               
                   
               
               
                 ATCGAAATGGCCAGAGAGAACCAGACCACACAGAAGGGCCAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAG 
               
               
                   
               
               
                 CTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGACGG 
               
               
                   
               
               
                 GATATGTACGTGGACCAAGAGCTGGACATCAACCGGCTGAGCGACTACGATGTGGACCATATCGTGCCCCAGAGCTTTCTGAAGGACGAC 
               
               
                   
               
               
                 TCCATCGATAACAAGGTCCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGATAACGTGCCCTCCGAAGAGGTGGTCAAGAAGATGAAG 
               
               
                   
               
               
                 AACTACTGGCGACAGCTGCTGAACGCCAAGCTGATTACCCAGCGGAAGTTCGATAACCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAA 
               
               
                   
               
               
                 CTTGATAAGGCCGGCTTCATTAAGCGGCAGCTGGTGGAAACCCGGCAGATCACCAAACACGTGGCACAGATTCTGGACTCCCGGATGAAC 
               
               
                   
               
               
                 ACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTCATCACCCTGAAGTCTAAGCTGGTGTCCGATTTCCGGAAGGATTTC 
               
               
                   
               
               
                 CAGTTCTACAAAGTGCGGGAAATCAACAACTACCATCACGCCCACGACGCCTACCTGAATGCCGTTGTTGGAACAGCCCTGATCAAGAAG 
               
               
                   
               
               
                 TATCCCAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAACAAGAGATCGGC 
               
               
                   
               
               
                 AAGGCTACCGCCAAGTACTTTTTCTACAGCAACATCATGAACTTTTTCAAGACAGAGATCACCCTGGCCAACGGCGAGATCCGGAAAAGA 
               
               
                   
               
               
                 CCCCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCAGAGATTTTGCCACAGTGCGGAAAGTGCTGAGCATGCCC 
               
               
                   
               
               
                 CAAGTGAATATCGTGAAGAAAACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCTAAGCGGAACAGCGATAAGCTGATC 
               
               
                   
               
               
                 GCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGATAGCCCTACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAA 
               
               
                   
               
               
                 AAGGGCAAGTCCAAAAAGCTCAAGAGCGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTTGAGAAGAACCCGATCGAC 
               
               
                   
               
               
                 TTTCTGGAAGCCAAGGGCTACAAAGAAGTCAAGAAGGACCTCATCATCAAGCTCCCCAAGTACAGCCTGTTCGAGCTGGAAAATGGCCGG 
               
               
                   
               
               
                 AAGCGGATGCTGGCCTCAGCAGGCGAACTGCAGAAAGGCAATGAACTGGCCCTGCCTAGCAAATACGTCAACTTCCTGTACCTGGCCAGC 
               
               
                   
               
               
                 CACTATGAGAAGCTGAAGGGCAGCCCCGAGGACAATGAGCAAAAGCAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATC 
               
               
                   
               
               
                 GAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAACCTGGATAAGGTGCTGTCTGCCTATAACAAGCACCGGGACAAG 
               
               
                   
               
               
                 CCTATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAACCTGGGAGCCCCTGCCGCCTTCAAGTACTTCGACACCACC 
               
               
                   
               
               
                 ATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACACTGATCCACCAGTCTATCACCGGCCTGTACGAAACCCGGATC 
               
               
                   
               
               
                 GACCTGTCTCAGCTCGGCGGCGATTCTGGTGGTTCTGGCGGAAGTGGCGGATCCACCAATCTGAGCGACATCATCGAAAAAGAGACAGGC 
               
               
                   
               
               
                 AAGCAGCTCGTGATCCAAGAATCCATCCTGATGCTGCCTGAAGAGGTTGAGGAAGTGATCGGCAACAAGCCTGAGTCCGACATCCTGGTG 
               
               
                   
               
               
                 CACACCGCCTACGATGAGAGCACCGATGAGAACGTCATGCTGCTGACAAGCGACGCCCCTGAGTACAAGCCTTGGGCTCTCGTGATTCAG 
               
               
                   
               
               
                 GACAGCAATGGGGAGAACAAGATCAAGATGCTGAGCGGAGGTAGCGGAGGCAGTGGCGGAAGCACAAACCTGTCTGATATCATTGAAAAA 
               
               
                   
               
               
                 GAAACCGGGAAGCAACTGGTCATTCAAGAGTCCATTCTCATGCTCCCGGAAGAAGTCGAGGAAGTCATTGGAAACAAACCCGAGAGCGAT 
               
               
                   
               
               
                 ATTCTGGTCCACACAGCCTATGACGAGTCTACAGACGAAAACGTGATGCTCCTGACCTCTGACGCTCCCGAGTATAAGCCCTGGGCACTT 
               
               
                   
               
               
                 GTTATCCAGGACTCTAACGGGGAAAACAAAATCAAAATGTTGTCCGGCGGCAGCAAGCGGACAGCCGATGGATCTGAGTTCGAGAGCCCC 
               
               
                   
               
               
                 AAGAAGAAACGGAAGGTgGAGtaa 
               
            
           
         
       
     
     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 CG to TA. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, e.g., converting A⋅T to G⋅C. 
     The term “base editor system” refers to a system for editing a nucleobase of a target nucleotide sequence. In various embodiments, the base editor (BE) system comprises (1) a polynucleotide programmable nucleotide binding domain and a deaminase domain for deaminating said 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 (1) a base editor (BE) comprising a polynucleotide programmable DNA binding domain and a deaminase domain for deaminating said nucleobase; and (2) a guide RNA in conjunction with the polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE). 
     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. 
     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 “deaminase” or “deaminase domain,” as used herein, refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase, catalyzing the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase or deaminase domain is a cytosine deaminase, catalyzing the hydrolytic deamination of cytosine to uracil. 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  E. coli, S. aureus, S. typhi, S. putrefaciens, H influenzae , or  C. crescentus . In some embodiments, the adenosine deaminase is a TadA deaminase. 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 (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); 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 “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 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. Examples of diseases include retinitis pigmentosa, Usher syndrome, sickle cell disease, beta-thalassemia, alpha-1 antitrypsin deficiency (A1AD), hepatic porphyria, medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, lysosomal acid lipase (LAL) deficiency, phenylketonuria, hemochromatosis, Von Gierke disease, Pompe disease, Gaucher disease, Hurler syndrome, cystic fibrosis, or chronic pain. In a particular embodiment, the disease is A1 AD. 
     By “effective amount” is meant the amount of an agent or active compound, e.g., a base editor as described herein, that is required to ameliorate the symptoms of a disease relative to an untreated patient or an individual without disease, i.e., a healthy individual. The effective amount of active compound(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 sufficient to introduce an alteration in a gene of interest in a cell (e.g., a cell in vitro or in vivo). 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 retinitis pigmentosa, Usher syndrome, sickle cell disease, beta-thalassemia, alpha-1 antitrypsin deficiency (A1AD), hepatic porphyria, medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, lysosomal acid lipase (LAL) deficiency, phenylketonuria, hemochromatosis, Von Gierke disease, Pompe disease, Gaucher disease, Hurler syndrome, cystic fibrosis, or chronic pain, or a symptom or condition thereof). Such therapeutic effect need not be sufficient to alter a pathogenic gene in all cells of a subject, tissue or organ, but only to alter the pathogenic gene in about 1%, 5%, 10%, 25%, 50%, 75% or more of the cells present in a subject, tissue or organ. In one embodiment, an effective amount is sufficient to ameliorate one or more symptoms of a disease (e.g., retinitis pigmentosa, Usher syndrome, sickle cell disease, beta-thalassemia, alpha-1 antitrypsin deficiency (A1AD), hepatic porphyria, medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, lysosomal acid lipase (LAL) deficiency, phenylketonuria, hemochromatosis, Von Gierke disease, Pompe disease, Gaucher disease, Hurler syndrome, cystic fibrosis, or chronic pain). 
     By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 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. 
     “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 terms “inhibitor of base repair”, “base repair inhibitor”, “IBR” or their grammatical equivalents refer to a protein that is capable in inhibiting the activity of a nucleic acid repair enzyme, for example a base excision repair 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 base repair inhibitor is an inhibitor of Endo V or 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 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. 
     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 or a cytidine deaminase). 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. 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 a 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 a RNA-binding portion of a deaminating component and a 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 a 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 a 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 a 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, a linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 16), which may also be referred to as the XTEN linker. In some embodiments, a linker comprises the amino acid sequence SGGS (SEQ ID NO: 17). In some embodiments, a linker comprises (SGGS) n  (SEQ ID NO: 18), (GGGS) n  (SEQ ID NO: 19), (GGGGS) n  (SEQ ID NO: 20), (G) n  (SEQ ID NO: 21), (EAAAK) n  (SEQ ID NO: 22), (GGS) n  (SEQ ID NO: 23), SGSETPGTSESATPES (SEQ ID NO: 16), or (XP) n  motif (SEQ ID NO: 24), or a combination of any of these, where n is independently an integer between 1 and 30, and where 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, 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: 25), PAPAPA (SEQ ID NO: 26), PAPAPAP (SEQ ID NO: 27), PAPAPAPA (SEQ ID NO: 28), P(AP) 4  (SEQ ID NO: 29), P(AP) 7  (SEQ ID NO: 30), P(AP) 10  (SEQ ID NO: 31). Such proline-rich linkers are also termed “rigid” linkers. 
     In some embodiments, the domains of a base editor are fused via a linker that comprises the amino acid sequence of SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 32), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 33), or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 34). In some embodiments, domains of the base editor are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 16), which may also be referred to as the XTEN linker. 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: 35). 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: 36). 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: 37). In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence 
     
       
         
           
               
            
               
                 (SEQ ID NO: 38) 
               
               
                 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG 
               
               
                   
               
               
                 TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS. 
               
            
           
         
       
     
     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 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 40) 
               
               
                   
                 KRTADGSEFESPKKKRKV,  
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 41) 
               
               
                   
                 KRPAATKKAGQAKKKK, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 42) 
               
               
                   
                 KKTELQTTNAENKTKKL,   
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 43) 
               
               
                   
                 KRGINDRNFWRGENGRKTR, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 44) 
               
               
                   
                 RKSGKIAAIVVKRPRK,   
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 45) 
               
               
                   
                 PKKKRKV, 
               
               
                   
                 or 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 46) 
               
               
                   
                 MDSLLMNRRKFLYQFKNVRWAKGRRETYLC. 
               
            
           
         
       
     
     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 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”, “polynucleotide”, and “polynucleic acid” 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 can be naturally occurring, for example, in the context of a genome, a transcript, mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecules. On the other hand, a nucleic acid molecule can 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, pyrrolopyrimidine, 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 (e.g., 2′-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, that guides the napDNAbp to a specific nucleic acid sequence. For example, 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). Examples of nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, and Cas12i. 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 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., a cytidine deaminase, a cytosine deaminase, an adenine deaminase, or an adenosine 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 (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. 
     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 or suspected of having a disease or disorder, for instance, but not restricted to alpha-1 antitrypsin deficiency (A1AD). 
     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 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 β-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” 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 DNA 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 Cas9 protein. 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). Non-limiting examples of nucleic acid programmable DNA binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a/Cpf1, Cas12b/C2c1, and Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, and Cas12i. Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, 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, Cas12h, 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, though they are not specifically listed in this disclosure. 
     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. 
     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, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably 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, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween. 
     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 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.” 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 (Csn1) from  Streptococcus pyogenes  (See, e.g., “Complete genome sequence of an M1 strain of  Streptococcus pyogenes .” Ferretti J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C, Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., 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., Chylinski K., Sharma C M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011). 
     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 (e.g., caused by cancer) can also be called a single-nucleotide alteration. 
     By “SERPINA1 polynucleotide” is meant a nucleic acid molecule encoding an A1AT protein or fragment thereof. The sequence of an exemplary SERPINA1 polynucleotide, which is available at NCBI Accession NO. NM_000295, is provided below: 
                        (SEQ ID NO: 47)              1 acaatgactc ctttcggtaa gtgcagtgga agctgtacac tgcccaggca aagcgtccgg                     61 gcagcgtagg cgggcgactc agatcccagc cagtggactt agcccctgtt tgctcctccg                121 ataactgggg tgaccttggt taatattcac cagcagcctc ccccgttgcc cctctggatc                181 cactgcttaa atacggacga ggacagggcc ctgtctcctc agcttcaggc accaccactg                241 acctgggaca gtgaatcgac aatgccgtct tctgtctcgt ggggcatcct cctgctggca                301 ggcctgtgct gcctggtccc tgtctccctg gctgaggatc cccagggaga tgctgcccag                361 aagacagata catcccacca tgatcaggat cacccaacct tcaacaagat cacccccaac                421 ctggctgagt tcgccttcag cctataccgc cagctggcac accagtccaa cagcaccaat                481 atcttcttct ccccagtgag catcgctaca gcctttgcaa tgctctccct ggggaccaag                541 gctgacactc acgatgaaat cctggagggc ctgaatttca acctcacgga gattccggag                601 gctcagatcc atgaaggctt ccaggaactc ctccgtaccc tcaaccagcc agacagccag                661 ctccagctga ccaccggcaa tggcctgttc ctcagcgagg gcctgaagct agtggataag                721 tttttggagg atgttaaaaa gttgtaccac tcagaagcct tcactgtcaa cttcggggac                781 accgaagagg ccaagaaaca gatcaacgat tacgtggaga agggtactca agggaaaatt                841 gtggatttgg tcaaggagct tgacagagac acagtttttg ctctggtgaa ttacatcttc                901 tttaaaggca aatgggagag accctttgaa gtcaaggaca ccgaggaaga ggacttccac                961 gtggaccagg tgaccaccgt gaaggtgcct atgatgaagc gtttaggcat gtttaacatc               1021 cagcactgta agaagctgtc cagctgggtg ctgctgatga aatacctggg caatgccacc               1081 gccatcttct tcctgcctga tgaggggaaa ctacagcacc tggaaaatga actcacccac               1141 gatatcatca ccaagttcct ggaaaatgaa gacagaaggt ctgccagctt acatttaccc               1201 aaactgtcca ttactggaac ctatgatctg aagagcgtcc tgggtcaact gggcatcact               1261 aaggtcttca gcaatggggc tgacctctcc ggggtcacag aggaggcacc cctgaagctc               1321 tccaaggccg tgcataaggc tgtgctgacc atcgac g aga aagggactga    agc   tgctggg               1381 gccatgtttt tagaggccat acccatgtct atcccccccg aggtcaagtt caacaaaccc               1441 tttgtcttct taatgattga acaaaatacc aagtctcccc tcttcatggg aaaagtggtg               1501 aatcccaccc aaaaataact gcctctcgct cctcaacccc tcccctccat ccctggcccc               1561 ctccctggat gacattaaag aagggttgag ctggtccctg cctgcatgtg actgtaaatc               1621 cctcccatgt tttctctgag tctccctttg cctgctgagg ctgtatgtgg gctccaggta               1681 acagtgctgt cttcgggccc cctgaactgt gttcatggag catctggctg ggtaggcaca               1741 tgctgggctt gaatccaggg gggactgaat cctcagctta cggacctggg cccatctgtt               1801 tctggagggc tccagtcttc cttgtcctgt cttggagtcc ccaagaagga atcacagggg               1861 aggaaccaga taccagccat gaccccaggc tccaccaagc atcttcatgt ccccctgctc               1921 atcccccact cccccccacc cagagttgct catcctgcca gggctggctg tgcccacccc               1981 aaggctgccc tcctgggggc cccagaactg cctgatcgtg ccgtggccca gttttgtggc               2041 atctgcagca acacaagaga gaggacaatg tcctcctctt gacccgctgt cacctaacca               2101 gactcgggcc ctgcacctct caggcacttc tggaaaatga ctgaggcaga ttcttcctga               2161 agcccattct ccatggggca acaaggacac ctattctgtc cttgtccttc catcgctgcc               2221 ccagaaagcc tcacatatct ccgtttagaa tcaggtccct tctccccaga tgaagaggag               2281 ggtctctgct ttgttttctc tatctcctcc tcagacttga ccaggcccag caggccccag               2341 aagaccatta ccctatatcc cttctcctcc ctagtcacat ggccataggc ctgctgatgg               2401 ctcaggaagg ccattgcaag gactcctcag ctatgggaga ggaagcacat cacccattga               2461 cccccgcaac ccctcccttt cctcctctga gtcccgactg gggccacatg cagcctgact               2521 tctttgtgcc tgttgctgtc cctgcagtct tcagagggcc accgcagctc cagtgccacg               2581 gcaggaggct gttcctgaat agcccctgtg gtaagggcca ggagagtcct tccatcctcc               2641 aaggccctgc taaaggacac agcagccagg aagtcccctg ggcccctagc tgaaggacag               2701 cctgctccct ccgtctctac caggaatggc cttgtcctat ggaaggcact gccccatccc               2761 aaactaatct aggaatcact gtctaaccac tcactgtcat gaatgtgtac ttaaaggatg               2821 aggttgagtc ataccaaata gtgatttcga tagttcaaaa tggtgaaatt agcaattcta               2881 catgattcag tctaatcaat ggataccgac tgtttcccac acaagtctcc tgttctctta               2941 agcttactca ctgacagcct ttcactctcc acaaatacat taaagatatg gccatcacca               3001 agccccctag gatgacacca gacctgagag tctgaagacc tggatccaag ttctgacttt               3061 tccccctgac agctgtgtga ccttcgtgaa gtcgccaaac ctctctgagc cccagtcatt               3121 gctagtaaga cctgcctttg agttggtatg atgttcaagt tagataacaa aatgtttata               3181 cccattagaa cagagaataa atagaactac atttcttgca             
The PAM sequence is shown in italics and double underlining, and the correct sequence after adenosine base editing is shown.
 
     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 preferred: embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred 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 a most preferred 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 a preferred 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 “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). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 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.       

     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. 
     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., a cytidine or an adenine deaminase). 
     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 ah, Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823 (2013);  Mali , P. et ah, RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, W. Y. et ah, Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et ah, RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, J. E. et ah, Genome engineering in  Saccharomyces cerevisiae  using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et ah 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). 
     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” 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. 
     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. 
     The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. 
     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. 
     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 and a nucleobase editing domain. A polynucleotide programmable nucleotide binding domain, 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 
     The term “polynucleotide programmable nucleotide binding domain” 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 Cas9 protein. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cpf1 protein. 
     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 cases, 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 cases, 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: 48) 
               
               
                 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 cases, 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 cases, 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., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. 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: 49). 
     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, Cas5h, 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, Cas12h, 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);  Psychoflexus 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 
     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 casn1 nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat) associated nuclease. An exemplary Cas9, is  Streptococcus pyogenes  Cas9 (spCas9), the amino acid sequence of which is provided below: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 50) 
               
               
                 MDKK YSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGA   
               
               
                   
               
               
                   LLFGSGET AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENP 
               
               
                   
               
               
                 INASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQG HSLHEQIANLAGSPAIKKGILQTVKIVDELVKV   
               
               
                   
               
               
                   MGHKPENIVIEMAR ENQTTQK GQKNSRERMKRIEEGIKELGSQILKEHPV   
               
               
                   
               
               
                 
                   ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDS 
                 
               
               
                   
               
               
                 
                   IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT 
                 
               
               
                   
               
               
                   K AERG GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR   
               
               
                   
               
               
                 
                   EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY 
                 
               
               
                   
               
               
                 
                   PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT 
                 
               
               
                   
               
               
                 
                   LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ 
                 
               
               
                   
               
               
                   T GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK 
               
               
                   
               
               
                 GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY 
               
               
                   
               
               
                 SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED 
               
               
                   
               
               
                 NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP 
               
               
                   
               
               
                 IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS 
               
               
                   
               
               
                 ITGLYETRIDLSQLGGD 
               
               
                 (single underline: HNH domain; double underline:  
               
               
                 RuvC domain) 
               
            
           
         
       
     
     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., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C, Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., 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., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. 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 aspects, 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, or a Cas9 nickase. 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 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: 51) 
                   
               
               
                 ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGC 
                   
               
               
                   
               
               
                 GGTGATCACTGATGATTATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATA 
               
               
                   
               
               
                 CAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGGCAGTGGA 
               
               
                   
               
               
                 GAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCG 
               
               
                   
               
               
                 GAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAG 
               
               
                   
               
               
                 ATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGC 
               
               
                   
               
               
                 ATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAAT 
               
               
                   
               
               
                 ATCCAACTATCTATCATCTGCGAAAAAAATTGGCAGATTCTACTGATAAAGCGGATT 
               
               
                   
               
               
                 TGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGAT 
               
               
                   
               
               
                 TGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGT 
               
               
                   
               
               
                 ACAAATCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTAGAGTAGATG 
               
               
                   
               
               
                 CTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTG 
               
               
                   
               
               
                 CTCAGCTCCCCGGTGAGAAGAGAAATGGCTTGTTTGGGAATCTCATTGCTTTGTCAT 
               
               
                   
               
               
                 TGGGATTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTAC 
               
               
                   
               
               
                 AGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAG 
               
               
                   
               
               
                 ATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTC 
               
               
                   
               
               
                 AGATATCCTAAGAGTAAATAGTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGA 
               
               
                   
               
               
                 TTAAGCGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGAC 
               
               
                   
               
               
                 AACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATAT 
               
               
                   
               
               
                 GCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACC 
               
               
                   
               
               
                 AATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAG 
               
               
                   
               
               
                 ATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACT 
               
               
                   
               
               
                 TGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAG 
               
               
                   
               
               
                 ACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTC 
               
               
                   
               
               
                 CATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACA 
               
               
                   
               
               
                 ATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTT 
               
               
                   
               
               
                 ATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAA 
               
               
                   
               
               
                 ACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATA 
               
               
                   
               
               
                 TGTTACTGAGGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCA 
               
               
                   
               
               
                 TTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAA 
               
               
                   
               
               
                 GATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGAT 
               
               
                   
               
               
                 AGATTTAATGCTTCATTAGGCGCCTACCATGATTTGCTAAAAATTATTAAAGATAAA 
               
               
                   
               
               
                 GATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTG 
               
               
                   
               
               
                 ACCTTATTTGAAGATAGGGGGATGATTGAGGAAAGACTTAAAACATATGCTCACCT 
               
               
                   
               
               
                 CTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGAC 
               
               
                   
               
               
                 GTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATA 
               
               
                   
               
               
                 TTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCAT 
               
               
                   
               
               
                 GATGATAGTTTGACATTTAAAGAAGATATTCAAAAAGCACAGGTGTCTGGACAAGG 
               
               
                   
               
               
                 CCATAGTTTACATGAACAGATTGCTAACTTAGCTGGCAGTCCTGCTATTAAAAAAGG 
               
               
                   
               
               
                 TATTTTACAGACTGTAAAAATTGTTGATGAACTGGTCAAAGTAATGGGGCATAAGC 
               
               
                   
               
               
                 CAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAG 
               
               
                   
               
               
                 AAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAA 
               
               
                   
               
               
                 GTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTC 
               
               
                   
               
               
                 TATCTCTATTATCTACAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATT 
               
               
                   
               
               
                 AATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCATTAAAGAC 
               
               
                   
               
               
                 GATTCAATAGACAATAAGGTACTAACGCGTTCTGATAAAAATCGTGGTAAATCGGA 
               
               
                   
               
               
                 TAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTC 
               
               
                   
               
               
                 TAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGT 
               
               
                   
               
               
                 GGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACT 
               
               
                   
               
               
                 CGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATA 
               
               
                   
               
               
                 CGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAAT 
               
               
                   
               
               
                 TAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATT 
               
               
                   
               
               
                 ACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGA 
               
               
                   
               
               
                 AATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTC 
               
               
                   
               
               
                 GTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTC 
               
               
                   
               
               
                 TTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAG 
               
               
                   
               
               
                 ATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGA 
               
               
                   
               
               
                 TAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATA 
               
               
                   
               
               
                 TTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCA 
               
               
                   
               
               
                 AAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATA 
               
               
                   
               
               
                 TGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGA 
               
               
                   
               
               
                 AAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTA 
               
               
                   
               
               
                 TGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATAT 
               
               
                   
               
               
                 AAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTA 
               
               
                   
               
               
                 GAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATG 
               
               
                   
               
               
                 AGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAA 
               
               
                   
               
               
                 AGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCAT 
               
               
                   
               
               
                 AAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATT 
               
               
                   
               
               
                 TTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAA 
               
               
                   
               
               
                 ACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGG 
               
               
                   
               
               
                 AGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTC 
               
               
                   
               
               
                 TACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGA 
               
               
                   
               
               
                 AACACGCATTGATTTGAGTCAGCTAGGAGGTGACTGA  
               
               
                   
               
               
                 (SEQ ID NO: 52) 
                   
               
               
                 MDKK YSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGALLFGSGET A 
                   
               
               
                   
               
               
                 EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF 
               
               
                   
               
               
                 GNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDN 
               
               
                   
               
               
                 SDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFG 
               
               
                   
               
               
                 NLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD 
               
               
                   
               
               
                 AILLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNG 
               
               
                   
               
               
                 YAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGE 
               
               
                   
               
               
                 LHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNF 
               
               
                   
               
               
                 EEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRK 
               
               
                   
               
               
                 PAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYH 
               
               
                   
               
               
                 DLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERLKTYAHLFDDKVMKQLKRRR 
               
               
                   
               
               
                 YTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS 
               
               
                   
               
               
                 GQG HSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMAR ENQTTQK GQ   
               
               
                   
               
               
                 
                   KNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRL 
                 
               
               
                   
               
               
                 
                   SDYDVDHIVPOSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKL 
                 
               
               
                   
               
               
                   ITQRKFDNLTK AERG GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI   
               
               
                   
               
               
                 
                   REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTAEIKKYPKLESEFV 
                 
               
               
                   
               
               
                 
                   YGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET 
                 
               
               
                   
               
               
                   GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQT GGFSKESILPKRNSDKLIARKKDWD 
               
               
                   
               
               
                 PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG 
               
               
                   
               
               
                 YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKL 
               
               
                   
               
               
                 KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQ 
               
               
                   
               
               
                 AENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG 
               
               
                   
               
               
                 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: 53) 
                   
               
               
                 ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGATGGGCT 
                   
               
               
                   
               
               
                 GTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACAC 
               
               
                   
               
               
                 AGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGA 
               
               
                   
               
               
                 AACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGC 
               
               
                   
               
               
                 AAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGA 
               
               
                   
               
               
                 CGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACA 
               
               
                   
               
               
                 TGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGT 
               
               
                   
               
               
                 ACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGAC 
               
               
                   
               
               
                 CTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTC 
               
               
                   
               
               
                 ATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTT 
               
               
                   
               
               
                 AGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGG 
               
               
                   
               
               
                 ATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTG 
               
               
                   
               
               
                 ATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCT 
               
               
                   
               
               
                 CTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAA 
               
               
                   
               
               
                 ATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAA 
               
               
                   
               
               
                 TTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATCC 
               
               
                   
               
               
                 TCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTT 
               
               
                   
               
               
                 CAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTA 
               
               
                   
               
               
                 GTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAA 
               
               
                   
               
               
                 CGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTTA 
               
               
                   
               
               
                 TCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAAT 
               
               
                   
               
               
                 CGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCA 
               
               
                   
               
               
                 AATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTT 
               
               
                   
               
               
                 CCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACT 
               
               
                   
               
               
                 ATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCC 
               
               
                   
               
               
                 GAAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGC 
               
               
                   
               
               
                 TCAATCGTTCATCGAGAGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAAG 
               
               
                   
               
               
                 TATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGA 
               
               
                   
               
               
                 AAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAG 
               
               
                   
               
               
                 AAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCA 
               
               
                   
               
               
                 ATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGG 
               
               
                   
               
               
                 GGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAAT 
               
               
                   
               
               
                 TAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAG 
               
               
                   
               
               
                 TGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACA 
               
               
                   
               
               
                 TACGCTCACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATAC 
               
               
                   
               
               
                 GGGCTGGGGACGATTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGT 
               
               
                   
               
               
                 GGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATG 
               
               
                   
               
               
                 CAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGT 
               
               
                   
               
               
                 TTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGC 
               
               
                   
               
               
                 CATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCA 
               
               
                   
               
               
                 TGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAAC 
               
               
                   
               
               
                 GACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGG 
               
               
                   
               
               
                 TATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAAT 
               
               
                   
               
               
                 TGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTT 
               
               
                   
               
               
                 GATCAGGAACTGGACATAAACCGTTTATCTGATTACGACGTCGATCACATTGTACCC 
               
               
                   
               
               
                 CAATCCTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAA 
               
               
                   
               
               
                 GAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAG 
               
               
                   
               
               
                 AACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAA 
               
               
                   
               
               
                 CTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTA 
               
               
                   
               
               
                 AACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATACTAGAT 
               
               
                   
               
               
                 TCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGT 
               
               
                   
               
               
                 AATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAA 
               
               
                   
               
               
                 AGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTAATGCCGTCG 
               
               
                   
               
               
                 TAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGT 
               
               
                   
               
               
                 GATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGG 
               
               
                   
               
               
                 CAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGA 
               
               
                   
               
               
                 AATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGG 
               
               
                   
               
               
                 AGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGT 
               
               
                   
               
               
                 TTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGT 
               
               
                   
               
               
                 TTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAA 
               
               
                   
               
               
                 AAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTC 
               
               
                   
               
               
                 TGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCA 
               
               
                   
               
               
                 AAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATC 
               
               
                   
               
               
                 GACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACT 
               
               
                   
               
               
                 ACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCG 
               
               
                   
               
               
                 CCGGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTC 
               
               
                   
               
               
                 CTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACA 
               
               
                   
               
               
                 GAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAA 
               
               
                   
               
               
                 TTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTA 
               
               
                   
               
               
                 AGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTA 
               
               
                   
               
               
                 TCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACAC 
               
               
                   
               
               
                 AACGATAGATCGCAAACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGA 
               
               
                   
               
               
                 TTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGG 
               
               
                   
               
               
                 GTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGA 
               
               
                   
               
               
                 CGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCTGCAG 
               
               
                   
               
               
                 GA  
               
               
                   
               
               
                 (SEQ ID NO: 54) 
                   
               
               
                 MDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET A 
                   
               
               
                   
               
               
                 EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF 
               
               
                   
               
               
                 GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDN 
               
               
                   
               
               
                 SDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF 
               
               
                   
               
               
                 GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLS 
               
               
                   
               
               
                 DAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN 
               
               
                   
               
               
                 GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLG 
               
               
                   
               
               
                 ELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWN 
               
               
                   
               
               
                 FEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMR 
               
               
                   
               
               
                 KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY 
               
               
                   
               
               
                 HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR 
               
               
                   
               
               
                 RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQV 
               
               
                   
               
               
                 SGQG DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA RENQTTQK 
               
               
                   
               
               
                 
                   GQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDI 
                 
               
               
                   
               
               
                 
                   NRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLL 
                 
               
               
                   
               
               
                   NAKLITQRKFDNLTK AERG GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEN   
               
               
                   
               
               
                 
                   DKTIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLE 
                 
               
               
                   
               
               
                 
                   SEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIET 
                 
               
               
                   
               
               
                   NGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQT GGFSKESILPKRNSDKLIARKK 
               
               
                   
               
               
                 DWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLE 
               
               
                   
               
               
                 AKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH 
               
               
                   
               
               
                 YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK 
               
               
                   
               
               
                 PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL 
               
               
                   
               
               
                 SQLGGD  
               
               
                 (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: 55) 
                   
               
               
                 ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGC 
                   
               
               
                   
               
               
                 GGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATA 
               
               
                   
               
               
                 CAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGA 
               
               
                   
               
               
                 GAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCG 
               
               
                   
               
               
                 GAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAG 
               
               
                   
               
               
                 ATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGC 
               
               
                   
               
               
                 ATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAAT 
               
               
                   
               
               
                 ATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATT 
               
               
                   
               
               
                 TGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGAT 
               
               
                   
               
               
                 TGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGT 
               
               
                   
               
               
                 ACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATG 
               
               
                   
               
               
                 CTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTG 
               
               
                   
               
               
                 CTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCAT 
               
               
                   
               
               
                 TGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTAC 
               
               
                   
               
               
                 AGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAG 
               
               
                   
               
               
                 ATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTC 
               
               
                   
               
               
                 AGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGA 
               
               
                   
               
               
                 TTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGAC 
               
               
                   
               
               
                 AACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATAT 
               
               
                   
               
               
                 GCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACC 
               
               
                   
               
               
                 AATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAG 
               
               
                   
               
               
                 ATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACT 
               
               
                   
               
               
                 TGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAG 
               
               
                   
               
               
                 ACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTC 
               
               
                   
               
               
                 CATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACA 
               
               
                   
               
               
                 ATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTT 
               
               
                   
               
               
                 ATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAA 
               
               
                   
               
               
                 ACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATA 
               
               
                   
               
               
                 TGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCA 
               
               
                   
               
               
                 TTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAA 
               
               
                   
               
               
                 GATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGAT 
               
               
                   
               
               
                 AGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAA 
               
               
                   
               
               
                 GATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTG 
               
               
                   
               
               
                 ACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCT 
               
               
                   
               
               
                 CTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGAC 
               
               
                   
               
               
                 GTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATA 
               
               
                   
               
               
                 TTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCAT 
               
               
                   
               
               
                 GATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGG 
               
               
                   
               
               
                 CGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGG 
               
               
                   
               
               
                 TATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATA 
               
               
                   
               
               
                 AGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGC 
               
               
                   
               
               
                 CAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAG 
               
               
                   
               
               
                 GAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAG 
               
               
                   
               
               
                 CTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGAT 
               
               
                   
               
               
                 ATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAA 
               
               
                   
               
               
                 GACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATC 
               
               
                   
               
               
                 GGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAA 
               
               
                   
               
               
                 CTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAA 
               
               
                   
               
               
                 CGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAA 
               
               
                   
               
               
                 ACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAA 
               
               
                   
               
               
                 ATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTA 
               
               
                   
               
               
                 AATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACA 
               
               
                   
               
               
                 ATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTA 
               
               
                   
               
               
                 AGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATG 
               
               
                   
               
               
                 TTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATAT 
               
               
                   
               
               
                 TTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGA 
               
               
                   
               
               
                 GAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTG 
               
               
                   
               
               
                 GGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCA 
               
               
                   
               
               
                 ATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTA 
               
               
                   
               
               
                 CCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAA 
               
               
                   
               
               
                 ATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGT 
               
               
                   
               
               
                 GGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACA 
               
               
                   
               
               
                 ATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGG 
               
               
                   
               
               
                 ATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGA 
               
               
                   
               
               
                 GTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGA 
               
               
                   
               
               
                 AATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTAT 
               
               
                   
               
               
                 GAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGC 
               
               
                   
               
               
                 AGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTG 
               
               
                   
               
               
                 TTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAG 
               
               
                   
               
               
                 ACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAAT 
               
               
                   
               
               
                 CTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATAT 
               
               
                   
               
               
                 ACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTT 
               
               
                   
               
               
                 TATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACTGA  
               
               
                   
               
               
                 (SEQ ID NO: 56) 
                   
               
               
                 MDKK YSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET A 
                   
               
               
                   
               
               
                 EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF 
               
               
                   
               
               
                 GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDN 
               
               
                   
               
               
                 SDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF 
               
               
                   
               
               
                 GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLS 
               
               
                   
               
               
                 DAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN 
               
               
                   
               
               
                 GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLG 
               
               
                   
               
               
                 ELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWN 
               
               
                   
               
               
                 FEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMR 
               
               
                   
               
               
                 KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY 
               
               
                   
               
               
                 HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR 
               
               
                   
               
               
                 RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQV 
               
               
                   
               
               
                 SGQG DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA RENQTTQK 
               
               
                   
               
               
                 
                   GQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDI 
                 
               
               
                   
               
               
                 
                   NRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLL 
                 
               
               
                   
               
               
                   NAKLITQRKFDNLTK AERG GLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEN   
               
               
                   
               
               
                 
                   DKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLE 
                 
               
               
                   
               
               
                 
                   SEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIET 
                 
               
               
                   
               
               
                   NGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQT GGFSKESILPKRNSDKLIARKK 
               
               
                   
               
               
                 DWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLE 
               
               
                   
               
               
                 AKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH 
               
               
                   
               
               
                 YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK 
               
               
                   
               
               
                 PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL 
               
               
                   
               
               
                 SQLGGD   
               
               
                 (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 torquisI  (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: 57)       MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA               LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR               LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD               LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP               INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP               NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI               LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI               FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR               KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY               YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK               NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD               LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI               IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ               LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD               SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV               MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP               VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDD               SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL               TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI               REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK               YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI               TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV               QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE               KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK               YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE               DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK               PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ               SITGLYETRIDLSQLGGD             
(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).
 
     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: 58) 
               
               
                 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, 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: 59) 
               
               
                 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  
               
            
           
         
       
     
     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. 
     An exemplary CasX ((uniprot.org/uniprot/FONN87; uniprot.org/uniprot/FONH53) tr|F0NN87|FONN87_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: 60) 
               
               
                 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAK 
               
               
                   
               
               
                 NNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFP 
               
               
                   
               
               
                 TTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLE 
               
               
                   
               
               
                 VEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNG 
               
               
                   
               
               
                 IVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGG 
               
               
                   
               
               
                 FSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG 
               
               
                   
               
               
                 SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG. 
               
            
           
         
       
     
     An exemplary CasX (&gt;tr|F0NH53|F0NH53_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: 61) 
               
               
                 MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAK 
               
               
                   
               
               
                 NNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFP 
               
               
                   
               
               
                 TTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLE 
               
               
                   
               
               
                 VEPHYLIMAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNG 
               
               
                   
               
               
                 IVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGG 
               
               
                   
               
               
                 FSIDLTKLLEKRDLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG 
               
               
                   
               
               
                 SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG. 
               
               
                   
               
               
                 Deltaproteobacteria CasX 
               
               
                 (SEQ ID NO: 62) 
               
               
                 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: 63) 
               
               
                   
                 MSKRHPRISGVKGYRLHAQRLEYTGKSGAMRTIKY 
               
               
                   
               
               
                   
                 PLYSSPSGGRTVPREIVSAINDDYVGLYGLSNFDD 
               
               
                   
               
               
                   
                 LYNAEKRNEEKVYSVLDFWYDCVQYGAVFSYTAPG 
               
               
                   
               
               
                   
                 LLKNVAEVRGGSYELTKTLKGSHLYDELQIDKVIK 
               
               
                   
               
               
                   
                 FLNKKEISRANGSLDKLKKDIIDCFKAEYRERHKD 
               
               
                   
               
               
                   
                 QCNKLADDIKNAKKDAGASLGERQKKLFRDFFGIS 
               
               
                   
               
               
                   
                 EQSENDKPSFTNPLNLTCCLLPFDTVNNNRNRGEV 
               
               
                   
               
               
                   
                 LFNKLKEYAQKLDKNEGSLEMWEYIGIGNSGTAFS 
               
               
                   
               
               
                   
                 NFLGEGFLGRLRENKITELKKAMMDITDAWRGQEQ 
               
               
                   
               
               
                   
                 EEELEKRLRILAALTIKLREPKFDNHWGGYRSDIN 
               
               
                   
               
               
                   
                 GKLSSWLQNYINQTVKIKEDLKGHKKDLKKAKEMI 
               
               
                   
               
               
                   
                 NRFGESDTKEEAVVSSLLESIEKIVPDDSADDEKP 
               
               
                   
               
               
                   
                 DIPAIAIYRRFLSDGRLTLNRFVQREDVQEALIKE 
               
               
                   
               
               
                   
                 RLEAEKKKKPKKRKKKSDAEDEKETIDFKELFPHL 
               
               
                   
               
               
                   
                 AKPLKLVPNFYGDSKRELYKKYKNAAIYTDALWKA 
               
               
                   
               
               
                   
                 VEKIYKSAFSSSLKNSFFDTDFDKDFFIKRLQKIF 
               
               
                   
               
               
                   
                 SVYRRFNTDKWKPIVKNSFAPYCDIVSLAENEVLY 
               
               
                   
               
               
                   
                 KPKQSRSRKSAAIDKNRVRLPSTENIAKAGIALAR 
               
               
                   
               
               
                   
                 ELSVAGFDWKDLLKKEEHEEYIDLIELHKTALALL 
               
               
                   
               
               
                   
                 LAVTETQLDISALDFVENGTVKDFMKTRDGNLVLE 
               
               
                   
               
               
                   
                 GRFLEMFSQSIVFSELRGLAGLMSRKEFITRSAIQ 
               
               
                   
               
               
                   
                 TMNGKQAELLYIPHEFQSAKITTPKEMSRAFLDLA 
               
               
                   
               
               
                   
                 PAEFATSLEPESLSEKSLLKLKQMRYYPHYFGYEL 
               
               
                   
               
               
                   
                 TRTGQGIDGGVAENALRLEKSPVKKREIKCKQYKT 
               
               
                   
               
               
                   
                 LGRGQNKIVLYVRSSYYQTQFLEWFLHRPKNVQTD 
               
               
                   
               
               
                   
                 VAVSGSFLIDEKKVKTRWNYDALTVALEPVSGSER 
               
               
                   
               
               
                   
                 VFVSQPFTIFPEKSAEEEGQRYLGIDIGEYGIAYT 
               
               
                   
               
               
                   
                 ALEITGDSAKILDQNFISDPQLKTLREEVKGLKLD 
               
               
                   
               
               
                   
                 QRRGTFAMPSTKIARIRESLVHSLRNRIHHLALKH 
               
               
                   
               
               
                   
                 KAKIVYELEVSRFEEGKQKIKKVYATLKKADVYSE 
               
               
                   
               
               
                   
                 IDADKNLQTTVWGKLAVASEISASYTSQFCGACKK 
               
               
                   
               
               
                   
                 LWRAEMQVDETITTQELIGTVRVIKGGTLIDAIKD 
               
               
                   
               
               
                   
                 FMRPPIFDENDTPFPKYRDFCDKHHISKKMRGNSC 
               
               
                   
               
               
                   
                 LFICPFCRANADADIQASQTIALLRYVKEEKKVED 
               
               
                   
               
               
                   
                 YFERFRKLKNIKVLGQMKKI. 
               
            
           
         
       
     
     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  Alicyclobacillus acidoterrestris  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. 
     A Cas12b/C2c1 ((uniprot.org/uniprot/TOD7A2 #2) sp|T0D7A2|C2C1_ALIAG CRISPR-associated endonuclease C2c1 OS= Alicyclobacillus acido - terrestris  (strain ATCC 49025/DSM 3922/CIP 106132/NCIMB 13137/GD3B) GN=c2c1 PE=1 SV=1) amino acid sequence is as follows: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 64) 
               
               
                   
                 MAVKSIKVKLRLDDMPEIRAGLWKLHKEVNAGVRY 
               
               
                   
               
               
                   
                 YTEWLSLLRQENLYRRSPNGDGEQECDKTAEECKA 
               
               
                   
               
               
                   
                 ELLERLRARQVENGHRGPAGSDDELLQLARQLYEL 
               
               
                   
               
               
                   
                 LVPQAIGAKGDAQQIARKFLSPLADKDAVGGLGIA 
               
               
                   
               
               
                   
                 KAGNKPRWVRMREAGEPGWEEEKEKAETRKSADRT 
               
               
                   
               
               
                   
                 ADVLRALADFGLKPLMRVYTDSEMSSVEWKPLRKG 
               
               
                   
               
               
                   
                 QAVRTWDRDMFQQAIERMMSWESWNQRVGQEYAKL 
               
               
                   
               
               
                   
                 VEQKNRFEQKNFVGQEHLVHLVNQLQQDMKEASPG 
               
               
                   
               
               
                   
                 LESKEQTAHYVTGRALRGSDKVFEKWGKLAPDAPF 
               
               
                   
               
               
                   
                 DLYDAEIKNVQRRNTRRFGSHDLFAKLAEPEYQAL 
               
               
                   
               
               
                   
                 WREDASFLTRYAVYNSILRKLNHAKMFATFTLPDA 
               
               
                   
               
               
                   
                 TAHPIWTRFDKLGGNLHQYTFLFNEFGERRHAIRF 
               
               
                   
               
               
                   
                 HKLLKVENGVAREVDDVTVPISMSEQLDNLLPRDP 
               
               
                   
               
               
                   
                 NEPIALYFRDYGAEQHFTGEFGGAKIQCRRDQLAH 
               
               
                   
               
               
                   
                 MHRRRGARDVYLNVSVRVQSQSEARGERRPPYAAV 
               
               
                   
               
               
                   
                 FRLVGDNHRAFVHFDKLSDYLAEHPDDGKLGSEGL 
               
               
                   
               
               
                   
                 LSGLRVMSVDLGLRTSASISVFRVARKDELKPNSK 
               
               
                   
               
               
                   
                 GRVPFFFPIKGNDNLVAVHERSQLLKLPGETESKD 
               
               
                   
               
               
                   
                 LRAIREERQRTLRQLRTQLAYLRLLVRCGSEDVGR 
               
               
                   
               
               
                   
                 RERSWAKLIEQPVDAANHMTPDWREAFENELQKLK 
               
               
                   
               
               
                   
                 SLHGICSDKEWMDAVYESVRRVWRHMGKQVRDWRK 
               
               
                   
               
               
                   
                 DVRSGERPKIRGYAKDVVGGNSIEQIEYLERQYKF 
               
               
                   
               
               
                   
                 LKSWSFFGKVSGQVIRAEKGSRFAITLREHIDHAK 
               
               
                   
               
               
                   
                 EDRLKKLADRIIMEALGYVYALDERGKGKWVAKYP 
               
               
                   
               
               
                   
                 PCQLILLEELSEYQFNNDRPPSENNQLMQWSHRGV 
               
               
                   
               
               
                   
                 FQELINQAQVHDLLVGTMYAAFSSRFDARTGAPGI 
               
               
                   
               
               
                   
                 RCRRVPARCTQEHNPEPFPWWLNKFVVEHTLDACP 
               
               
                   
               
               
                   
                 LRADDLIPTGEGEIFVSPFSAEEGDFHQIHADLNA 
               
               
                   
               
               
                   
                 AQNLQQRLWSDFDISQIRLRCDWGEVDGELVLIPR 
               
               
                   
               
               
                   
                 LTGKRTADSYSNKVFYTNTGVTYYERERGKKRRKV 
               
               
                   
               
               
                   
                 FAQEKLSEEEAELLVEADEAREKSVVLMRDPSGII 
               
               
                   
               
               
                   
                 NRGNWTRQKEFWSMVNQRIEGYLVKQIRSRVPLQD 
               
               
                   
               
               
                   
                 SACENTGDI. 
               
               
                   
               
               
                   
                 hCas12b ( Bacillus   hisashii ) NCBI 
               
               
                   
                 Reference Sequence: WP_095142515 
               
               
                   
                 (SEQ ID NO: 65) 
               
               
                   
                 MAPKKKRKVGIHGVPAAATRSFILKIEPNEEVKKG 
               
               
                   
               
               
                   
                 LWKTHEVLNHGIAYYMNILKLIRQEAIYEHHEQDP 
               
               
                   
               
               
                   
                 KNPKKVSKAEIQAELWDFVLKMQKCNSFTHEVDKD 
               
               
                   
               
               
                   
                 EVFNILRELYEELVPSSVEKKGEANQLSNKFLYPL 
               
               
                   
               
               
                   
                 VDPNSQSGKGTASSGRKPRWYNLKIAGDPSWEEEK 
               
               
                   
               
               
                   
                 KKWEEDKKKDPLAKILGKLAEYGLIPLFIPYTDSN 
               
               
                   
               
               
                   
                 EPIVKEIKWMEKSRNQSVRRLDKDMFIQALERFLS 
               
               
                   
               
               
                   
                 WESWNLKVKEEYEKVEKEYKTLEERIKEDIQALKA 
               
               
                   
               
               
                   
                 LEQYEKERQEQLLRDTLNTNEYRLSKRGLRGWREI 
               
               
                   
               
               
                   
                 IQKWLKMDENEPSEKYLEVFKDYQRKHPREAGDYS 
               
               
                   
               
               
                   
                 VYEFLSKKENHFIWRNHPEYPYLYATFCEIDKKKK 
               
               
                   
               
               
                   
                 DAKQQATFTLADPINHPLWVRFEERSGSNLNKYRI 
               
               
                   
               
               
                   
                 LTEQLHTEKLKKKLTVQLDRLIYPTESGGWEEKGK 
               
               
                   
               
               
                   
                 VDIVLLPSRQFYNQIFLDIEEKGKHAFTYKDESIK 
               
               
                   
               
               
                   
                 FPLKGTLGGARVQFDRDHLRRYPHKVESGNVGRIY 
               
               
                   
               
               
                   
                 FNMTVNIEPTESPVSKSLKIHRDDFPKVVNFKPKE 
               
               
                   
               
               
                   
                 LTEWIKDSKGKKLKSGIESLEIGLRVMSIDLGQRQ 
               
               
                   
               
               
                   
                 AAAASIFEVVDQKPDIEGKLFFPIKGTELYAVHRA 
               
               
                   
               
               
                   
                 SFNIKLPGETLVKSREVLRKAREDNLKLMNQKLNF 
               
               
                   
               
               
                   
                 LRNVLHFQQFEDITEREKRVTKWISRQENSDVPLV 
               
               
                   
               
               
                   
                 YQDELIQIRELMYKPYKDWVAFLKQLHKRLEVEIG 
               
               
                   
               
               
                   
                 KEVKHWRKSLSDGRKGLYGISLKNIDEIDRTRKFL 
               
               
                   
               
               
                   
                 LRWSLRPTEPGEVRRLEPGQRFAIDQLNHLNALKE 
               
               
                   
               
               
                   
                 DRLKKMANTIIMHALGYCYDVRKKKWQAKNPACQI 
               
               
                   
               
               
                   
                 ILFEDLSNYNPYEERSRFENSKLMKWSRREIPRQV 
               
               
                   
               
               
                   
                 ALQGEIYGLQVGEVGAQFSSRFHAKTGSPGIRCSV 
               
               
                   
               
               
                   
                 VTKEKLQDNRFFKNLQREGRLTLDKIAVLKEGDLY 
               
               
                   
               
               
                   
                 PDKGGEKFISLSKDRKCVTTHADINAAQNLQKRFW 
               
               
                   
               
               
                   
                 TRTHGFYKVYCKAYQVDGQTVYIPESKDQKQKIIE 
               
               
                   
               
               
                   
                 EFGEGYFILKDGVYEWVNAGKLKIKKGSSKQSSSE 
               
               
                   
               
               
                   
                 LVDSDILKDSFDLASELKGEKLMLYRDPSGNVFPS 
               
               
                   
               
               
                   
                 DKWMAAGVFFGKLERILISKLTNQYSISTIEDDSS 
               
               
                   
               
               
                   
                 KQSMKRPAATKKAGQAKKKK 
               
            
           
         
       
     
     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: 66) 
               
               
                   
                 MAIRSIKLKMKTNSGTDSIYLRKALWRTHQLINEG 
               
               
                   
               
               
                   
                 IAYYMNLLTLYRQEAIGDKTKEAYQAELINIIRNQ 
               
               
                   
               
               
                   
                 QRNNGSSEEHGSDQEILALLRQLYELIIPSSIGES 
               
               
                   
               
               
                   
                 GDANQLGNKFLYPLVDPNSQSGKGTSNAGRKPRWK 
               
               
                   
               
               
                   
                 RLKEEGNPDWELEKKKDEERKAKDPTVKIFDNLNK 
               
               
                   
               
               
                   
                 YGLLPLFPLFTNIQKDIEWLPLGKRQSVRKWDKDM 
               
               
                   
               
               
                   
                 FIQAIERLLSWESWNRRVADEYKQLKEKTESYYKE 
               
               
                   
               
               
                   
                 HLTGGEEWIEKIRKFEKERNMELEKNAFAPNDGYF 
               
               
                   
               
               
                   
                 ITSRQIRGWDRVYEKWSKLPESASPEELWKVVAEQ 
               
               
                   
               
               
                   
                 QNKMSEGFGDPKVFSFLANRENRDIWRGHSERIYH 
               
               
                   
               
               
                   
                 IAAYNGLQKKLSRTKEQATFTLPDAIEHPLWIRYE 
               
               
                   
               
               
                   
                 SPGGTNLNLFKLEEKQKKNYYVTLSKIIWPSEEKW 
               
               
                   
               
               
                   
                 IEKENIEIPLAPSIQFNRQIKLKQHVKGKQEISFS 
               
               
                   
               
               
                   
                 DYSSRISLDGVLGGSRIQFNRKYIKNHKELLGEGD 
               
               
                   
               
               
                   
                 IGPVFFNLVVDVAPLQETRNGRLQSPIGKALKVIS 
               
               
                   
               
               
                   
                 SDFSKVIDYKPKELMDWMNTGSASNSFGVASLLEG 
               
               
                   
               
               
                   
                 MRVMSIDMGQRTSASVSIFEVVKELPKDQEQKLFY 
               
               
                   
               
               
                   
                 SINDTELFAIHKRSFLLNLPGEVVTKNNKQQRQER 
               
               
                   
               
               
                   
                 RKKRQFVRSQIRMLANVLRLETKKTPDERKKAIHK 
               
               
                   
               
               
                   
                 LMEIVQSYDSWTASQKEVWEKELNLLTNMAAFNDE 
               
               
                   
               
               
                   
                 IWKESLVELHHRIEPYVGQIVSKWRKGLSEGRKNL 
               
               
                   
               
               
                   
                 AGISMWNIDELEDTRRLLISWSKRSRTPGEANRIE 
               
               
                   
               
               
                   
                 TDEPFGSSLLQHIQNVKDDRLKQMANLIIMTALGF 
               
               
                   
               
               
                   
                 KYDKEEKDRYKRWKETYPACQIILFENLNRYLFNL 
               
               
                   
               
               
                   
                 DRSRRENSRLMKWAHRSIPRTVSMQGEMFGLQVGD 
               
               
                   
               
               
                   
                 VRSEYSSRFHAKTGAPGIRCHALTEEDLKAGSNTL 
               
               
                   
               
               
                   
                 KRLIEDGFINESELAYLKKGDIIPSQGGELFVTLS 
               
               
                   
               
               
                   
                 KRYKKDSDNNELTVIHADINAAQNLQKRFWQQNSE 
               
               
                   
               
               
                   
                 VYRVPCQLARMGEDKLYIPKSQTETIKKYFGKGSF 
               
               
                   
               
               
                   
                 VKNNTEQEVYKWEKSEKMKIKTDTTFDLQDLDGFE 
               
               
                   
               
               
                   
                 DISKTIELAQEQQKKYLTMFRDPSGYFFNNETWRP 
               
               
                   
               
               
                   
                 QKEYWSIVNNIIKSCLKKKILSNKVEL 
               
            
           
         
       
     
     The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Cas9 undergoes a second 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 cases, efficiency can be expressed in terms of percentage of successful HDR. For example, a surveyor nuclease assay can be used 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 cases, 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 Icleaves 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 cases, 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 cases, 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 cases, 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 cases, 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 cases, 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 cases, 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 cases, 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 cases, 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 cases, 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 cases, 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 cases, 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 cases, 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 cases, 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 cases, 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 cases, 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 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 cases, 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. 
     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. 
     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. DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, and Cas12i. One example of a programmable polynucleotide-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. 
     Also useful in the present compositions and methods are nuclease-inactive Cpf1 (dCpf1) variants that may be used as a guide nucleotide sequence-programmable polynucleotide-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 nucleotide binding protein 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. 
     The amino acid sequence of wild type  Francisella novicida  Cpf1 follows. D917, E1006, and D1255 are bolded and underlined. 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 67) 
               
               
                   
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARG 
               
               
                   
               
               
                   
                 LILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVC 
               
               
                   
               
               
                   
                 ISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTI 
               
               
                   
               
               
                   
                 KKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLIL 
               
               
                   
               
               
                   
                 WLKQSKDNGIELFKANSDITDIDEALEIIKSFKGW 
               
               
                   
               
               
                   
                 TTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPK 
               
               
                   
               
               
                   
                 FLENKAKYESLKDKAPEAINYEQIKKDLAEELTFD 
               
               
                   
               
               
                   
                 IDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITK 
               
               
                   
               
               
                   
                 FNTIIGGKFVNGENTKRKGINEYINLYSQQINDKT 
               
               
                   
               
               
                   
                 LKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT 
               
               
                   
               
               
                   
                 TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQK 
               
               
                   
               
               
                   
                 LDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEY 
               
               
                   
               
               
                   
                 ITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLET 
               
               
                   
               
               
                   
                 IKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFD 
               
               
                   
               
               
                   
                 EIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKA 
               
               
                   
               
               
                   
                 IKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEH 
               
               
                   
               
               
                   
                 FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEK 
               
               
                   
               
               
                   
                 FKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYL 
               
               
                   
               
               
                   
                 GVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGA 
               
               
                   
               
               
                   
                 NKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN 
               
               
                   
               
               
                   
                 GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWK 
               
               
                   
               
               
                   
                 DFGFRFSDTQRYNSIDEFYREVENQGYKLTFENIS 
               
               
                   
               
               
                   
                 ESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHT 
               
               
                   
               
               
                   
                 LYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKK 
               
               
                   
               
               
                   
                 ITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTE 
               
               
                   
               
               
                   
                 DKFFFHCPITINFKSSGANKFNDEINLLLKEKAND 
               
               
                   
               
               
                   
                 VHILSI   D   RGERHLAYYTLVDGKGNIIKQDTFNIIG 
               
               
                   
               
               
                   
                 NDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEM 
               
               
                   
               
               
                   
                 KEGYLSQVVHEIAKLVIEYNAIVVF   E   DLNFGFKRG 
               
               
                   
               
               
                   
                 RFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG 
               
               
                   
               
               
                   
                 VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKI 
               
               
                   
               
               
                   
                 CPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD 
               
               
                   
               
               
                   
                 KGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFR 
               
               
                   
               
               
                   
                 NSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGEC 
               
               
                   
               
               
                   
                 IKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTE 
               
               
                   
               
               
                   
                 LDYLISPVADVNGNFFDSRQAPKNMPQDA   D   ANGAY 
               
               
                   
               
               
                   
                 HIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFV 
               
               
                   
               
               
                   
                 QNRNN. 
               
            
           
         
       
     
     The amino acid sequence of  Francisella novicida  Cpf1 D917A follows. (A917, E1006, and D1255 are bolded and underlined). 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 68) 
               
               
                   
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARG 
               
               
                   
               
               
                   
                 LILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVC 
               
               
                   
               
               
                   
                 ISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTI 
               
               
                   
               
               
                   
                 KKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLIL 
               
               
                   
               
               
                   
                 WLKQSKDNGIELFKANSDITDIDEALEIIKSFKGW 
               
               
                   
               
               
                   
                 TTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPK 
               
               
                   
               
               
                   
                 FLENKAKYESLKDKAPEAINYEQIKKDLAEELTFD 
               
               
                   
               
               
                   
                 IDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITK 
               
               
                   
               
               
                   
                 FNTIIGGKFVNGENTKRKGINEYINLYSQQINDKT 
               
               
                   
               
               
                   
                 LKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT 
               
               
                   
               
               
                   
                 TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQK 
               
               
                   
               
               
                   
                 LDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEY 
               
               
                   
               
               
                   
                 ITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLET 
               
               
                   
               
               
                   
                 IKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFD 
               
               
                   
               
               
                   
                 EIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKA 
               
               
                   
               
               
                   
                 IKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEH 
               
               
                   
               
               
                   
                 FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEK 
               
               
                   
               
               
                   
                 FKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYL 
               
               
                   
               
               
                   
                 GVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGA 
               
               
                   
               
               
                   
                 NKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN 
               
               
                   
               
               
                   
                 GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWK 
               
               
                   
               
               
                   
                 DFGFRFSDTQRYNSIDEFYREVENQGYKLTFENIS 
               
               
                   
               
               
                   
                 ESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHT 
               
               
                   
               
               
                   
                 LYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKK 
               
               
                   
               
               
                   
                 ITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTE 
               
               
                   
               
               
                   
                 DKFFFHCPITINFKSSGANKFNDEINLLLKEKAND 
               
               
                   
               
               
                   
                 VHILSI   A   RGERHLAYYTLVDGKGNIIKQDTFNIIG 
               
               
                   
               
               
                   
                 NDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEM 
               
               
                   
               
               
                   
                 KEGYLSQVVHEIAKLVIEYNAIVVF   E   DLNFGFKRG 
               
               
                   
               
               
                   
                 RFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG 
               
               
                   
               
               
                   
                 VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKI 
               
               
                   
               
               
                   
                 CPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD 
               
               
                   
               
               
                   
                 KGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFR 
               
               
                   
               
               
                   
                 NSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGEC 
               
               
                   
               
               
                   
                 IKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTE 
               
               
                   
               
               
                   
                 LDYLISPVADVNGNFFDSRQAPKNMPQDA   D   ANGAY 
               
               
                   
               
               
                   
                 HIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFV 
               
               
                   
               
               
                   
                 QNRNN. 
               
            
           
         
       
     
     The amino acid sequence of  Francisella novicida  Cpf1 E1006A follows. (D917, A1006, and D1255 are bolded and underlined). 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 69) 
               
               
                   
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARG 
               
               
                   
               
               
                   
                 LILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVC 
               
               
                   
               
               
                   
                 ISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTI 
               
               
                   
               
               
                   
                 KKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLIL 
               
               
                   
               
               
                   
                 WLKQSKDNGIELFKANSDITDIDEALEIIKSFKGW 
               
               
                   
               
               
                   
                 TTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPK 
               
               
                   
               
               
                   
                 FLENKAKYESLKDKAPEAINYEQIKKDLAEELTFD 
               
               
                   
               
               
                   
                 IDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITK 
               
               
                   
               
               
                   
                 FNTIIGGKFVNGENTKRKGINEYINLYSQQINDKT 
               
               
                   
               
               
                   
                 LKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT 
               
               
                   
               
               
                   
                 TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQK 
               
               
                   
               
               
                   
                 LDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEY 
               
               
                   
               
               
                   
                 ITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLET 
               
               
                   
               
               
                   
                 IKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFD 
               
               
                   
               
               
                   
                 EIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKA 
               
               
                   
               
               
                   
                 IKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEH 
               
               
                   
               
               
                   
                 FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEK 
               
               
                   
               
               
                   
                 FKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYL 
               
               
                   
               
               
                   
                 GVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGA 
               
               
                   
               
               
                   
                 NKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN 
               
               
                   
               
               
                   
                 GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWK 
               
               
                   
               
               
                   
                 DFGFRFSDTQRYNSIDEFYREVENQGYKLTFENIS 
               
               
                   
               
               
                   
                 ESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHT 
               
               
                   
               
               
                   
                 LYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKK 
               
               
                   
               
               
                   
                 ITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTE 
               
               
                   
               
               
                   
                 DKFFFHCPITINFKSSGANKFNDEINLLLKEKAND 
               
               
                   
               
               
                   
                 VHILSI   D   RGERHLAYYTLVDGKGNIIKQDTFNIIG 
               
               
                   
               
               
                   
                 NDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEM 
               
               
                   
               
               
                   
                 KEGYLSQVVHEIAKLVIEYNAIVVF   A   DLNFGFKRG 
               
               
                   
               
               
                   
                 RFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG 
               
               
                   
               
               
                   
                 VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKI 
               
               
                   
               
               
                   
                 CPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD 
               
               
                   
               
               
                   
                 KGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFR 
               
               
                   
               
               
                   
                 NSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGEC 
               
               
                   
               
               
                   
                 IKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTE 
               
               
                   
               
               
                   
                 LDYLISPVADVNGNFFDSRQAPKNMPQDA   D   ANGAY 
               
               
                   
               
               
                   
                 HIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFV 
               
               
                   
               
               
                   
                 QNRNN. 
               
            
           
         
       
     
     The amino acid sequence of  Francisella novicida  Cpf1 D1255A follows. (D917, E1006, and A1255 mutation positions are bolded and underlined). 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 70) 
               
               
                   
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARG 
               
               
                   
               
               
                   
                 LILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVC 
               
               
                   
               
               
                   
                 ISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTI 
               
               
                   
               
               
                   
                 KKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLIL 
               
               
                   
               
               
                   
                 WLKQSKDNGIELFKANSDITDIDEALEIIKSFKGW 
               
               
                   
               
               
                   
                 TTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPK 
               
               
                   
               
               
                   
                 FLENKAKYESLKDKAPEAINYEQIKKDLAEELTFD 
               
               
                   
               
               
                   
                 IDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITK 
               
               
                   
               
               
                   
                 FNTIIGGKFVNGENTKRKGINEYINLYSQQINDKT 
               
               
                   
               
               
                   
                 LKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT 
               
               
                   
               
               
                   
                 TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQK 
               
               
                   
               
               
                   
                 LDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEY 
               
               
                   
               
               
                   
                 ITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLET 
               
               
                   
               
               
                   
                 IKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFD 
               
               
                   
               
               
                   
                 EIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKA 
               
               
                   
               
               
                   
                 IKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEH 
               
               
                   
               
               
                   
                 FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEK 
               
               
                   
               
               
                   
                 FKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYL 
               
               
                   
               
               
                   
                 GVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGA 
               
               
                   
               
               
                   
                 NKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN 
               
               
                   
               
               
                   
                 GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWK 
               
               
                   
               
               
                   
                 DFGFRFSDTQRYNSIDEFYREVENQGYKLTFENIS 
               
               
                   
               
               
                   
                 ESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHT 
               
               
                   
               
               
                   
                 LYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKK 
               
               
                   
               
               
                   
                 ITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTE 
               
               
                   
               
               
                   
                 DKFFFHCPITINFKSSGANKFNDEINLLLKEKAND 
               
               
                   
               
               
                   
                 VHILSI   D   RGERHLAYYTLVDGKGNIIKQDTFNIIG 
               
               
                   
               
               
                   
                 NDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEM 
               
               
                   
               
               
                   
                 KEGYLSQVVHEIAKLVIEYNAIVVF   E   DLNFGFKRG 
               
               
                   
               
               
                   
                 RFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG 
               
               
                   
               
               
                   
                 VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKI 
               
               
                   
               
               
                   
                 CPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD 
               
               
                   
               
               
                   
                 KGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFR 
               
               
                   
               
               
                   
                 NSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGEC 
               
               
                   
               
               
                   
                 IKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTE 
               
               
                   
               
               
                   
                 LDYLISPVADVNGNFFDSRQAPKNMPQDA   A   ANGAY 
               
               
                   
               
               
                   
                 HIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFV 
               
               
                   
               
               
                   
                 QNRNN 
               
            
           
         
       
     
     The amino acid sequence of  Francisella novicida  Cpf1 D917A/E1006A follows. (A917, A1006, and D1255 are bolded and underlined). 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 71) 
               
               
                   
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARG 
               
               
                   
               
               
                   
                 LILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVC 
               
               
                   
               
               
                   
                 ISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTI 
               
               
                   
               
               
                   
                 KKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLIL 
               
               
                   
               
               
                   
                 WLKQSKDNGIELFKANSDITDIDEALEIIKSFKGW 
               
               
                   
               
               
                   
                 TTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPK 
               
               
                   
               
               
                   
                 FLENKAKYESLKDKAPEAINYEQIKKDLAEELTFD 
               
               
                   
               
               
                   
                 IDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITK 
               
               
                   
               
               
                   
                 FNTIIGGKFVNGENTKRKGINEYINLYSQQINDKT 
               
               
                   
               
               
                   
                 LKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT 
               
               
                   
               
               
                   
                 TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQK 
               
               
                   
               
               
                   
                 LDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEY 
               
               
                   
               
               
                   
                 ITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLET 
               
               
                   
               
               
                   
                 IKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFD 
               
               
                   
               
               
                   
                 EIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKA 
               
               
                   
               
               
                   
                 IKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEH 
               
               
                   
               
               
                   
                 FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEK 
               
               
                   
               
               
                   
                 FKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYL 
               
               
                   
               
               
                   
                 GVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGA 
               
               
                   
               
               
                   
                 NKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN 
               
               
                   
               
               
                   
                 GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWK 
               
               
                   
               
               
                   
                 DFGFRFSDTQRYNSIDEFYREVENQGYKLTFENIS 
               
               
                   
               
               
                   
                 ESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHT 
               
               
                   
               
               
                   
                 LYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKK 
               
               
                   
               
               
                   
                 ITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTE 
               
               
                   
               
               
                   
                 DKFFFHCPITINFKSSGANKFNDEINLLLKEKAND 
               
               
                   
               
               
                   
                 VHILSI   A   RGERHLAYYTLVDGKGNIIKQDTFNIIG 
               
               
                   
               
               
                   
                 NDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEM 
               
               
                   
               
               
                   
                 KEGYLSQVVHEIAKLVIEYNAIVVF   A   DLNFGFKRG 
               
               
                   
               
               
                   
                 RFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG 
               
               
                   
               
               
                   
                 VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKI 
               
               
                   
               
               
                   
                 CPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD 
               
               
                   
               
               
                   
                 KGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFR 
               
               
                   
               
               
                   
                 NSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGEC 
               
               
                   
               
               
                   
                 IKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTE 
               
               
                   
               
               
                   
                 LDYLISPVADVNGNFFDSRQAPKNMPQDA   D   ANGAY 
               
               
                   
               
               
                   
                 HIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFV 
               
               
                   
               
               
                   
                 QNRNN. 
               
            
           
         
       
     
     The amino acid sequence of  Francisella novicida  Cpf1 D917A/D1255A follows. (A917, E1006, and A1255 are bolded and underlined). 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 72) 
               
               
                   
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARG 
               
               
                   
               
               
                   
                 LILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVC 
               
               
                   
               
               
                   
                 ISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTI 
               
               
                   
               
               
                   
                 KKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLIL 
               
               
                   
               
               
                   
                 WLKQSKDNGIELFKANSDITDIDEALEIIKSFKGW 
               
               
                   
               
               
                   
                 TTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPK 
               
               
                   
               
               
                   
                 FLENKAKYESLKDKAPEAINYEQIKKDLAEELTFD 
               
               
                   
               
               
                   
                 IDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITK 
               
               
                   
               
               
                   
                 FNTIIGGKFVNGENTKRKGINEYINLYSQQINDKT 
               
               
                   
               
               
                   
                 LKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT 
               
               
                   
               
               
                   
                 TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQK 
               
               
                   
               
               
                   
                 LDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEY 
               
               
                   
               
               
                   
                 ITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLET 
               
               
                   
               
               
                   
                 IKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFD 
               
               
                   
               
               
                   
                 EIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKA 
               
               
                   
               
               
                   
                 IKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEH 
               
               
                   
               
               
                   
                 FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEK 
               
               
                   
               
               
                   
                 FKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYL 
               
               
                   
               
               
                   
                 GVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGA 
               
               
                   
               
               
                   
                 NKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN 
               
               
                   
               
               
                   
                 GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWK 
               
               
                   
               
               
                   
                 DFGFRFSDTQRYNSIDEFYREVENQGYKLTFENIS 
               
               
                   
               
               
                   
                 ESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHT 
               
               
                   
               
               
                   
                 LYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKK 
               
               
                   
               
               
                   
                 ITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTE 
               
               
                   
               
               
                   
                 DKFFFHCPITINFKSSGANKFNDEINLLLKEKAND 
               
               
                   
               
               
                   
                 VHILSI   A   RGERHLAYYTLVDGKGNIIKQDTFNIIG 
               
               
                   
               
               
                   
                 NDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEM 
               
               
                   
               
               
                   
                 KEGYLSQVVHEIAKLVIEYNAIVVF   E   DLNFGFKRG 
               
               
                   
               
               
                   
                 RFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG 
               
               
                   
               
               
                   
                 VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKI 
               
               
                   
               
               
                   
                 CPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD 
               
               
                   
               
               
                   
                 KGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFR 
               
               
                   
               
               
                   
                 NSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGEC 
               
               
                   
               
               
                   
                 IKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTE 
               
               
                   
               
               
                   
                 LDYLISPVADVNGNFFDSRQAPKNMPQDA   A   ANGAY 
               
               
                   
               
               
                   
                 HIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFV 
               
               
                   
               
               
                   
                 QNRNN. 
               
            
           
         
       
     
     The amino acid sequence of  Francisella novicida  Cpf1 E1006A/D1255A follows. (D917, A1006, and A1255 are bolded and underlined). 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 73) 
               
               
                   
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARG 
               
               
                   
               
               
                   
                 LILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVC 
               
               
                   
               
               
                   
                 ISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTI 
               
               
                   
               
               
                   
                 KKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLIL 
               
               
                   
               
               
                   
                 WLKQSKDNGIELFKANSDITDIDEALEIIKSFKGW 
               
               
                   
               
               
                   
                 TTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPK 
               
               
                   
               
               
                   
                 FLENKAKYESLKDKAPEAINYEQIKKDLAEELTFD 
               
               
                   
               
               
                   
                 IDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITK 
               
               
                   
               
               
                   
                 FNTIIGGKFVNGENTKRKGINEYINLYSQQINDKT 
               
               
                   
               
               
                   
                 LKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT 
               
               
                   
               
               
                   
                 TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQK 
               
               
                   
               
               
                   
                 LDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEY 
               
               
                   
               
               
                   
                 ITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLET 
               
               
                   
               
               
                   
                 IKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFD 
               
               
                   
               
               
                   
                 EIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKA 
               
               
                   
               
               
                   
                 IKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEH 
               
               
                   
               
               
                   
                 FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEK 
               
               
                   
               
               
                   
                 FKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYL 
               
               
                   
               
               
                   
                 GVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGA 
               
               
                   
               
               
                   
                 NKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN 
               
               
                   
               
               
                   
                 GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWK 
               
               
                   
               
               
                   
                 DFGFRFSDTQRYNSIDEFYREVENQGYKLTFENIS 
               
               
                   
               
               
                   
                 ESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHT 
               
               
                   
               
               
                   
                 LYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKK 
               
               
                   
               
               
                   
                 ITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTE 
               
               
                   
               
               
                   
                 DKFFFHCPITINFKSSGANKFNDEINLLLKEKAND 
               
               
                   
               
               
                   
                 VHILSI   D   RGERHLAYYTLVDGKGNIIKQDTFNIIG 
               
               
                   
               
               
                   
                 NDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEM 
               
               
                   
               
               
                   
                 KEGYLSQVVHEIAKLVIEYNAIVVF   A   DLNFGFKRG 
               
               
                   
               
               
                   
                 RFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG 
               
               
                   
               
               
                   
                 VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKI 
               
               
                   
               
               
                   
                 CPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLD 
               
               
                   
               
               
                   
                 KGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFR 
               
               
                   
               
               
                   
                 NSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGEC 
               
               
                   
               
               
                   
                 IKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTE 
               
               
                   
               
               
                   
                 LDYLISPVADVNGNFFDSRQAPKNMPQDA   A   ANGAY 
               
               
                   
               
               
                   
                 HIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFV 
               
               
                   
               
               
                   
                 QNRNN. 
               
            
           
         
       
     
     The amino acid sequence of  Francisella novicida  Cpf1 D917A/E1006A/D1255A follows. (A917, A1006, and A1255 are bolded and underlined). 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 74) 
               
               
                   
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARG 
               
               
                   
                   
               
               
                   
                 LILDDEKRAKDYKKAKQIIDKYHQFFIEEILSSVC 
               
               
                   
                   
               
               
                   
                 ISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTI 
               
               
                   
                   
               
               
                   
                 KKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLIL 
               
               
                   
                   
               
               
                   
                 WLKQSKDNGIELFKANSDITDIDEALEIIKSFKGW 
               
               
                   
                   
               
               
                   
                 TTYFKGFHENRKNVYSSNDIPTSIIYRIVDDNLPK 
               
               
                   
                   
               
               
                   
                 FLENKAKYESLKDKAPEAINYEQIKKDLAEELTFD 
               
               
                   
                   
               
               
                   
                 IDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITK 
               
               
                   
                   
               
               
                   
                 FNTIIGGKFVNGENTKRKGINEYINLYSQQINDKT 
               
               
                   
                   
               
               
                   
                 LKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT 
               
               
                   
                   
               
               
                   
                 TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQK 
               
               
                   
                   
               
               
                   
                 LDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEY 
               
               
                   
                   
               
               
                   
                 ITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLET 
               
               
                   
                   
               
               
                   
                 IKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFD 
               
               
                   
                   
               
               
                   
                 EIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKA 
               
               
                   
                   
               
               
                   
                 IKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEH 
               
               
                   
                   
               
               
                   
                 FYLVFEECYFELANIVPLYNKIRNYITQKPYSDEK 
               
               
                   
                   
               
               
                   
                 FKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYL 
               
               
                   
                   
               
               
                   
                 GVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGA 
               
               
                   
                   
               
               
                   
                 NKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN 
               
               
                   
                   
               
               
                   
                 GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWK 
               
               
                   
                   
               
               
                   
                 DFGFRFSDTQRYNSIDEFYREVENQGYKLTFENIS 
               
               
                   
                   
               
               
                   
                 ESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHT 
               
               
                   
                   
               
               
                   
                 LYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKK 
               
               
                   
                   
               
               
                   
                 ITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTE 
               
               
                   
                   
               
               
                   
                 DKFFFHCPITINFKSSGANKFNDEINLLLKEKAND 
               
               
                   
                   
               
               
                   
                 VHILSI   A   RGERHLAYY 
               
               
                   
                   
               
               
                   
                 TLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIE 
               
               
                   
                   
               
               
                   
                 KDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLV 
               
               
                   
                   
               
               
                   
                 IEYNAIVVF   A   DLNFGFKRGRFKVEKQVYQKLEKML 
               
               
                   
                   
               
               
                   
                 IEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKK 
               
               
                   
                   
               
               
                   
                 MGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYES 
               
               
                   
                   
               
               
                   
                 VSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDK 
               
               
                   
                   
               
               
                   
                 AAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPT 
               
               
                   
                   
               
               
                   
                 KELEKLLKDYSIEYGHGECIKAAICGESDKKFFAK 
               
               
                   
                   
               
               
                   
                 LTSVLNTILQMRNSKTGTELDYLISPVADVNGNFF 
               
               
                   
                   
               
               
                   
                 DSRQAPKNMPQDA   A   ANGAYHIGLKGLMLLGRIKNN 
               
               
                   
                   
               
               
                   
                 QEGKKLNLVIKNEEYFEFVQNRNN. 
               
            
           
         
       
     
     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 Cas 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 domain 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. 
     The amino acid sequence of an exemplary SaCas9 is as follows: 
     KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRR HRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHN VNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKE AKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHC TYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTL KQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIY QSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNR LKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKN SKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPL EDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETF KKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSY FRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKL DKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPN RELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQ KLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDD YPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKL KKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRP PRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG (SEQ ID NO: 75). In this sequence, residue N579, which is underlined and in bold, may be mutated (e.g., to a A579) to yield a SaCas9 nickase. 
     The amino acid sequence of an exemplary SaCas9n is as follows: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 76) 
               
               
                   
                 KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLF 
               
               
                   
               
               
                   
                 KEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLL 
               
               
                   
               
               
                   
                 FDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFS 
               
               
                   
               
               
                   
                 AALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRN 
               
               
                   
               
               
                   
                 SKALEEKYVAELQLERLKKDGEVRGSINRFKTSDY 
               
               
                   
               
               
                   
                 VKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTY 
               
               
                   
               
               
                   
                 YEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRS 
               
               
                   
               
               
                   
                 VKYAYNADLYNALNDLNNLVITRDENEKLEYYEKF 
               
               
                   
               
               
                   
                 QIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVT 
               
               
                   
               
               
                   
                 STGKPEFTNLKVYHDIKDITARKEIIENAELLDQI 
               
               
                   
               
               
                   
                 AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNL 
               
               
                   
               
               
                   
                 KGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRL 
               
               
                   
               
               
                   
                 KLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQ 
               
               
                   
               
               
                   
                 SIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMI 
               
               
                   
               
               
                   
                 NEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKL 
               
               
                   
               
               
                   
                 HDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPR 
               
               
                   
               
               
                   
                 SVSFDNSFNNKVLVKQEE   A   SKKGNRTPFQYLSSSD 
               
               
                   
               
               
                   
                 SKISYETFKKHILNLAKGKGRISKTKKEYLLEERD 
               
               
                   
               
               
                   
                 INRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRV 
               
               
                   
               
               
                   
                 NNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH 
               
               
                   
               
               
                   
                 AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEK 
               
               
                   
               
               
                   
                 QAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKY 
               
               
                   
               
               
                   
                 SHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLN 
               
               
                   
               
               
                   
                 GLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLK 
               
               
                   
               
               
                   
                 LIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPV 
               
               
                   
               
               
                   
                 IKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKP 
               
               
                   
               
               
                   
                 YRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKC 
               
               
                   
               
               
                   
                 YEEAKKLKKISNQAEFIASFYNNDLIKINGELYRV 
               
               
                   
               
               
                   
                 IGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRI 
               
               
                   
               
               
                   
                 IKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK 
               
               
                   
               
               
                   
                 KG. 
               
            
           
         
       
     
     In this sequence, residue A579, which can be mutated from N579 to yield a SaCas9 nickase, is underlined and in bold. 
     The amino acid sequences of an exemplary SaKKH Cas9 is as follows: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 77) 
               
               
                   
                 KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLF 
               
               
                   
               
               
                   
                 KEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLL 
               
               
                   
               
               
                   
                 FDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFS 
               
               
                   
               
               
                   
                 AALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRN 
               
               
                   
               
               
                   
                 SKALEEKYVAELQLERLKKDGEVRGSINRFKTSDY 
               
               
                   
               
               
                   
                 VKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTY 
               
               
                   
               
               
                   
                 YEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRS 
               
               
                   
               
               
                   
                 VKYAYNADLYNALNDLNNLVITRDENEKLEYYEKF 
               
               
                   
               
               
                   
                 QIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVT 
               
               
                   
               
               
                   
                 STGKPEFTNLKVYHDIKDITARKEIIENAELLDQI 
               
               
                   
               
               
                   
                 AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNL 
               
               
                   
               
               
                   
                 KGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRL 
               
               
                   
               
               
                   
                 KLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQ 
               
               
                   
               
               
                   
                 SIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMI 
               
               
                   
               
               
                   
                 NEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKL 
               
               
                   
               
               
                   
                 HDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPR 
               
               
                   
               
               
                   
                 SVSFDNSFNNKVLVKQEE   A   SKKGNRTPFQYLSSSD 
               
               
                   
               
               
                   
                 SKISYETFKKHILNLAKGKGRISKTKKEYLLEERD 
               
               
                   
               
               
                   
                 INRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRV 
               
               
                   
               
               
                   
                 NNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH 
               
               
                   
               
               
                   
                 AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEK 
               
               
                   
               
               
                   
                 QAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKY 
               
               
                   
               
               
                   
                 SHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLN 
               
               
                   
               
               
                   
                 GLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLK 
               
               
                   
               
               
                   
                 LIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPV 
               
               
                   
               
               
                   
                 IKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKP 
               
               
                   
               
               
                   
                 YRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKC 
               
               
                   
               
               
                   
                 YEEAKKLKKISNQAEFIASFYKNDLIKINGELYRV 
               
               
                   
               
               
                   
                 IGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHI 
               
               
                   
               
               
                   
                 IKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK 
               
               
                   
               
               
                   
                 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. 
     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 the sugar-phosphate backbone of a DNA, relative to a corresponding wild-type Cas9 domain. High fidelity Cas9 domains that have decreased electrostatic interactions with the sugar-phosphate backbone of DNA can have less off-target effects. In some embodiments, the Cas9 domain (e.g., a wild type Cas9 domain) comprises one or more mutations that decrease the association between the Cas9 domain and the 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 the sugar-phosphate backbone of 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 Underline 
       
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 78) 
               
               
                   
                 MDKKYSIGL   A   IGTNSVGWAVITDEYKVPSKKFKVL 
               
               
                   
               
               
                   
                 GNTDRHSIKKNLIGALLFDSGETAEATRLKRTARR 
               
               
                   
               
               
                   
                 RYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF 
               
               
                   
               
               
                   
                 LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRK 
               
               
                   
               
               
                   
                 KLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLN 
               
               
                   
               
               
                   
                 PDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKA 
               
               
                   
               
               
                   
                 ILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS 
               
               
                   
               
               
                   
                 LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA 
               
               
                   
               
               
                   
                 QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA 
               
               
                   
               
               
                   
                 PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                   
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG 
               
               
                   
               
               
                   
                 TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELH 
               
               
                   
               
               
                   
                 AILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL 
               
               
                   
               
               
                   
                 ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQS 
               
               
                   
               
               
                   
                 FIERMT   A   FDKNLPNEKVLPKHSLLYEYFTVYNELT 
               
               
                   
               
               
                   
                 KVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT 
               
               
                   
               
               
                   
                 VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYH 
               
               
                   
               
               
                   
                 DLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE 
               
               
                   
               
               
                   
                 MIEERLKTYAHLFDDKVMKQLKRRRYTGWG   A   LSRK 
               
               
                   
               
               
                   
                 LINGIRDKQSGKTILDFLKSDGFANRNFM   A   LIHDD 
               
               
                   
               
               
                   
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKK 
               
               
                   
               
               
                   
                 GILQTVKVVDELVKVMGRHKPENIVIEMARENQTT 
               
               
                   
               
               
                   
                 QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQ 
               
               
                   
               
               
                   
                 LQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH 
               
               
                   
               
               
                   
                 IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV 
               
               
                   
               
               
                   
                 VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSE 
               
               
                   
               
               
                   
                 LDKAGFIKRQLVETR   A   ITKHVAQILDSRMNTKYDE 
               
               
                   
               
               
                   
                 NDKLIREVKVITLKSKLVSDFRKDFQFYKVREINN 
               
               
                   
               
               
                   
                 YHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKV 
               
               
                   
               
               
                   
                 YDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                   
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRK 
               
               
                   
               
               
                   
                 VLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI 
               
               
                   
               
               
                   
                 ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSK 
               
               
                   
               
               
                   
                 KLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEV 
               
               
                   
               
               
                   
                 KKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE 
               
               
                   
               
               
                   
                 LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE 
               
               
                   
               
               
                   
                 QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYN 
               
               
                   
               
               
                   
                 KHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTT 
               
               
                   
               
               
                   
                 IDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQL 
               
               
                   
               
               
                   
                 GGD 
               
            
           
         
       
     
     Guide Polynucleotides 
     As used herein, the term “guide polynucleotide(s)” refer to 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. As used herein, the term “guide RNA (gRNA)” and its grammatical equivalents can refer to an RNA which can be specific for a target DNA and can form a complex with Cas protein. 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 cases, the guide polynucleotide comprises natural nucleotides (e.g., adenosine). In some cases, the guide polynucleotide comprises non-natural (or unnatural) nucleotides (e.g., peptide nucleic acid or nucleotide analogs). In some cases, 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 cases, 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 complementarily. 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 cases, 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 cases, a guide can target exon 1 or 2 of a gene, in other cases; 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 cases, 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, a 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 cases, 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 publically 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. Said 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 cases, 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 cases, 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 cases, a modification is permanent. In other cases, a modification is transient. In some cases, 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. 
     A modification can also be a phosphorothioate substitute. In some cases, 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 cases, 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 cases, phosphorothioate bonds can be added throughout an entire gRNA to reduce attack by endonucleases. 
     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 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. 
     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 D9X mutation, or a corresponding mutation in any of the amino acid sequences provided herein, wherein X is any amino acid except for D. In some embodiments, the SpCas9 comprises a D9A mutation, 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 T1336X 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 D1135E, R1335Q, and T1336R mutation, 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 T1336R mutation, 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 T1336X 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 R1335Q, and a T1336R mutation, 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 T1336R mutation, 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 G1217X, a R1335X, and a T1336X 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 G1217R, a R1335Q, and a T1336R mutation, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises a D1135V, a G1217R, a R1335Q, and a T1336R mutation, or corresponding mutations in any of the amino acid sequences provided herein. 
     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. 
     The amino acid sequence of an exemplary PAM-binding SpCas9 is as follows: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 79) 
               
               
                   
                 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVL 
               
               
                   
               
               
                   
                 GNTDRHSIKKNLIGALLFDSGETAEATRLKRTARR 
               
               
                   
               
               
                   
                 RYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF 
               
               
                   
               
               
                   
                 LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRK 
               
               
                   
               
               
                   
                 KLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLN 
               
               
                   
               
               
                   
                 PDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKA 
               
               
                   
               
               
                   
                 ILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS 
               
               
                   
               
               
                   
                 LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA 
               
               
                   
               
               
                   
                 QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA 
               
               
                   
               
               
                   
                 PLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                   
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG 
               
               
                   
               
               
                   
                 TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELH 
               
               
                   
               
               
                   
                 AILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL 
               
               
                   
               
               
                   
                 ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQS 
               
               
                   
               
               
                   
                 FIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELT 
               
               
                   
               
               
                   
                 KVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT 
               
               
                   
               
               
                   
                 VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYH 
               
               
                   
               
               
                   
                 DLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE 
               
               
                   
               
               
                   
                 MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRK 
               
               
                   
               
               
                   
                 LINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                   
                 SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKK 
               
               
                   
               
               
                   
                 GILQTVKVVDELVKVMGRHKPENIVIEMARENQTT 
               
               
                   
               
               
                   
                 QKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQ 
               
               
                   
               
               
                   
                 LQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH 
               
               
                   
               
               
                   
                 IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEV 
               
               
                   
               
               
                   
                 VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSE 
               
               
                   
               
               
                   
                 LDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDE 
               
               
                   
               
               
                   
                 NDKLIREVKVITLKSKLVSDFRKDFQFYKVREINN 
               
               
                   
               
               
                   
                 YHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKV 
               
               
                   
               
               
                   
                 YDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI 
               
               
                   
               
               
                   
                 TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRK 
               
               
                   
               
               
                   
                 VLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI 
               
               
                   
               
               
                   
                 ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSK 
               
               
                   
               
               
                   
                 KLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEV 
               
               
                   
               
               
                   
                 KKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE 
               
               
                   
               
               
                   
                 LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE 
               
               
                   
               
               
                   
                 QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYN 
               
               
                   
               
               
                   
                 KHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTT 
               
               
                   
               
               
                   
                 IDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQL 
               
               
                   
               
               
                   
                 GGD. 
               
            
           
         
       
     
     The amino acid sequence of an exemplary PAM-binding SpCas9n is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 80) 
               
               
                 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: 
     MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFVEEDKKHERHPIFG NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFG NLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD AILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNG YAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGE LHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNF EEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRK PAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYH DLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRR YTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQK GQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDI NRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLL NAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEN DKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLE SEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIET NGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKK DWDPKKYGGF E SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLE AKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK Q Y R STKEVLDATLIHQSITGLYETRIDL SQLGGD (SEQ ID NO: 81). In this sequence, residues E1135, Q1335 and R1337, which can be mutated from D1135, R1335, and T1337 to yield a SpEQR Cas9, are underlined and in bold. 
     The amino acid sequence of an exemplary PAM-binding SpVQR Cas9 is as follows: 
     MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDN SDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLS DAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLG ELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWN FEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMR KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQV SGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQK GQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDI NRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLL NAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDEN DKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLE SEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIET NGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKK DWDPKKYGGF V SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLE AKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASH YEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK Q Y R STKEVLDATLIHQSITGLYETRIDL SQLGGD (SEQ ID NO: 82). In this sequence, residues V1135, Q1335, and R1336, which can be mutated from D1135, R1335, 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: 83) 
               
               
                 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   ELQKGNELALPSKYVNFLYLASHYEKLKGSPE 
               
               
                   
               
               
                 DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK 
               
               
                   
               
               
                 PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK   E   Y   R   STKEVLDATLIHQ 
               
               
                   
               
               
                 SITGLYETRIDLSQLGGD. 
               
            
           
         
       
     
     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. 
     Exemplary SpyMacCas9 
     
       
         
           
               
            
               
                 (SEQ ID NO: 84) 
               
               
                 MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGA 
               
               
                   
               
               
                 LLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR 
               
               
                   
               
               
                 LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKAD 
               
               
                   
               
               
                 LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENP 
               
               
                   
               
               
                 INASRVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFGNLIALSLGLTP 
               
               
                   
               
               
                 NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 
               
               
                   
               
               
                 LLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI 
               
               
                   
               
               
                 FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR 
               
               
                   
               
               
                 KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY 
               
               
                   
               
               
                 YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK 
               
               
                   
               
               
                 NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD 
               
               
                   
               
               
                 LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGAYHDLLKI 
               
               
                   
               
               
                 IKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEERLKTYAHLFDDKVMKQ 
               
               
                   
               
               
                 LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD 
               
               
                   
               
               
                 SLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKV 
               
               
                   
               
               
                 MGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV 
               
               
                   
               
               
                 ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDS 
               
               
                   
               
               
                 IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT 
               
               
                   
               
               
                 KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR 
               
               
                   
               
               
                 EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY 
               
               
                   
               
               
                 PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT 
               
               
                   
               
               
                 LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEIQ 
               
               
                   
               
               
                 TVGQNGGLFDDNPKSPLEVTPSKLVPLKKELNPKKYGGYQKPTTAYPVLL 
               
               
                   
               
               
                 ITDTKQLIPISVMNKKQFEQNPVKFLRDRGYQQVGKNDFIKLPKYTLVDI 
               
               
                   
               
               
                 GDGIKRLWASSKEIHKGNQLVVSKKSQILLYHAHHLDSDLSNDYLQNHNQ 
               
               
                   
               
               
                 QFDVLFNEIISFSKKCKLGKEHIQKIENVYSNKKNSASIEELAESFIKLL 
               
               
                   
               
               
                 GFTQLGATSPFNFLGVKLNQKQYKGKKDYILPCTEGTLIRQSITGLYETR 
               
               
                   
               
               
                 VDLSKIGED. 
               
            
           
         
       
     
     In some cases, 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 cases, 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 cases, 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 cases, 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. 
     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 cases, 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 cases, a different endonuclease can be used to target certain genomic targets. In some cases, 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 kilo base 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 cases, a Cas protein can target a different PAM sequence. In some cases, a target gene can be adjacent to a Cas9 PAM, 5′-NGG, for example. In other cases, 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 meningitidis  (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 cases, an adjacent cut can be or can be about 3 base pairs upstream of a PAM. In some cases, an adjacent cut can be or can be about 10 base pairs upstream of a PAM. In some cases, 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. 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: 85), KRTADGSEFESPKKKRKV (SEQ ID NO: 40), KRPAATKKAGQAKKKK (SEQ ID NO: 41), KKTELQTTNAENKTKKL (SEQ ID NO: 42), KRGINDRNFWRGENGRKTR (SEQ ID NO: 43), RKSGKIAAIVVKRPRKPKKKRKV (SEQ ID NO: 86), or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 46). 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: 304), 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: 85) 
               
               
                   
                 PKKKRKVEGADKRTADGSEFES PKKKRKV. 
               
            
           
         
       
     
     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. 
     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. 
     In some embodiments, an NLS comprises the amino acid sequence 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 85) 
               
               
                   
                 PKKKRKVEGADKRTADGSEFES PKKKRKV, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 40) 
               
               
                   
                 KRTADGSEFESPKKKRKV, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 41) 
               
               
                   
                 KRPAATKKAGQAKKKK, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 42) 
               
               
                   
                 KKTELQTTNAENKTKKL, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 43) 
               
               
                   
                 KRGINDRNFWRGENGRKTR, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 86) 
               
               
                   
                 RKSGKIAAIVVKRPRKPKKKRKV, 
               
               
                   
                 or 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 46) 
               
               
                   
                 MDSLLMNRRKFLYQFKNVRWAKGRRETYLC. 
               
            
           
         
       
     
     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: 304), 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: 85) 
               
               
                   
                 PKKKRKVEGADKRTADGSEFES PKKKRKV 
               
            
           
         
       
     
     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. 
     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. 
     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., 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 component of the base editor can then edit a target base. 
     In some embodiments, the nucleobase editing domain is a deaminase domain. In some cases, a deaminase domain can be a cytosine deaminase or a cytidine deaminase. In some embodiments, the terms “cytosine deaminase” and “cytidine deaminase” can be used interchangeably. In some cases, a deaminase domain can be an adenine deaminase or an adenosine deaminase. 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. 
     C to T Editing 
     In some embodiments, a base editor disclosed herein comprises a fusion protein comprising 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 where the polynucleotide is double-stranded (e.g. DNA), the uridine base can then be substituted with a thymidine base (e.g. by 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 cytosine (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, completing the C-to-G base editing event). 
     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 NAGPB domain comprises a Cas9 domain, several nucleotides can be left unpaired during formation of the Cas9-gRNA-target DNA complex, resulting in formation of a Cas9 “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., 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. APOBEC is a family of 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 (“APOBEC3E” now refers to this), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and Activation-induced (cytidine) deaminase. 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 should 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 amino acid and nucleic acid sequences of PmCDA1 are shown herein below. 
     &gt;tr|A5H718|A5H718_PETMA Cytosine deaminase OS= Petromyzon marinus  OX=7757 PE=2 SV=1 amino acid sequence: 
                    (SEQ ID NO: 87)       MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFW               GYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADC               AEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNV               MVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKIL               HTTKSPAV            
Nucleic acid sequence: &gt;EF094822.1  Petromyzon marinus  isolate PmCDA.21 cytosine deaminase mRNA, complete cds:
 
                    (SEQ ID NO: 88)       TGACACGACACAGCCGTGTATATGAGGAAGGGTAGCTGGATGGGGGGGGG               GGGAATACGTTCAGAGAGGACATTAGCGAGCGTCTTGTTGGTGGCCTTGA               GTCTAGACACCTGCAGACATGACCGACGCTGAGTACGTGAGAATCCATGA               GAAGTTGGACATCTACACGTTTAAGAAACAGTTTTTCAACAACAAAAAAT               CCGTGTCGCATAGATGCTACGTTCTCTTTGAATTAAAACGACGGGGTGAA               CGTAGAGCGTGTTTTTGGGGCTATGCTGTGAATAAACCACAGAGCGGGAC               AGAACGTGGAATTCACGCCGAAATCTTTAGCATTAGAAAAGTCGAAGAAT               ACCTGCGCGACAACCCCGGACAATTCACGATAAATTGGTACTCATCCTGG               AGTCCTTGTGCAGATTGCGCTGAAAAGATCTTAGAATGGTATAACCAGGA               GCTGCGGGGGAACGGCCACACTTTGAAAATCTGGGCTTGCAAACTCTATT               ACGAGAAAAATGCGAGGAATCAAATTGGGCTGTGGAACCTCAGAGATAAC               GGGGTTGGGTTGAATGTAATGGTAAGTGAACACTACCAATGTTGCAGGAA               AATATTCATCCAATCGTCGCACAATCAATTGAATGAGAATAGATGGCTTG               AGAAGACTTTGAAGCGAGCTGAAAAACGACGGAGCGAGTTGTCCATTATG               ATTCAGGTAAAAATACTCCACACCACTAAGAGTCCTGCTGTTTAAGAGGC               TATGCGGATGGTTTTC            
The amino acid and nucleic acid sequences of the coding sequence (CDS) of human activation-induced cytidine deaminase (AID) are shown below.
 
&gt;tr|Q6QJ80|Q6QJ80_HUMAN Activation-induced cytidine deaminase OS= Homo sapiens  OX=9606 GN=AICDA PE=2 SV=1 amino acid sequence:
 
                    (SEQ ID NO: 89)       MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLR               NKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRG               NPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKAPV            
The amino acid and nucleic acid sequences of the coding sequence (CDS) of human activation-induced cytidine deaminase (AID) are shown below.
 
&gt;tr|Q6QJ80|Q6QJ80_HUMAN Activation-induced cytidine deaminase OS= Homo sapiens  OX=9606 GN=AICDA PE=2 SV=1 amino acid sequence:
 
                    (SEQ ID NO: 90)       MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLR               NKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRG               NPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKAPV            
Nucleic acid sequence: &gt;NG_011588.1:5001-15681  Homo sapiens  activation induced cytidine deaminase (AICDA), RefSeqGene (LRG_17) on chromosome 12:
 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 91) 
                   
               
               
                 AGAGAACCATCATTAATTGAAGTGAGATTTTTCTGGCCTGAGACTTGCAGGGAGGCAAGAAGACACTCTG 
                   
               
               
                   
               
               
                 GACACCACTATGGACAGGTAAAGAGGCAGTCTTCTCGTGGGTGATTGCACTGGCCTTCCTCTCAGAGCAA 
               
               
                   
               
               
                 ATCTGAGTAATGAGACTGGTAGCTATCCCTTTCTCTCATGTAACTGTCTGACTGATAAGATCAGCTTGAT 
               
               
                   
               
               
                 CAATATGCATATATATTTTTTGATCTGTCTCCTTTTCTTCTATTCAGATCTTATACGCTGTCAGCCCAAT 
               
               
                   
               
               
                 TCTTTCTGTTTCAGACTTCTCTTGATTTCCCTCTTTTTCATGTGGCAAAAGAAGTAGTGCGTACAATGTA 
               
               
                   
               
               
                 CTGATTCGTCCTGAGATTTGTACCATGGTTGAAACTAATTTATGGTAATAATATTAACATAGCAAATCTT 
               
               
                   
               
               
                 TAGAGACTCAAATCATGAAAAGGTAATAGCAGTACTGTACTAAAAACGGTAGTGCTAATTTTCGTAATAA 
               
               
                   
               
               
                 TTTTGTAAATATTCAACAGTAAAACAACTTGAAGACACACTTTCCTAGGGAGGCGTTACTGAAATAATTT 
               
               
                   
               
               
                 AGCTATAGTAAGAAAATTTGTAATTTTAGAAATGCCAAGCATTCTAAATTAATTGCTTGAAAGTCACTAT 
               
               
                   
               
               
                 GATTGTGTCCATTATAAGGAGACAAATTCATTCAAGCAAGTTATTTAATGTTAAAGGCCCAATTGTTAGG 
               
               
                   
               
               
                 CAGTTAATGGCACTTTTACTATTAACTAATCTTTCCATTTGTTCAGACGTAGCTTAACTTACCTCTTAGG 
               
               
                   
               
               
                 TGTGAATTTGGTTAAGGTCCTCATAATGTCTTTATGTGCAGTTTTTGATAGGTTATTGTCATAGAACTTA 
               
               
                   
               
               
                 TTCTATTCCTACATTTATGATTACTATGGATGTATGAGAATAACACCTAATCCTTATACTTTACCTCAAT 
               
               
                   
               
               
                 TTAACTCCTTTATAAAGAACTTACATTACAGAATAAAGATTTTTTAAAAATATATTTTTTTGTAGAGACA 
               
               
                   
               
               
                 GGGTCTTAGCCCAGCCGAGGCTGGTCTCTAAGTCCTGGCCCAAGCGATCCTCCTGCCTGGGCCTCCTAAA 
               
               
                   
               
               
                 GTGCTGGAATTATAGACATGAGCCATCACATCCAATATACAGAATAAAGATTTTTAATGGAGGATTTAAT 
               
               
                   
               
               
                 GTTCTTCAGAAAATTTTCTTGAGGTCAGACAATGTCAAATGTCTCCTCAGTTTACACTGAGATTTTGAAA 
               
               
                   
               
               
                 ACAAGTCTGAGCTATAGGTCCTTGTGAAGGGTCCATTGGAAATACTTGTTCAAAGTAAAATGGAAAGCAA 
               
               
                   
               
               
                 AGGTAAAATCAGCAGTTGAAATTCAGAGAAAGACAGAAAAGGAGAAAAGATGAAATTCAACAGGACAGAA 
               
               
                   
               
               
                 GGGAAATATATTATCATTAAGGAGGACAGTATCTGTAGAGCTCATTAGTGATGGCAAAATGACTTGGTCA 
               
               
                   
               
               
                 GGATTATTTTTAACCCGCTTGTTTCTGGTTTGCACGGCTGGGGATGCAGCTAGGGTTCTGCCTCAGGGAG 
               
               
                   
               
               
                 CACAGCTGTCCAGAGCAGCTGTCAGCCTGCAAGCCTGAAACACTCCCTCGGTAAAGTCCTTCCTACTCAG 
               
               
                   
               
               
                 GACAGAAATGACGAGAACAGGGAGCTGGAAACAGGCCCCTAACCAGAGAAGGGAAGTAATGGATCAACAA 
               
               
                   
               
               
                 AGTTAACTAGCAGGTCAGGATCACGCAATTCATTTCACTCTGACTGGTAACATGTGACAGAAACAGTGTA 
               
               
                   
               
               
                 GGCTTATTGTATTTTCATGTAGAGTAGGACCCAAAAATCCACCCAAAGTCCTTTATCTATGCCACATCCT 
               
               
                   
               
               
                 TCTTATCTATACTTCCAGGACACTTTTTCTTCCTTATGATAAGGCTCTCTCTCTCTCCACACACACACAC 
               
               
                   
               
               
                 ACACACACACACACACACACACACACACACACAAACACACACCCCGCCAACCAAGGTGCATGTAAAAAGA 
               
               
                   
               
               
                 TGTAGATTCCTCTGCCTTTCTCATCTACACAGCCCAGGAGGGTAAGTTAATATAAGAGGGATTTATTGGT 
               
               
                   
               
               
                 AAGAGATGATGCTTAATCTGTTTAACACTGGGCCTCAAAGAGAGAATTTCTTTTCTTCTGTACTTATTAA 
               
               
                   
               
               
                 GCACCTATTATGTGTTGAGCTTATATATACAAAGGGTTATTATATGCTAATATAGTAATAGTAATGGTGG 
               
               
                   
               
               
                 TTGGTACTATGGTAATTACCATAAAAATTATTATCCTTTTAAAATAAAGCTAATTATTATTGGATCTTTT 
               
               
                   
               
               
                 TTAGTATTCATTTTATGTTTTTTATGTTTTTGATTTTTTAAAAGACAATCTCACCCTGTTACCCAGGCTG 
               
               
                   
               
               
                 GAGTGCAGTGGTGCAATCATAGCTTTCTGCAGTCTTGAACTCCTGGGCTCAAGCAATCCTCCTGCCTTGG 
               
               
                   
               
               
                 CCTCCCAAAGTGTTGGGATACAGTCATGAGCCACTGCATCTGGCCTAGGATCCATTTAGATTAAAATATG 
               
               
                   
               
               
                 CATTTTAAATTTTAAAATAATATGGCTAATTTTTACCTTATGTAATGTGTATACTGGCAATAAATCTAGT 
               
               
                   
               
               
                 TTGCTGCCTAAAGTTTAAAGTGCTTTCCAGTAAGCTTCATGTACGTGAGGGGAGACATTTAAAGTGAAAC 
               
               
                   
               
               
                 AGACAGCCAGGTGTGGTGGCTCACGCCTGTAATCCCAGCACTCTGGGAGGCTGAGGTGGGTGGATCGCTT 
               
               
                   
               
               
                 GAGCCCTGGAGTTCAAGACCAGCCTGAGCAACATGGCAAAACGCTGTTTCTATAACAAAAATTAGCCGGG 
               
               
                   
               
               
                 CATGGTGGCATGTGCCTGTGGTCCCAGCTACTAGGGGGCTGAGGCAGGAGAATCGTTGGAGCCCAGGAGG 
               
               
                   
               
               
                 TCAAGGCTGCACTGAGCAGTGCTTGCGCCACTGCACTCCAGCCTGGGTGACAGGACCAGACCTTGCCTCA 
               
               
                   
               
               
                 AAAAAATAAGAAGAAAAATTAAAAATAAATGGAAACAACTACAAAGAGCTGTTGTCCTAGATGAGCTACT 
               
               
                   
               
               
                 TAGTTAGGCTGATATTTTGGTATTTAACTTTTAAAGTCAGGGTCTGTCACCTGCACTACATTATTAAAAT 
               
               
                   
               
               
                 ATCAATTCTCAATGTATATCCACACAAAGACTGGTACGTGAATGTTCATAGTACCTTTATTCACAAAACC 
               
               
                   
               
               
                 CCAAAGTAGAGACTATCCAAATATCCATCAACAAGTGAACAAATAAACAAAATGTGCTATATCCATGCAA 
               
               
                   
               
               
                 TGGAATACCACCCTGCAGTACAAAGAAGCTACTTGGGGATGAATCCCAAAGTCATGACGCTAAATGAAAG 
               
               
                   
               
               
                 AGTCAGACATGAAGGAGGAGATAATGTATGCCATACGAAATTCTAGAAAATGAAAGTAACTTATAGTTAC 
               
               
                   
               
               
                 AGAAAGCAAATCAGGGCAGGCATAGAGGCTCACACCTGTAATCCCAGCACTTTGAGAGGCCACGTGGGAA 
               
               
                   
               
               
                 GATTGCTAGAACTCAGGAGTTCAAGACCAGCCTGGGCAACACAGTGAAACTCCATTCTCCACAAAAATGG 
               
               
                   
               
               
                 GAAAAAAAGAAAGCAAATCAGTGGTTGTCCTGTGGGGAGGGGAAGGACTGCAAAGAGGGAAGAAGCTCTG 
               
               
                   
               
               
                 GTGGGGTGAGGGTGGTGATTCAGGTTCTGTATCCTGACTGTGGTAGCAGTTTGGGGTGTTTACATCCAAA 
               
               
                   
               
               
                 AATATTCGTAGAATTATGCATCTTAAATGGGTGGAGTTTACTGTATGTAAATTATACCTCAATGTAAGAA 
               
               
                   
               
               
                 AAAATAATGTGTAAGAAAACTTTCAATTCTCTTGCCAGCAAACGTTATTCAAATTCCTGAGCCCTTTACT 
               
               
                   
               
               
                 TCGCAAATTCTCTGCACTTCTGCCCCGTACCATTAGGTGACAGCACTAGCTCCACAAATTGGATAAATGC 
               
               
                   
               
               
                 ATTTCTGGAAAAGACTAGGGACAAAATCCAGGCATCACTTGTGCTTTCATATCAACCATGCTGTACAGCT 
               
               
                   
               
               
                 TGTGTTGCTGTCTGCAGCTGCAATGGGGACTCTTGATTTCTTTAAGGAAACTTGGGTTACCAGAGTATTT 
               
               
                   
               
               
                 CCACAAATGCTATTCAAATTAGTGCTTATGATATGCAAGACACTGTGCTAGGAGCCAGAAAACAAAGAGG 
               
               
                   
               
               
                 AGGAGAAATCAGTCATTATGTGGGAACAACATAGCAAGATATTTAGATCATTTTGACTAGTTAAAAAAGC 
               
               
                   
               
               
                 AGCAGAGTACAAAATCACACATGCAATCAGTATAATCCAAATCATGTAAATATGTGCCTGTAGAAAGACT 
               
               
                   
               
               
                 AGAGGAATAAACACAAGAATCTTAACAGTCATTGTCATTAGACACTAAGTCTAATTATTATTATTAGACA 
               
               
                   
               
               
                 CTATGATATTTGAGATTTAAAAAATCTTTAATATTTTAAAATTTAGAGCTCTTCTATTTTTCCATAGTAT 
               
               
                   
               
               
                 TCAAGTTTGACAATGATCAAGTATTACTCTTTCTTTTTTTTTTTTTTTTTTTTTTTTTGAGATGGAGTTT 
               
               
                   
               
               
                 TGGTCTTGTTGCCCATGCTGGAGTGGAATGGCATGACCATAGCTCACTGCAACCTCCACCTCCTGGGTTC 
               
               
                   
               
               
                 AAGCAAAGCTGTCGCCTCAGCCTCCCGGGTAGATGGGATTACAGGCGCCCACCACCACACTCGGCTAATG 
               
               
                   
               
               
                 TTTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCAAACTCCTGACCTCAGAGG 
               
               
                   
               
               
                 ATCCACCTGCCTCAGCCTCCCAAAGTGCTGGGATTACAGATGTAGGCCACTGCGCCCGGCCAAGTATTGC 
               
               
                   
               
               
                 TCTTATACATTAAAAAACAGGTGTGAGCCACTGCGCCCAGCCAGGTATTGCTCTTATACATTAAAAAATA 
               
               
                   
               
               
                 GGCCGGTGCAGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAAGCCAAGGCGGGCAGAACACCCGAGGT 
               
               
                   
               
               
                 CAGGAGTCCAAGGCCAGCCTGGCCAAGATGGTGAAACCCCGTCTCTATTAAAAATACAAACATTACCTGG 
               
               
                   
               
               
                 GCATGATGGTGGGCGCCTGTAATCCCAGCTACTCAGGAGGCTGAGGCAGGAGGATCCGCGGAGCCTGGCA 
               
               
                   
               
               
                 GATCTGCCTGAGCCTGGGAGGTTGAGGCTACAGTAAGCCAAGATCATGCCAGTATACTTCAGCCTGGGCG 
               
               
                   
               
               
                 ACAAAGTGAGACCGTAACAAAAAAAAAAAAATTTAAAAAAAGAAATTTAGATCAAGATCCAACTGTAAAA 
               
               
                   
               
               
                 AGTGGCCTAAACACCACATTAAAGAGTTTGGAGTTTATTCTGCAGGCAGAAGAGAACCATCAGGGGGTCT 
               
               
                   
               
               
                 TCAGCATGGGAATGGCATGGTGCACCTGGTTTTTGTGAGATCATGGTGGTGACAGTGTGGGGAATGTTAT 
               
               
                   
               
               
                 TTTGGAGGGACTGGAGGCAGACAGACCGGTTAAAAGGCCAGCACAACAGATAAGGAGGAAGAAGATGAGG 
               
               
                   
               
               
                 GCTTGGACCGAAGCAGAGAAGAGCAAACAGGGAAGGTACAAATTCAAGAAATATTGGGGGGTTTGAATCA 
               
               
                   
               
               
                 ACACATTTAGATGATTAATTAAATATGAGGACTGAGGAATAAGAAATGAGTCAAGGATGGTTCCAGGCTG 
               
               
                   
               
               
                 CTAGGCTGCTTACCTGAGGTGGCAAAGTCGGGAGGAGTGGCAGTTTAGGACAGGGGGCAGTTGAGGAATA 
               
               
                   
               
               
                 TTGTTTTGATCATTTTGAGTTTGAGGTACAAGTTGGACACTTAGGTAAAGACTGGAGGGGAAATCTGAAT 
               
               
                   
               
               
                 ATACAATTATGGGACTGAGGAACAAGTTTATTTTATTTTTTGTTTCGTTTTCTTGTTGAAGAACAAATTT 
               
               
                   
               
               
                 AATTGTAATCCCAAGTCATCAGCATCTAGAAGACAGTGGCAGGAGGTGACTGTCTTGTGGGTAAGGGTTT 
               
               
                   
               
               
                 GGGGTCCTTGATGAGTATCTCTCAATTGGCCTTAAATATAAGCAGGAAAAGGAGTTTATGATGGATTCCA 
               
               
                   
               
               
                 GGCTCAGCAGGGCTCAGGAGGGCTCAGGCAGCCAGCAGAGGAAGTCAGAGCATCTTCTTTGGTTTAGCCC 
               
               
                   
               
               
                 AAGTAATGACTTCCTTAAAAAGCTGAAGGAAAATCCAGAGTGACCAGATTATAAACTGTACTCTTGCATT 
               
               
                   
               
               
                 TTCTCTCCCTCCTCTCACCCACAGCCTCTTGATGAACCGGAGGAAGTTTCTTTACCAATTCAAAAATGTC 
               
               
                   
               
               
                 CGCTGGGCTAAGGGTCGGCGTGAGACCTACCTGTGCTACGTAGTGAAGAGGCGTGACAGTGCTACATCCT 
               
               
                   
               
               
                 TTTCACTGGACTTTGGTTATCTTCGCAATAAGGTATCAATTAAAGTCGGCTTTGCAAGCAGTTTAATGGT 
               
               
                   
               
               
                 CAACTGTGAGTGCTTTTAGAGCCACCTGCTGATGGTATTACTTCCATCCTTTTTTGGCATTTGTGTCTCT 
               
               
                   
               
               
                 ATCACATTCCTCAAATCCTTTTTTTTATTTCTTTTTCCATGTCCATGCACCCATATTAGACATGGCCCAA 
               
               
                   
               
               
                 AATATGTGATTTAATTCCTCCCCAGTAATGCTGGGCACCCTAATACCACTCCTTCCTTCAGTGCCAAGAA 
               
               
                   
               
               
                 CAACTGCTCCCAAACTGTTTACCAGCTTTCCTCAGCATCTGAATTGCCTTTGAGATTAATTAAGCTAAAA 
               
               
                   
               
               
                 GCATTTTTATATGGGAGAATATTATCAGCTTGTCCAAGCAAAAATTTTAAATGTGAAAAACAAATTGTGT 
               
               
                   
               
               
                 CTTAAGCATTTTTGAAAATTAAGGAAGAAGAATTTGGGAAAAAATTAACGGTGGCTCAATTCTGTCTTCC 
               
               
                   
               
               
                 AAATGATTTCTTTTCCCTCCTACTCACATGGGTCGTAGGCCAGTGAATACATTCAACATGGTGATCCCCA 
               
               
                   
               
               
                 GAAAACTCAGAGAAGCCTCGGCTGATGATTAATTAAATTGATCTTTCGGCTACCCGAGAGAATTACATTT 
               
               
                   
               
               
                 CCAAGAGACTTCTTCACCAAAATCCAGATGGGTTTACATAAACTTCTGCCCACGGGTATCTCCTCTCTCC 
               
               
                   
               
               
                 TAACACGCTGTGACGTCTGGGCTTGGTGGAATCTCAGGGAAGCATCCGTGGGGTGGAAGGTCATCGTCTG 
               
               
                   
               
               
                 GCTCGTTGTTTGATGGTTATATTACCATGCAATTTTCTTTGCCTACATTTGTATTGAATACATCCCAATC 
               
               
                   
               
               
                 TCCTTCCTATTCGGTGACATGACACATTCTATTTCAGAAGGCTTTGATTTTATCAAGCACTTTCATTTAC 
               
               
                   
               
               
                 TTCTCATGGCAGTGCCTATTACTTCTCTTACAATACCCATCTGTCTGCTTTACCAAAATCTATTTCCCCT 
               
               
                   
               
               
                 TTTCAGATCCTCCCAAATGGTCCTCATAAACTGTCCTGCCTCCACCTAGTGGTCCAGGTATATTTCCACA 
               
               
                   
               
               
                 ATGTTACATCAACAGGCACTTCTAGCCATTTTCCTTCTCAAAAGGTGCAAAAAGCAACTTCATAAACACA 
               
               
                   
               
               
                 AATTAAATCTTCGGTGAGGTAGTGTGATGCTGCTTCCTCCCAACTCAGCGCACTTCGTCTTCCTCATTCC 
               
               
                   
               
               
                 ACAAAAACCCATAGCCTTCCTTCACTCTGCAGGACTAGTGCTGCCAAGGGTTCAGCTCTACCTACTGGTG 
               
               
                   
               
               
                 TGCTCTTTTGAGCAAGTTGCTTAGCCTCTCTGTAACACAAGGACAATAGCTGCAAGCATCCCCAAAGATC 
               
               
                   
               
               
                 ATTGCAGGAGACAATGACTAAGGCTACCAGAGCCGCAATAAAAGTCAGTGAATTTTAGCGTGGTCCTCTC 
               
               
                   
               
               
                 TGTCTCTCCAGAACGGCTGCCACGTGGAATTGCTCTTCCTCCGCTACATCTCGGACTGGGACCTAGACCC 
               
               
                   
               
               
                 TGGCCGCTGCTACCGCGTCACCTGGTTCACCTCCTGGAGCCCCTGCTACGACTGTGCCCGACATGTGGCC 
               
               
                   
               
               
                 GACTTTCTGCGAGGGAACCCCAACCTCAGTCTGAGGATCTTCACCGCGCGCCTCTACTTCTGTGAGGACC 
               
               
                   
               
               
                 GCAAGGCTGAGCCCGAGGGGCTGCGGCGGCTGCACCGCGCCGGGGTGCAAATAGCCATCATGACCTTCAA 
               
               
                   
               
               
                 AGGTGCGAAAGGGCCTTCCGCGCAGGCGCAGTGCAGCAGCCCGCATTCGGGATTGCGATGCGGAATGAAT 
               
               
                   
               
               
                 GAGTTAGTGGGGAAGCTCGAGGGGAAGAAGTGGGCGGGGATTCTGGTTCACCTCTGGAGCCGAAATTAAA 
               
               
                   
               
               
                 GATTAGAAGCAGAGAAAAGAGTGAATGGCTCAGAGACAAGGCCCCGAGGAAATGAGAAAATGGGGCCAGG 
               
               
                   
               
               
                 GTTGCTTCTTTCCCCTCGATTTGGAACCTGAACTGTCTTCTACCCCCATATCCCCGCCTTTTTTTCCTTT 
               
               
                   
               
               
                 TTTTTTTTTTGAAGATTATTTTTACTGCTGGAATACTTTTGTAGAAAACCACGAAAGAACTTTCAAAGCC 
               
               
                   
               
               
                 TGGGAAGGGCTGCATGAAAATTCAGTTCGTCTCTCCAGACAGCTTCGGCGCATCCTTTTGGTAAGGGGCT 
               
               
                   
               
               
                 TCCTCGCTTTTTAAATTTTCTTTCTTTCTCTACAGTCTTTTTTGGAGTTTCGTATATTTCTTATATTTTC 
               
               
                   
               
               
                 TTATTGTTCAATCACTCTCAGTTTTCATCTGATGAAAACTTTATTTCTCCTCCACATCAGCTTTTTCTTC 
               
               
                   
               
               
                 TGCTGTTTCACCATTCAGAGCCCTCTGCTAAGGTTCCTTTTCCCTCCCTTTTCTTTCTTTTGTTGTTTCA 
               
               
                   
               
               
                 CATCTTTAAATTTCTGTCTCTCCCCAGGGTTGCGTTTCCTTCCTGGTCAGAATTCTTTTCTCCTTTTTTT 
               
               
                   
               
               
                 TTTTTTTTTTTTTTTTTTTTAAACAAACAAACAAAAAACCCAAAAAAACTCTTTCCCAATTTACTTTCTT 
               
               
                   
               
               
                 CCAACATGTTACAAAGCCATCCACTCAGTTTAGAAGACTCTCCGGCCCCACCGACCCCCAACCTCGTTTT 
               
               
                   
               
               
                 GAAGCCATTCACTCAATTTGCTTCTCTCTTTCTCTACAGCCCCTGTATGAGGTTGATGACTTACGAGACG 
               
               
                   
               
               
                 CATTTCGTACTTTGGGACTTTGATAGCAACTTCCAGGAATGTCACACACGATGAAATATCTCTGCTGAAG 
               
               
                   
               
               
                 ACAGTGGATAAAAAACAGTCCTTCAAGTCTTCTCTGTTTTTATTCTTCAACTCTCACTTTCTTAGAGTTT 
               
               
                   
               
               
                 ACAGAAAAAATATTTATATACGACTCTTTAAAAAGATCTATGTCTTGAAAATAGAGAAGGAACACAGGTC 
               
               
                   
               
               
                 TGGCCAGGGACGTGCTGCAATTGGTGCAGTTTTGAATGCAACATTGTCCCCTACTGGGAATAACAGAACT 
               
               
                   
               
               
                 GCAGGACCTGGGAGCATCCTAAAGTGTCAACGTTTTTCTATGACTTTTAGGTAGGATGAGAGCAGAAGGT 
               
               
                   
               
               
                 AGATCCTAAAAAGCATGGTGAGAGGATCAAATGTTTTTATATCAACATCCTTTATTATTTGATTCATTTG 
               
               
                   
               
               
                 AGTTAACAGTGGTGTTAGTGATAGATTTTTCTATTCTTTTCCCTTGACGTTTACTTTCAAGTAACACAAA 
               
               
                   
               
               
                 CTCTTCCATCAGGCCATGATCTATAGGACCTCCTAATGAGAGTATCTGGGTGATTGTGACCCCAAACCAT 
               
               
                   
               
               
                 CTCTCCAAAGCATTAATATCCAATCATGCGCTGTATGTTTTAATCAGCAGAAGCATGTTTTTATGTTTGT 
               
               
                   
               
               
                 ACAAAAGAAGATTGTTATGGGTGGGGATGGAGGTATAGACCATGCATGGTCACCTTCAAGCTACTTTAAT 
               
               
                   
               
               
                 AAAGGATCTTAAAATGGGCAGGAGGACTGTGAACAAGACACCCTAATAATGGGTTGATGTCTGAAGTAGC 
               
               
                   
               
               
                 AAATCTTCTGGAAACGCAAACTCTTTTAAGGAAGTCCCTAATTTAGAAACACCCACAAACTTCACATATC 
               
               
                   
               
               
                 ATAATTAGCAAACAATTGGAAGGAAGTTGCTTGAATGTTGGGGAGAGGAAAATCTATTGGCTCTCGTGGG 
               
               
                   
               
               
                 TCTCTTCATCTCAGAAATGCCAATCAGGTCAAGGTTTGCTACATTTTGTATGTGTGTGATGCTTCTCCCA 
               
               
                   
               
               
                 AAGGTATATTAACTATATAAGAGAGTTGTGACAAAACAGAATGATAAAGCTGCGAACCGTGGCACACGCT 
               
               
                   
               
               
                 CATAGTTCTAGCTGCTTGGGAGGTTGAGGAGGGAGGATGGCTTGAACACAGGTGTTCAAGGCCAGCCTGG 
               
               
                   
               
               
                 GCAACATAACAAGATCCTGTCTCTCAAAAAAAAAAAAAAAAAAAAGAAAGAGAGAGGGCCGGGCGTGGTG 
               
               
                   
               
               
                 GCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGCCGGGCGGATCACCTGTGGTCAGGAGTTTGAGA 
               
               
                   
               
               
                 CCAGCCTGGCCAACATGGCAAAACCCCGTCTGTACTCAAAATGCAAAAATTAGCCAGGCGTGGTAGCAGG 
               
               
                   
               
               
                 CACCTGTAATCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCAGGAGGTGGAGGTTGCA 
               
               
                   
               
               
                 GTAAGCTGAGATCGTGCCGTTGCACTCCAGCCTGGGCGACAAGAGCAAGACTCTGTCTCAGAAAAAAAAA 
               
               
                   
               
               
                 AAAAAAAGAGAGAGAGAGAGAAAGAGAACAATATTTGGGAGAGAAGGATGGGGAAGCATTGCAAGGAAAT 
               
               
                   
               
               
                 TGTGCTTTATCCAACAAAATGTAAGGAGCCAATAAGGGATCCCTATTTGTCTCTTTTGGTGTCTATTTGT 
               
               
                   
               
               
                 CCCTAACAACTGTCTTTGACAGTGAGAAAAATATTCAGAATAACCATATCCCTGTGCCGTTATTACCTAG 
               
               
                   
               
               
                 CAACCCTTGCAATGAAGATGAGCAGATCCACAGGAAAACTTGAATGCACAACTGTCTTATTTTAATCTTA 
               
               
                   
               
               
                 TTGTACATAAGTTTGTAAAAGAGTTAAAAATTGTTACTTCATGTATTCATTTATATTTTATATTATTTTG 
               
               
                   
               
               
                 CGTCTAATGATTTTTTATTAACATGATTTCCTTTTCTGATATATTGAAATGGAGTCTCAAAGCTTCATAA 
               
               
                   
               
               
                 ATTTATAACTTTAGAAATGATTCTAATAACAACGTATGTAATTGTAACATTGCAGTAATGGTGCTACGAA 
               
               
                   
               
               
                 GCCATTTCTCTTGATTTTTAGTAAACTTTTATGACAGCAAATTTGCTTCTGGCTCACTTTCAATCAGTTA 
               
               
                   
               
               
                 AATAAATGATAAATAATTTTGGAAGCTGTGAAGATAAAATACCAAATAAAATAATATAAAAGTGATTTAT 
               
               
                   
               
               
                 ATGAAGTTAAAATAAAAAATCAGTATGATGGAATAAACTTG 
               
            
           
         
       
     
     Other exemplary deaminases that can be fused to Cas9 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 (nuclear localization sequence, without nuclear export signal, cytoplasmic localizing signal). 
     
       
         
           
               
               
            
               
                 Human AID: 
                   
               
               
                 (SEQ ID NO: 92) 
                   
               
               
                   MDSLLMNRRKFLYQFKNVRWAKGRRETYLC YVVKRRDSATSFSLDFGYLRNKNGCH 
                   
               
               
                 VELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYF 
               
               
                 CEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLS 
               
               
                 RQLRRILLP LYEVDDLRDAFRTLGL   
               
               
                 (underline: nuclear localization sequence; double underline: nuclear export signal) 
               
               
                   
               
               
                 Mouse AID: 
               
               
                 (SEQ ID NO: 93) 
                   
               
               
                   MDSLLMKQKKFLYHFKNVRWAKGRHETYLC YVVKRRDSATSCSLDFGHLRNKSGCH 
                   
               
               
                 VELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVAEFLRWNPNLSLRIFTARLYF 
               
               
                 CEDRKAEPEGLRRLHRAGVQIGIMTFKDYFYCWNTFVENRERTFKAWEGLHENSVRLT 
               
               
                 RQLRRTEEP LYEVDDLRDAFRMLGF   
               
               
                 (underline: nuclear localization sequence; double underline: nuclear export signal) 
               
               
                   
               
               
                 Dog AID: 
               
               
                 (SEQ ID NO: 94) 
                   
               
               
                   MDSLLMKQRKFLYHFKNVRWAKGRHETYLC YVVKRRDSATSFSLDFGHLRNKSGCHV 
                   
               
               
                 ELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFAARLYFC 
               
               
                 EDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENREKTFKAWEGLHENSVRLSR 
               
               
                 QLRRILLP LYEVDDLRDAFRTLGL   
               
               
                 (underline: nuclear localization sequence; double underline: nuclear export signal) 
               
               
                   
               
               
                 Bovine AID: 
               
               
                 (SEQ ID NO: 95) 
                   
               
               
                   MDSLLKKQRQFLYQFKNVRWAKGRHETYLC YVVKRRDSPTSFSLDFGHLRNKAGCHV 
                   
               
               
                 ELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFTARLYFC 
               
               
                 DKERKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLS 
               
               
                 RQLRRILLP LYEVDDLRDAFRTLGL   
               
               
                 (underline: nuclear localization sequence; double underline: nuclear export signal) 
               
               
                   
               
               
                 Rat AID 
               
               
                 (SEQ ID NO: 96) 
                   
               
               
                   MAVGSKPKAALVGPHWERERIWCFLC STGLGTQQTGQTSRWLRPAATQDPVSPPRSLL 
                   
               
               
                 MKQRKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGYLRNKSGCHVELLFL 
               
               
                 RYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLTGWGALP 
               
               
                 AGEMSPARPSDYFYCWNTFV ENHERTFKAWEGLHENSVRLSRRLRRILLPLYEVDDLR   
               
               
                 
                   DAFRTLGL 
                 
               
               
                 (underline: nuclear localization sequence; double underline: nuclear export signal) 
               
               
                   
               
               
                 Mouse APOBEC-3 
               
               
                 (SEQ ID NO: 97) 
                   
               
               
                 MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLGYAKGRKDTFLCYEVTRKDCDSPVSL 
                   
               
               
                 HHGVFKNKDNI HAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFEC AEQIVRFLATHH 
               
               
                 NLSLDIFSSRLYNVQDPETQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFR 
               
               
                 PWKRLLTNFRYQDSKLQEILRPCYIPVPSSSSSTLSNICLTKGLPETRFCVEGRRMDPLSE 
               
               
                 EEFYSQFYNQRVKHLCYYHRMKPYLCYQLEQFNGQAPLKGCLLSEKGKQ HAEILFLDKI   
               
               
                   RSMELSQVTITCYLTWSPCPNC AWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSL 
               
               
                 WQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLRRIKESWGLQDL 
               
               
                 VNDFGNLQLGPPMS 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Rat APOBEC-3: 
               
               
                 (SEQ ID NO: 98) 
                   
               
               
                 MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNRLRYAIDRKDTFLCYEVTRKDCDSPVSL 
                   
               
               
                 HHGVFKNKDNI HAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFEC AEQVLRFLATH 
               
               
                 HNLSLDIFSSRLYNIRDPENQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFR 
               
               
                 PWKKLLTNFRYQDSKLQEILRPCYIPVPSSSSSTLSNICLTKGLPETRFCVERRRVHLLSE 
               
               
                 EEFYSQFYNQRVKHLCYYHGVKPYLCYQLEQFNGQAPLKGCLLSEKGKQ HAEILFLDKI   
               
               
                   RSMELSQVIITCYLTWSPCPNC AWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSL 
               
               
                 WQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLHRIKESWGLQDL 
               
               
                 VNDFGNLQLGPPMS 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Rhesus macaque APOBEC-3G: 
               
               
                 (SEQ ID NO: 99) 
                   
               
               
                 
                   MVEPMDPRTFVSNFNNRPILSGLNTVWLCCEVKTKDPSGPPLDAKIFQGKVYSKAKY H   
                 
                   
               
               
                     PEM     RFLRWFHKWRQLHHDQEYKVTWYVSWSPCTRC ANSVATFLAKDPKVTLTIFVARLY 
               
               
                 YFWKPDYQQALRILCQKRGGPHATMKIMNYNEFQDCWNKFVDGRGKPFKPRNNLPKH 
               
               
                 YTLLQATLGELLRHLMDPGTFTSNFNNKPWVSGQHETYLCYKVERLHNDTWVPLNQH 
               
               
                 RGFLRNQAPNIHGFPKGR HAELCFLDLIPFWKLDGQQYRVTCFTSWSPCFSC AQEMAKFIS 
               
               
                 NNEHVSLCIFAARIYDDQGRYQEGLRALHRDGAKIAMMNYSEFEYCWDTFVDRQGRPF 
               
               
                 QPWDGLDEHSQALSGRLRAI 
               
               
                 (italic: nucleic acid editing domain; underline: cytoplasmic localization signal) 
               
               
                   
               
               
                 Chimpanzee APOBEC-3G: 
               
               
                 (SEQ ID NO: 100) 
                   
               
               
                 
                   MKPHFRNPVERMYQDTFSDNFYNRPILSHRNTVWLCYEVKTKGPSRPPLDAKIFRGQV 
                 
                   
               
               
                   Y SKLKY HPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKC TRDVATFLAEDPKVTLTI 
               
               
                 FVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPW 
               
               
                 NNLPKYYILLHIMLGEILRHSMDPPTFTSNFNNELWVRGRHETYLCYEVERLHNDTWVL 
               
               
                 LNQRRGFLCNQAPHKHGFLEGR HAELCFLDVIPFWKLDLHQDYRVTCFTSWSPCFSC AQE 
               
               
                 MAKFISNNKHVSLCIFAARIYDDQGRCQEGLRTLAKAGAKISIMTYSEFKHCWDTFVDH 
               
               
                 QGCPFQPWDGLEEHSQALSGRLRAILQNQGN 
               
               
                 (italic: nucleic acid editing domain; underline: cytoplasmic localization signal) 
               
               
                   
               
               
                 Green monkey APOBEC-3G: 
               
               
                 (SEQ ID NO: 101) 
                   
               
               
                 
                   MNPQIRNMVEQMEPDIFVYYFNNRPILSGRNTVWLCYEVKTKDPSGPPLDANIFQGKLY 
                 
                   
               
               
                   P EAKD HPEMKFLHWFRKWRQLHRDQEYEVTWYVSWSPCTRC ANSVAYFLAEDPKVTLTIF 
               
               
                 VARLYYFWKPDYQQALRILCQERGGPHATMKIMNYNEFQHCWNEFVDGQGKPFKPRK 
               
               
                 NLPKHYTLLHATLGELLRHVMDPGTFTSNFNNKPWVSGQRETYLCYKVERSHNDTWV 
               
               
                 LLNQHRGFLRNQAPDRHGFPKGR HAELCFLDLIPFWKLDDQQYRVTCFTSWSPCFSC AQK 
               
               
                 MAKFISNNKHVSLCIFAARIYDDQGRCQEGLRTLHRDGAKIAVMNYSEFEYCWDTFVD 
               
               
                 RQGRPFQPWDGLDEHSQALSGRLRAI 
               
               
                 (italic: nucleic acid editing domain; underline: cytoplasmic localization signal) 
               
               
                   
               
               
                 Human APOBEC-3G: 
               
               
                 (SEQ ID NO: 102) 
                   
               
               
                 
                   MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQV 
                 
                   
               
               
                   YS ELKY HPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKC FRDMAYFLAEDPKVTLTI 
               
               
                 FVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPW 
               
               
                 NNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWV 
               
               
                 EENQRRGFECNQAPGKHGFEEGR HAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSC AQ 
               
               
                 EMAKFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVD 
               
               
                 HQGCPFOPWDGLDEHSQDLSGRLRAILQNQEN 
               
               
                 (italic: nucleic acid editing domain; underline: cytoplasmic localization signal) 
               
               
                   
               
               
                 Human APOBEC-3F: 
               
               
                 (SEQ ID NO: 103) 
                   
               
               
                 MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPRLDAKIFRGQV 
                   
               
               
                 YSQPEH HAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDC VAKLAEFLAEHPNVTLTIS 
               
               
                 AARLYYYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYSEGQPFMPWYKFD 
               
               
                 DNYAFLHRTLKEILRNPMEAMYPHIFYFHFKNLRKAYGRNESWLCFTMEVVKHHSPVS 
               
               
                 WKRGVFRNQVDPETH CHAERCFLSWFCDDILSPNTNYEVTWYTSWSPCPEC AGEVAEF 
               
               
                 LARHSNVNLTIFTARLYYFWDTDYQEGLRSLSQEGASVEIMGYKDFKYCWENFVYND 
               
               
                 DEPFKPWKGLKYNFLFLDSKLQEILE 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Human APOBEC-3B: 
               
               
                 (SEQ ID NO: 104) 
                   
               
               
                 MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGQ 
                   
               
               
                 VYFKPQY HAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDC VAKLAEFLSEHPNVTLTI 
               
               
                 SAARLYYYWERDYRRALCRLSQAGARVTIMDYEEFAYCWENFVYNEGQQFMPWYKF 
               
               
                 DENYAFLHRTLKEILRYLMDPDTFTFNFNNDPLVLRRRQTYLCYEVERLDNGTWVLMD 
               
               
                 QHMGFECNEARNEECGFY GRHAELRFLDLVPSLQLDPAQIYRVTWFLSWSPCFSWGC AGE 
               
               
                 VRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVY 
               
               
                 RQGCPFQPWDGLEEHSQALSGRLRAILQNQGN 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Rat APOBEC-3B: 
               
               
                 (SEQ ID NO: 105) 
                   
               
               
                 MQPQGLGPNAGMGPVCLGCSHRRPYSPIRNPLKKLYQQTFYFHFKNVRYAWGRKNNF 
                   
               
               
                 LCYEVNGMDCALPVPLRQGVFRKQGHIHAELCFIYWFHDKVLRVLSPMEEFKVTWYM 
               
               
                 SWSPCSKCAEQVARFLAAHRNLSLAIFSSRLYYYLRNPNYQQKLCRLIQEGVHVAAMD 
               
               
                 LPEFKKCWNKFVDNDGQPFRPWMRLRINFSFYDCKLQEIFSRMNLLREDVFYLQFNNS 
               
               
                 HRVKPVQNRYYRRKSYLCYQLERANGQEPLKGYLLYKKGEQHVEILFLEKMRSMELS 
               
               
                 QVRITCYLTWSPCPNCARQLAAFKKDHPDLILRIYTSRLYFWRKKFQKGLCTLWRSGIH 
               
               
                 VDVMDLPQFADCWTNFVNPQRPFRPWNELEKNSWRIQRRLRRIKESWGL 
               
               
                   
               
               
                 Bovine APOBEC-3B: 
               
               
                 (SEQ ID NO: 106) 
                   
               
               
                 DGWEVAFRSGTVLKAGVLGVSMTEGWAGSGHPGQGACVWTPGTRNTMNLLREVLFK 
                   
               
               
                 QQFGNQPRVPAPYYRRKTYLCYQLKQRNDLTLDRGCFRNKKQRHAERFIDKINSLDLN 
               
               
                 PSQSYKIICYITWSPCPNCANELVNFITRNNHLKLEIFASRLYFHWIKSFKMGLQDLQNA 
               
               
                 GISVAVMTHTEFEDCWEQFVDNQSRPFQPWDKLEQYSASIRRRLQRILTAPI 
               
               
                   
               
               
                 Chimpanzee APOBEC-3B: 
               
               
                 (SEQ ID NO: 107) 
                   
               
               
                 MNPQIRNPMEWMYQRTFYYNFENEPILYGRSYTWLCYEVKIRRGHSNLLWDTGVFRG 
                   
               
               
                 QMYSQPEHHAEMCFLSWFCGNQLSAYKCFQITWFVSWTPCPDCVAKLAKFLAEHPNV 
               
               
                 TLTISAARLYYYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYNEGQPFMPW 
               
               
                 YKFDDNYAFLHRTLKEIIRHLMDPDTFTFNFNNDPLVLRRHQTYLCYEVERLDNGTWV 
               
               
                 LMDQHMGFLCNEAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSW 
               
               
                 GCAGQVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYC 
               
               
                 WDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQVRASSLCMVPHRPPPPPQSPGPCLP 
               
               
                 LCSEPPLGSLLPTGRPAPSLPFLLTASFSFPPPASLPPLPSLSLSPGHLPVPSFHSLTSCSIQP 
               
               
                 PCSSRIRETEGWASVSKEGRDLG 
               
               
                   
               
               
                 Human APOBEC-3C: 
               
               
                 (SEQ ID NO: 108) 
                   
               
               
                 MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVFRN 
                   
               
               
                 QVDSETH CHAERCFLSWFCDDILSPNTKYQVTWYTSWSPCPDC AGEVAEFLARHSNVNLT 
               
               
                 IFTARLYYFQYPCYQEGLRSLSQEGVAVEIMDYEDFKYCWENFVYNDNEPFKPWKGLK 
               
               
                 TNFRLLKRRLRESLQ 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Gorilla APOBEC-3C 
               
               
                 (SEQ ID NO: 109) 
                   
               
               
                 MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVFRN 
                   
               
               
                 QVDSETHCHAERCFLSWECDDILSPNTNYOVTWYTSWSPCPECAGEVAEFLARHSNVNLTI 
               
               
                 FTARLYYFQDTDYQEGLRSLSQEGVAVKIMDYKDFKYCWENFVYNDDEPFKPWKGLK 
               
               
                 YNFRFLKRRLQEILE 
               
               
                   
               
               
                 Human APOBEC-3 A: 
               
               
                 (SEQ ID NO: 110) 
                   
               
               
                 MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQ 
                   
               
               
                 AKNEECGFYGR HAELRFLDLVPSLQLDPAQIYRVTWFLSWSPCFSWGC AGEVRAFEQENY 
               
               
                 HVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQP 
               
               
                 WDGLDEHSQALSGRLRAILQNQGN 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Rhesus macaque APOBEC-3A: 
               
               
                 (SEQ ID NO: 111) 
                   
               
               
                 MDGSPASRPRHLMDPNTFTFNFNNDLSVRGRHQTYLCYEVERLDNGTWVPMDERRGF 
                   
               
               
                 LCNKAKNVPCGDYGC HVELRFLCEVPSWQLDPAQTYRVTWFISWSPC FRRGCAGQVRVF 
               
               
                 LQENKHVRLRIFAARIYDYDPLYQEALRTLRDAGAQVSIMTYEEFKHCWDTFVDRQGR 
               
               
                 PFQPWDGLDEHSQALSGRLRAILQNQGN 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Bovine APOBEC-3A: 
               
               
                 (SEQ ID NO: 112) 
                   
               
               
                 MDEYTFTENFNNQGWPSKTYLCYEMERLDGDATIPLDEYKGFVRNKGLDQPEKPC HAE   
                   
               
               
                   LYFLGKIHSWNLDRNQHYRLTCFISWSPC YDCAQKLTTFLKENHHISLHILASRIYTHNRFG 
               
               
                 CHQSGLCELQAAGARITIMTFEDFKHCWETFVDHKGKPFQPWEGLNVKSQALCTELQA 
               
               
                 ILKTQQN 
               
               
                 (italic: nucleic acid editing domain 
               
               
                   
               
               
                 Human APOBEC-3H: 
               
               
                 (SEQ ID NO: 113) 
                   
               
               
                 MALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTPQNGSTPTRGYFENKKKC HAEICF   
                   
               
               
                   INEIKSMGLDETQCYQVTCYLTWSPCSSC AWELVDFIKAHDHLNLGIFASRLYYHWCKPQ 
               
               
                 QKGLRLLCGSQVPVEVMGFPKFADCWENFVDHEKPLSFNPYKMLEELDKNSRAIKRRL 
               
               
                 ERIKIPGVRAQGRYMDILCDAEV 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Rhesus macaque APOBEC-3H: 
               
               
                 (SEQ ID NO: 114) 
                   
               
               
                 MALLTAKTFSLQFNNKRRVNKPYYPRKALLCYQLTPQNGSTPTRGHLKNKKKDHAEIR 
                   
               
               
                 FINKIKSMGLDETQCYQVTCYLTWSPCPSCAGELVDFIKAHRHLNLRIFASRLYYHWRP 
               
               
                 NYQEGLLLLCGSQVPVEVMGLPEFTDCWENFVDHKEPPSFNPSEKLEELDKNSQAIKRR 
               
               
                 LERIKSRSVDVLENGLRSLQLGPVTPSSSIRNSR 
               
               
                   
               
               
                 Human APOBEC-3D: 
               
               
                 (SEQ ID NO: 115) 
                   
               
               
                 MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGP 
                   
               
               
                 VLPKRQSNHRQEVYFRFEN HAEMCFLSWFCGNRLPANRRFQITWFVSWNPC LPCVVKVT 
               
               
                 KFLAEHPNVTLTISAARLYYYRDRDWRWVLLRLHKAGARVKIMDYEDFAYCWENFVC 
               
               
                 NEGQPFMPWYKFDDNYASLHRTLKEILRNPMEAMYPHIFYFHFKNLLKACGRNESWLC 
               
               
                 FTMEVTKHHSAVFRKRGVFRNQVDPETHC HAERCFLSWFCDDILSPNTNYEVTWYTSWSP   
               
               
                   CPEC AGEVAEFLARHSNVNLTIFTARLCYFWDTDYQEGLCSLSQEGASVKIMGYKDFV 
               
               
                 SCWKNFVYSDDEPFKPWKGLQTNFRLLKRRLREILQ 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Human APOBEC-1: 
               
               
                 (SEQ ID NO: 116) 
                   
               
               
                 MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTT 
                   
               
               
                 NHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARL 
               
               
                 FWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWM 
               
               
                 MLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR 
               
               
                   
               
               
                 Mouse APOBEC-1: 
               
               
                 (SEQ ID NO: 117) 
                   
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSVWRHTSQNTS 
                   
               
               
                 NHVEVNFLEKFTTERYFRPNTRCSITWFLSWSPCGECSRAITEFLSRHPYVTLFIYIARLY 
               
               
                 HHTDQRNRQGLRDLISSGVTIQIMTEQEYCYCWRNFVNYPPSNEAYWPRYPHLWVKLY 
               
               
                 VLELYCIILGLPPCLKILRRKQPQLTFFTITLQTCHYQRIPPHLLWATGLK 
               
               
                   
               
               
                 Rat APOBEC-1: 
               
               
                 (SEQ ID NO: 118) 
                   
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNK 
                   
               
               
                 HVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHH 
               
               
                 ADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL 
               
               
                 ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK 
               
               
                   
               
               
                 Human APOBEC-2: 
               
               
                 (SEQ ID NO: 119) 
                   
               
               
                 MAQKEEAAVATEAASQNGEDLENLDDPEKLKELIELPPFEIVTGERLPANFFKFQFRNV 
                   
               
               
                 EYSSGRNKTFLCYVVEAQGKGGQVQASRGYLEDEHAAAHAEEAFFNTILPAFDPALRY 
               
               
                 NVTWYVSSSPCAACADRIIKTLSKTKNLRLLILVGRLFMWEEPEIQAALKKLKEAGCKL 
               
               
                 RIMKPQDFEYVWQNFVEQEEGESKAFQPWEDIQENFLYYEEKLADILK 
               
               
                   
               
               
                 Mouse APOBEC-2: 
               
               
                 (SEQ ID NO: 120) 
                   
               
               
                 MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFRNV 
                   
               
               
                 EYSSGRNKTFLCYVVEVQSKGGQAQATQGYLEDEHAGAHAEEAFFNTILPAFDPALKY 
               
               
                 NVTWYVSSSPCAACADRILKTLSKTKNLRLLILVSRLFMWEEPEVQAALKKLKEAGCK 
               
               
                 LRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK 
               
               
                   
               
               
                 Rat APOBEC-2: 
               
               
                 (SEQ ID NO: 121) 
                   
               
               
                 MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFRNV 
                   
               
               
                 EYSSGRNKTFLCYVVEAQSKGGQVQATQGYLEDEHAGAHAEEAFFNTILPAFDPALKY 
               
               
                 NVTWYVSSSPCAACADRILKTLSKTKNLRLLILVSRLFMWEEPEVQAALKKLKEAGCK 
               
               
                 LRIMKPQDFEYLWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK 
               
               
                   
               
               
                 Bovine APOBEC-2: 
               
               
                 (SEQ ID NO: 122) 
                   
               
               
                 MAQKEEAAAAAEPASQNGEEVENLEDPEKLKELIELPPFEIVTGERLPAHYFKFQFRNV 
                   
               
               
                 EYSSGRNKTFLCYVVEAQSKGGQVQASRGYLEDEHATNHAEEAFFNSIMPTFDPALRY 
               
               
                 MVTWYVSSSPCAACADRIVKTLNKTKNLRLLILVGRLFMWEEPEIQAALRKLKEAGCR 
               
               
                 LRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK 
               
               
                   
               
               
                   Petromyzon marinus  CDA1 (pmCDAl): 
               
               
                 (SEQ ID NO: 123) 
                   
               
               
                 MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNK 
                   
               
               
                 PQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRG 
               
               
                 NGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQ 
               
               
                 LNENRWLEKTLKRAEKRRSELSFMIQVKILHTTKSPAV 
               
               
                   
               
               
                 Human APOBEC3G D316R D317R 
               
               
                 (SEQ ID NO: 124) 
                   
               
               
                 MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQ 
                   
               
               
                 VYSELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDP 
               
               
                 KVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKFNYDEFQHCWSKFVYSQ 
               
               
                 RELFEPWNNLPKYYILLHFMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVER 
               
               
                 MHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTC 
               
               
                 FTSWSPCFSCAQEMAKFISKKHVSLCIFTARIYRRQGRCQEGLRTLAEAGAKISFT 
               
               
                 YSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN 
               
               
                   
               
               
                 Human APOBEC3G chain A: 
               
               
                 (SEQ ID NO: 125) 
                   
               
               
                 MDPPTFTFNFNNEPWWGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHG 
                   
               
               
                 FLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCI 
               
               
                 FTARIYDDQGRCQEGLRTLAEAGAKISFTYSEFKHCWDTFVDHQGCPFQPWDGLD 
               
               
                 EHSQDLSGRLRAILQ 
               
               
                   
               
               
                 Human APOBEC3G chain A D120R D121R: 
               
               
                 (SEQ ID NO: 126) 
                   
               
               
                 MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHG 
                   
               
               
                 FLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCI 
               
               
                 FTARIYRRQGRCQEGLRTLAEAGAKISFMTYSEFKHCWDTFVDHQGCPFQPWDGLDE 
               
               
                 HSQDLSGRLRAILQ 
               
            
           
         
       
     
     Some aspects of the present disclosure are based on the recognition that modulating the deaminase domain catalytic activity of any of the fusion proteins described herein, for example by making point mutations in the deaminase domain, affect the processivity of the fusion proteins (e.g., base editors). For example, mutations that reduce, but do not eliminate, the catalytic activity of a deaminase domain within a base editing fusion protein can make it less likely that the deaminase domain will catalyze the deamination of a residue adjacent to a target residue, thereby narrowing the deamination window. The ability to narrow the deamination window can prevent unwanted deamination of residues adjacent to specific target residues, which can decrease or prevent off-target effects. 
     For example, in some embodiments, an APOBEC deaminase incorporated into a base editor can comprise one or more mutations selected from the group consisting of H121X, H122X, R126X, R126X, R118X, W90X, W90X, and R132X of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase, wherein X is any amino acid. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise one or more mutations selected from the group consisting of H121R, H122R, R126A, R126E, R118A, W90A, W90Y, and R132E of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. 
     In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise one or more mutations selected from the group consisting of D316X, D317X, R320X, R320X, R313X, W285X, W285X, R326X of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase, wherein X is any amino acid. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising one or more mutations selected from the group consisting of D316R, D317R, R320A, R320E, R313A, W285A, W285Y, R326E of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. 
     In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise a H121R and a H122R mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a R126A mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a R126E mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a R118A mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W90A mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W90Y mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a R132E mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W90Y and a R126E mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a R126E and a R132E mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W90Y and a R132E mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W90Y, R126E, and R132E mutation of rAPOBEC1, or one or more corresponding mutations in another APOBEC deaminase. 
     In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a D316R and a D317R mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, any of the fusion proteins provided herein comprise an APOBEC deaminase comprising a R320A mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a R320E mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a R313A mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W285A mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W285Y mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a R326E mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W285Y and a R320E mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a R320E and a R326E mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W285Y and a R326E mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. In some embodiments, an APOBEC deaminase incorporated into a base editor can comprise an APOBEC deaminase comprising a W285Y, R320E, and R326E mutation of hAPOBEC3G, or one or more corresponding mutations in another APOBEC deaminase. 
     A number of 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. 
     Details of C to T nucleobase editing proteins are described in International PCT Application No. PCT/US2016/058344 (WO2017/070632) 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. 
     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, I157F, 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. 
     TadA 
     In particular embodiments, the TadA is any one of the TadA described in PCT/US2017/045381 (WO2018/027078), which is incorporated herein by reference in its entirety. 
     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. The relevant sequences follow: 
     MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTA HAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGA AGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 127), which is termed “the TadA reference sequence” or wild type TadA (TadA(wt)). 
     
       
         
           
               
            
               
                 TadA7.10: 
               
               
                 (SEQ ID NO: 128) 
               
               
                 MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG 
               
               
                   
               
               
                 LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG 
               
               
                   
               
               
                 RVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFR 
               
               
                   
               
               
                 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 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: 129) 
               
               
                 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: 130) 
               
               
                 MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRET 
               
               
                   
               
               
                 LQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIP 
               
               
                   
               
               
                 RVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFK 
               
               
                   
               
               
                 NLRANKKSTN 
               
               
                   
               
               
                   Bacillus subtilis  TadA: 
               
               
                 (SEQ ID NO: 131) 
               
               
                 MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRS 
               
               
                   
               
               
                 IAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVF 
               
               
                   
               
               
                 GAFDPKGGC SGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELR 
               
               
                   
               
               
                 KKKKAARKNLSE 
               
               
                   
               
               
                   Salmonella typhimurium  ( S. typhimurium ) TadA: 
               
               
                 (SEQ ID NO: 132) 
               
               
                 MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHR 
               
               
                   
               
               
                 VIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVM 
               
               
                   
               
               
                 CAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRD 
               
               
                   
               
               
                 ECATLLSDFFRMRRQEIKALKKADRAEGAGPAV 
               
               
                   
               
               
                   Shewanella putrefaciens  ( S. putrefaciens ) TadA: 
               
               
                 (SEQ ID NO: 133) 
               
               
                 MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTA 
               
               
                   
               
               
                 HAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGA 
               
               
                   
               
               
                 RDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEK 
               
               
                   
               
               
                 KALKLAQRAQQGIE 
               
               
                   
               
               
                   Haemophilus influenzae  F3031 ( H. influenzae ) TadA: 
               
               
                 (SEQ ID NO: 134) 
               
               
                 MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWN 
               
               
                   
               
               
                 LSIVQSDPTAHAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILH 
               
               
                   
               
               
                 SRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLS 
               
               
                   
               
               
                 TFFQKRREEKKIEKALLKSLSDK 
               
               
                   
               
               
                   Caulobacter crescentus  ( C. crescentus ) TadA: 
               
               
                 (SEQ ID NO: 135) 
               
               
                 MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGN 
               
               
                   
               
               
                 GPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISH 
               
               
                   
               
               
                 ARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLR 
               
               
                   
               
               
                 GFFRARRKAKI 
               
               
                   
               
               
                   Geobacter sulfurreducens  ( G. sulfurreducens ) TadA: 
               
               
                 (SEQ ID NO: 136) 
               
               
                 MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHN 
               
               
                   
               
               
                 LREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIIL 
               
               
                   
               
               
                 ARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLS 
               
               
                   
               
               
                 DFFRDLRRRKKAKATPALFIDERKVPPEP 
               
            
           
         
       
     
     An embodiment of  E. Coli  TadA (ecTadA) includes the following: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 137) 
               
               
                 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. 
     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 E55V; D108N, A106V, and D147Y; D108N, E55V, and D147Y; A106V, E55V, and D 147Y; and D108N, A106V, E55V, 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, 1951, 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, M611, 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, R126X, 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, M611, 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, R126W, 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, R24W, 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 I157X 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 I157F 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, R07K, 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, R07K, 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, S146X, 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_R24W_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), 
     (D103A_D104N), 
     (G22P_D103A_D104N), 
     (G22P_D103A_D104N_S138 A), 
     (D103A_D104N_S138A), 
     (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). 
     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 a naturally-occurring cytidine deaminase that includes one or more mutations corresponding to any of the mutations provided herein. 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 cytidine deaminase that corresponds to any of the mutations described herein. 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. It should be appreciated that cytidine 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 cytidine 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 cytidine deaminases provided herein. In some embodiments, the cytidine 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. 
     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 D317R mutation. In some embodiments, the deaminase is a fragment of the human APOBEC3G and comprising mutations corresponding to the D316R 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. 
     Cas9 Domains with Reduced 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); Nishimasu, H., et al., “Engineered CRISPR-Cas9 nuclease with expanded targeting space”  Science.  2018 Sep. 21; 361(6408):1259-1262, Chatterjee, P., et al., Minimal PAM specificity of a highly similar SpCas9 ortholog”  Sci Adv.  2018 Oct. 24; 4(10):eaau0766. doi: 10.1126/sciadv.aau0766, the entire contents of each are hereby incorporated by reference. Several PAM variants are described in Table 1 below. Several non-limiting examples of PAM variants are described at Table 1 below: 
                     TABLE 1                  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           SpyMacCas9   NAA           Cpfl   5′ (TTTV)                        
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 SERPINA1). 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. SERPINA1). 
     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 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. 
     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 cases, 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 a 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 cases, 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 cases, uracil DNA glyocosylase (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 cases, 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. 
     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 cases, 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). 
     Base Editor System 
     The base editor system provided herein comprises the steps of: (a) contacting a target nucleotide sequence of a polynucleotide (e.g., a double-stranded DNA or RNA, a single-stranded DNA or RNA) of a subject with a base editor system comprising a nucleobase editor (e.g., an adenosine base editor or a cytidine 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 the target region; (c) converting a first nucleobase of the target nucleobase pair in a single strand of the target region to a second nucleobase; and (d) cutting no more than one strand of the 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, the 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 a new 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 a cytosine base editor (CBE). 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 cases, a deaminase domain can be a cytosine deaminase or a cytidine deaminase. In some embodiments, the terms “cytosine deaminase” and “cytidine deaminase” can be used interchangeably. In some cases, a deaminase domain can be an adenine deaminase or an adenosine deaminase. 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. 
     In some embodiments, a nucleobase editor system may comprise more than one base editing component. For example, a nucleobase editor system may include more than one deaminase. In some embodiments, a nuclease base editor system may include one or more cytidine deaminase and/or 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 nucleobase component 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, 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 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 a 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 a 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 a RNA recognition motif. 
     In some embodiments, the base editor inhibits base excision repair 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 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. 
     In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, non-limiting exemplary CBE is BE1 (APOBEC1-XTEN-dCas9), BE2 (APOBEC1-XTEN-dCas9-UGI), BE3 (APOBEC1-XTEN-dCas9(A840H)-UGI), BE3-Gam, saBE3, saBE4-Gam, BE4, BE4-Gam, saBE4, or saB4E-Gam. BE4 extends the APOBEC1-Cas9n(D10A) linker to 32 amino acids and the Cas9n-UGI linker to 9 amino acids, and appends a second copy of UGI to the C terminus of the construct with another 9 amino acid linker into a single base editor construct. The base editors saBE3 and saBE4 have the  S. pyogene  Cas9n(D10A) replaced with the smaller  S. aureus  Cas9n(D10A). BE3-Gam, saBE3-Gam, BE4-Gam, and saBE4-Gam have 174 residues of Gam protein fused to the N-terminus of BE3, saBE3, BE4, and saBE4 via the 16 amino acid XTEN linker. 
     In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the adenosine base editor can deaminate adenine in DNA. In some embodiments, ABE is generated by replacing APOBEC1 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: 138)) 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 I157F). 
     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 2. 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 2. 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, ABE7.9, or ABE7.10, as shown in below Table 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Genotypes of ABEs 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 23 
                 26 
                 36 
                 37 
                 48 
                 49 
                 51 
                 72 
                 84 
                 87 
                 105 
                 108 
                 123 
                 125 
                 142 
                 145 
                 147 
                 152 
                 155 
                 156 
                 157 
                 16 
               
               
                   
               
               
                 ABE0.1 
                 W 
                 R 
                 H 
                 N 
                 P 
                   
                 R 
                 N 
                 L 
                 S 
                 A 
                 D 
                 H 
                 G 
                 A 
                 S 
                 D 
                 R 
                 E 
                 1 
                 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 
                 I 
                 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 
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                 ABE7.10 
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     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 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 cases, 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., a cytidine deaminase domain or 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 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. In some embodiments, a linker joins a dCas9 and a second domain (e.g., cytidine deaminase, 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: 16), 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: 18), (GGGS)n (SEQ ID NO: 19), (GGGGS)n (SEQ ID NO: 20), and (G)n (SEQ ID NO: 21), to more rigid linkers of the form (EAAAK)n (SEQ ID NO: 22), (GGS)n (SEQ ID NO: 23), SGSETPGTSESATPES (SEQ ID NO: 16) (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 (SEQ ID NO: 24), or a combination of any of these, in order to achieve the optimal length for activity for the nucleobase editor, 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 comprises a (GGS)n motif, wherein n is 1, 3, or 7 (SEQ ID NO: 139). 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: 16). 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: 25), PAPAPA (SEQ ID NO: 26), PAPAPAP (SEQ ID NO: 27), PAPAPAPA (SEQ ID NO: 28), P(AP) 4  (SEQ ID NO: 29), P(AP) 7  (SEQ ID NO: 30), P(AP) 10  (SEQ ID NO: 31) (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. 
     The domains of the base editor disclosed herein can be arranged in any order. Non-limiting examples of a base editor comprising a fusion protein comprising e.g., a polynucleotide-programmable nucleotide-binding domain and a deaminase domain can be arranged as following:
         NH 2 -[nucleobase editing domain]-Linker1-[e.g., Cas9 derived domain]-COOH;   NH 2 -[e.g., cytidine deaminase]-Linker1-[e.g., Cas9 derived domain]-COOH;   NH 2 -[e.g., cytidine deaminase]-Linker1-[e.g., Cas9 derived domain]-Linker2-[UGI]-COOH;   NH 2 -[e.g., APOBEC]-Linker1-[e.g., Cas9 derived domain]-COOH;   NH 2 -[e.g., cytidine deaminase]-Linker1-[e.g., Cas9 derived domain]-COOH;   NH 2 -[e.g., APOBEC]-Linker1-[e.g. Cas9 derived domain]-COOH;   NH 2 -[e.g., APOBEC]-Linker1-[e.g. Cas9 derived domain]-Linker2-[UGI]-COOH   NH 2 -[e.g., adenosine deaminase]-[e.g., Cas9 derived domain]-COOH;   NH 2 -[e.g., Cas9 derived domain]-[e.g., adenosine deaminase]-COOH;   NH 2 -[e.g., adenosine deaminase]-[e.g., Cas9 derived domain]-[inosine BER inhibitor]-COOH;   NH 2 -[e.g., adenosine deaminase]-[inosine BER inhibitor]-[e.g., Cas9 derived domain]-COOH;   NH 2 -[inosine BER inhibitor]-[e.g., adenosine deaminase]-[e.g., Cas9 derived domain]-COOH;   NH 2 -[e.g., Cas9 derived domain]-[e.g., adenosine deaminase]-[inosine BER inhibitor]-COOH;   NH 2 -[e.g., Cas9 derived domain]-[inosine BER inhibitor]-[e.g., adenosine deaminase]-COOH; or   NH 2 -[inosine BER inhibitor]-[e.g., Cas9 derived domain]-[e.g., adenosine deaminase]-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:
         NH 2 -[APOBEC1]-Linker1-[Cas9(D10A)]-Linker2-[UGI]-COOH;   NH 2 -[CDA1]-Linker1-[Cas9(D10A)]-Linker2-[UGI]-COOH;   NH 2 -[AID]-Linker1-[Cas9(D10A)]-Linker2-[UGI]-COOH;   NH 2 -[APOBEC1]-Linker1-[Cas9(D10A)]-Linker2-[SSB]-COOH;   NH 2 -[UGI]-Linker1-[ABOBEC1]-Linker2-[Cas9(D10A)]-COOH;   NH 2 -[APOBEC1]-Linker1-[Cas9(D10A)]-Linker2-[UGI]-Linker3-[UGI]-COOH;   NH 2 -[Cas9(D10A)]-Linker1-[CDA1]-Linker2-[UGI]-COOH;   NH 2 -[Gam]-Linker1-[APOBEC1]-Linker2-[Cas9(D10A)]-Linker3-[UGI]-COOH;   NH 2 -[Gam]-Linker1-[APOBEC1]-Linker2-[Cas9(D10A)]-Linker3-[UGI]-Linker4-[UGI]-COOH;   NH 2 -[APOBEC1]-Linker1-[dCas9(D10A, H840A)]-Linker2-[UGI]-COOH; or   NH 2 -[APOBEC1]-Linker1-[dCas9(D10A, H840A)]-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 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 a deaminase domain (e.g., cytidine deaminase and/or adenosine deaminase), a uracil glycosylase inhibitor (UGI) domain, epitope tags, reporter gene sequences, and/or protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Additional domains can be a heterologous functional domain. Such heterologous functional domains can confer a function activity, such as DNA methylation, DNA damage, DNA repair, 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 conferred can include methyltransferase activity, demethylase activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, remodeling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthase activity, synthetase activity, and demyristoylation activity, or any combination thereof. 
     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 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 system as provided herein provides a new way to edit 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 base editors provided herein are capable of modifying a specific nucleotide base without generating a significant proportion of indels. The term “indel(s)”, 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 target nucleotide sequence. In certain embodiments, any of the base editors provided herein are capable of generating a greater proportion of intended modifications (e.g., point mutations or deaminations) versus indels. 
     In some embodiments, any of base editor system provided herein results 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. 
     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. 
     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, the base editors provided herein are capable of generating a ratio of intended point 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 point 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 8.5:1, at least 9:1, at least 10:1, at least 11:1, at least 12:1, at least 13:1, at least 14:1, 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. 
     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 require 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. 
     The methods provided herein comprises the steps of: (a) contacting a target nucleotide sequence of a polynucleotide of a subject (e.g., a double-stranded DNA sequence) with a base editor system comprising a nucleobase editor (e.g., an adenosine base editor or a cytidine 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 the target region; (c) editing a first nucleobase of the target nucleobase pair in a single strand of the target region to a second nucleobase; and (d) cutting no more than one strand of the target region, 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 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. 
     Methods of Using Base Editors 
     The correction of point mutations in disease-associated genes and alleles opens up 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. 
     In some embodiments, the disease is a proliferative disease. In some embodiments, the disease is a genetic disease. In some embodiments, the disease is a neoplastic disease. In some embodiments, the disease is a metabolic disease. In some embodiments, the disease is a lysosomal storage disease. Exemplary suitable diseases and disorders include, without limitation, retinitis pigmentosa (e.g., adRP-PRPF3, adRP-RHO), Usher syndrome type 1F, sickle cell disease, alpha-1 antitrypsin deficiency (A1AD), hepatic porphyria, MCAD deficiency, LAL deficiency, phenylketonuria (PKU), hemochromatosis, Von Gierke disease (GSD1a), Pompe disease (GSDII), Gaucher disease, Hurler syndrome (MPS1), cystic fibrosis, homocystinuria (HCU) or chronic pain. Other diseases that can be treated by correcting a point mutation or introducing a deactivating mutation into a disease-associated gene are known to those of skill in the art, and the disclosure is not limited in this respect. Provided are methods for the treatment of additional diseases or disorders, e.g., diseases or disorders that are associated or caused by a point mutation that can be corrected by deaminase mediated gene editing. Such diseases are described herein, and additional 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. 
     In a certain aspect, methods are provided for the treatment of A1AD, which is associated or caused by a point mutation (e.g., in the SERPINA1 gene encoding the A1AT protein) and can be corrected by deaminase mediated gene editing. 
     It will be understood that the numbering of the specific positions or residues in the respective sequences, e.g., polynucleotide or amino acid sequences of a disease-related gene or its encoded protein, respectively, depends on the particular protein and numbering scheme used. Numbering can be different, e.g., in precursors of a mature protein and the mature protein itself, and differences in sequences from species to species can 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. 
     Provided herein are methods of using the 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 Cas9 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 wherein the 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 wherein the 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 adenine of 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 Cas9 or a Cpf1 protein) 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. 
     Use of Nucleobase Editors to Target Nucleotides in the SERPINA1 Gene 
     The suitability of nucleobase editors that target a nucleotide in the SERPINA1 gene is evaluated as described herein. In one embodiment, a single cell of interest is transfected, transduced, or otherwise modified with a nucleic acid molecule or molecules encoding a nucleobase editor described herein together with a small amount of a vector encoding a reporter (e.g., GFP). These cells can be immortalized human cell lines, such as 293T, K562 or U20S. Alternatively, primary human cells may be used. Cells may also be obtained from a subject or individual, such as from tissue biopsy, surgery, blood, plasma, serum, or other biological fluid. Such cells may be relevant to the eventual cell target, 
     Delivery may be performed using a viral vector as further described below. 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 target gene to detect alterations in the 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., hepatocytes) in conjunction with a guide RNA that is used to target a nucleic acid sequence, e.g., a SERPINA1 polynucleotide harboring AIAD-associated mutations, thereby altering the target gene, i.e., SERPINA1. 
     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 some embodiments, the one or more alterations introduced into the SERPINA1 or SERPINC1 gene are as presented in Tables 3A and 3B infra. 
     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. 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 polynucleotide programmable nucleotide binding domain (e.g., Cas9) 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 can 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. 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 (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, the editing of the plurality of nucleobase pairs in one or more genes result in formation of at least one intended mutation. In some embodiments, the formation of the at least one intended mutation results in a precise correction of a disease causing mutation. It should be appreciated that the characteristics of the multiplex editing of the base editors as described herein can be applied to any of combination of the methods of using the base editor provided herein. 
     Precise Correction of Pathogenic Mutations 
     In some embodiments, the intended mutation is a precise correction of a pathogenic mutation or a disease-causing mutation in a gene associated with a disease or pathology. The pathogenic mutation can be a pathogenic single nucleotide polymorphism (SNP) or can be caused by a SNP. For example, the pathogenic mutation can be an amino acid change in a protein encoded by a gene. In another example, the pathogenic mutation can be a pathogenic SNP in a gene. The precise correction can revert the pathogenic mutation back to its wild-type state. In some embodiments, the pathogenic mutation is a G→A point mutation associated with a disease or disorder, wherein the deamination of the mutant A base with an A-to-G base editor (ABE) results in a sequence that is not associated with a disease or disorder. In some embodiments, the pathogenic mutation is a C→T point mutation. The C→T point mutation can be corrected, for example, by targeting an A-to-G base editor (ABE) to the opposite strand and editing the complement A of the pathogenic T nucleobase. In some embodiments, the pathogenic mutation is a T→C point mutation associated with a disease or disorder, and wherein the deamination of the mutant C base with a C-to-T base editor (BE or CBE) results in a sequence that is not associated with a disease or disorder. In some embodiments, the pathogenic mutation is an A→G point mutation. The A→G point mutation can be corrected, for example, by targeting a CBE to the opposite strand and editing the complement C of the pathogenic G nucleobase. Non-limiting exemplary pathogenic mutations or disease-causing mutations are listed in Tables 3A and 3B herein, along with the base editor that can be used to correct the mutation by editing the pathogenic mutation back to its wild-type state. The indicated base editor can be targeted to a pathogenic SNP, or to the complement of the pathogenic SNP. Details of the nomenclature of the description of mutations and other sequence variations are described in den Dunnen, J. T. and Antonarakis, S. E., “Mutation Nomenclature Extensions and Suggestions to Describe Complex Mutations: A Discussion.” Human Mutation 15:712 (2000), the entire contents of which are incorporated by reference herein. 
     
       
         
           
               
             
               
                 TABLE 3A 
               
             
            
               
                   
               
               
                 Precise correction of pathogenic mutations in 
               
               
                 SERPINA1 or SERPINC1 genes 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Patho- 
                   
                 SEQ 
                   
                   
               
               
                   
                 genic 
                 Base 
                 ID 
                 gRNA Targeting  
                   
               
               
                 Gene 
                 Mutation 
                 Editor 
                 NO: 
                 Sequence 
                 PAM 
               
               
                   
               
               
                 SERPINA1 
                 E342K 
                 ABE 
                 140 
                 GACAAGAAAGGGACUGAAGC 
                 NGC 
               
               
                   
               
               
                 SERPINA1 
                 E342K 
                 ABE 
                 141 
                 AUCGACAAGAAAGGGACUGA 
                 NGC 
               
               
                   
               
               
                 SERPINC1 
                 R48C 
                 ABE 
                 142 
                 ACACACCGGUUGGUGGCCUC 
                 NGG 
               
               
                   
                 (R79C) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3B 
               
             
            
               
                   
               
               
                 Precise correction of pathogenic mutations in disease-associated genes 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 SEQ 
                   
                   
               
               
                   
                 Pathogenic 
                 Base 
                 ID 
                   
                   
               
               
                 Gene 
                 Mutation 
                 Editor 
                 NO: 
                 gRNA Targeting Sequence 
                 PAM 
               
               
                   
               
               
                 ABCA4 
                 A1038V 
                 ABE 
                 143 
                 CUCCAGCUGGACCUCCUCCU 
                 GGG 
               
               
                   
               
               
                 ABCA4 
                 A1038V 
                 ABE 
                 144 
                 UCCCAGGAGGAGGUCCAGCU 
                 NGA 
               
               
                   
               
               
                 ABCA4 
                 L541P 
                 CBE 
                 145 
                 CUCUCCACUGGAGGAAAACA 
                 NGT 
               
               
                   
               
               
                 ABCA4 
                 G1961E 
                 ABE 
                 146 
                 CUGUGUGUCGAAGUUCGCCC 
                 TGG 
               
               
                   
               
               
                 ABCA4 
                 G1961E 
                 ABE 
                 147 
                 UGUGUGUCGAAGUUCGCCCU 
                 GGAG 
               
               
                   
               
               
                 ABCA4 
                 G1961E 
                 ABE 
                 148 
                 GUCGAAGUUCGCCCUGGAGA 
                 NGT 
               
               
                   
               
               
                 ABCA4 
                 G1961E 
                 ABE 
                 149 
                 UGUCGAAGUUCGCCCUGGAG 
                 NGG 
               
               
                   
               
               
                 ABCC6 
                 R1141* 
                 ABE 
                 150 
                 GUUCAGAAUGCCCGGACCAC 
                 NGT 
               
               
                   
               
               
                 ACADM 
                 K329E 
                 CBE 
                 151 
                 UCAACUUCCAUUGCCAUUUC 
                 NGC 
               
               
                   
               
               
                 ACADM 
                 K329E 
                 CBE 
                 152 
                 CUUCCAUUGCCAUUUCAGCC 
                 NGC 
               
               
                   
               
               
                 ADA 
                 G216R 
                 ABE 
                 153 
                 GCCAGGGAGGUGGGCUCGGC 
                 NGA 
               
               
                   
               
               
                 ADA 
                 G216R 
                 ABE 
                 154 
                 CCACGCCAGGGAGGUGGGCU 
                 NGG 
               
               
                   
               
               
                 ADA 
                 Q3* 
                 ABE 
                 155 
                 UCUAGGCCAUGGUGCCCUCG 
                 NGCG 
               
               
                   
               
               
                 AGXT 
                 G170R 
                 ABE 
                 156 
                 GCUUCAGGGAACUCUGCCAC 
                 NGG 
               
               
                   
               
               
                 ARH 
                 Q136* 
                 ABE 
                 157 
                 UGCUAGCUCUGGGCGAUGU 
                 NGC 
               
               
                   
                   
                   
                   
                 A 
                   
               
               
                   
               
               
                 ARSA 
                 P426L 
                 ABE 
                 158 
                 GCAGGGGCUCAUGAGCAGUC 
                 NGA 
               
               
                   
               
               
                 ARSB 
                 Y210C 
                 CBE 
                 159 
                 GAACACAUAUUUUUAUAUC 
                 NGT 
               
               
                   
                   
                   
                   
                 C 
                   
               
               
                   
               
               
                 ASS 
                 G390R 
                 ABE 
                 160 
                 AUGCCACCAGGUUCAUCAAC 
                 NNNRR 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                   
               
               
                 ATP2A2 
                 N767S 
                 CBE 
                 161 
                 CGCUGGACGAGAUGAGGUA 
                 NGG 
               
               
                   
                   
                   
                   
                 G 
                   
               
               
                   
               
               
                 ATP2A2 
                 N767S 
                 CBE 
                 162 
                 CCCCGACGCUGGACGAGAUG 
                 NGG 
               
               
                   
               
               
                 ATP2A2 
                 N767S 
                 CBE 
                 163 
                 ACGCUGGACGAGAUGAGGU 
                 NNGRR 
               
               
                   
                   
                   
                   
                 A 
                 T 
               
               
                   
               
               
                 CBS 
                 T191M 
                 ABE 
                 164 
                 GUGGGCAUCCUCACAAUCUC 
                 NGC 
               
               
                   
               
               
                 CFTR 
                 G551D 
                 ABE 
                 165 
                 CUGAGUGGAGAUCAACGAG 
                 NNGRR 
               
               
                   
                   
                   
                   
                 C 
                 T 
               
               
                   
               
               
                 CFTR 
                 W1282* 
                 ABE 
                 166 
                 CAACAGUGAAGGAAAGCCU 
                 NGG 
               
               
                   
                   
                   
                   
                 U 
                   
               
               
                   
               
               
                 CFTR 
                 R553* 
                 ABE 
                 167 
                 GCUCAUUGACCUCCACUCAG 
                 NNNRR 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                   
               
               
                 CFTR 
                 R117H 
                 ABE 
                 168 
                 CACUCUAUCGCGAUUUAUCU 
                 NGG 
               
               
                   
               
               
                 CHM 
                 R270* 
                 ABE 
                 169 
                 CUCAUCCUUCUCGAAAUGCA 
                 NGA 
               
               
                   
               
               
                 CHM 
                 A117A 
                 CBE 
                 170 
                 CUGCAGCGCACCAGCUUCUU 
                 NGA 
               
               
                   
               
               
                 CLN2 
                 R208* 
                 ABE 
                 171 
                 UAUCACUUACGGAUCACAGA 
                 NGG 
               
               
                   
               
               
                 COCH 
                 G88E 
                 ABE 
                 172 
                 GGGAACCUGUACGAGUCUA 
                 NGC 
               
               
                   
                   
                   
                   
                 U 
                   
               
               
                   
               
               
                 CPT2 
                 S113L 
                 ABE 
                 173 
                 UACCCAAAAUGUAGCUUGU 
                 NGT 
               
               
                   
                   
                   
                   
                 A 
                   
               
               
                   
               
               
                 CX30 
                 T5M 
                 ABE 
                 174 
                 UGCAGCAUCCCCCAAUCCAU 
                 NGCG 
               
               
                   
               
               
                 DFNB59 
                 R183W 
                 ABE 
                 175 
                 UUACCCACAUUGCUUCCCCU 
                 NGA 
               
               
                   
               
               
                 E47 
                 E555K 
                 ABE 
                 176 
                 GCGGAAGCGGGUGCGCGUGC 
                 NGG 
               
               
                   
               
               
                 F11 
                 E117* 
                 ABE 
                 177 
                 UUGGCAUUAUUGAGCACUC 
                 NGG 
               
               
                   
                   
                   
                   
                 U 
                   
               
               
                   
               
               
                 F11 
                 F283L 
                 CBE 
                 178 
                 UGAUCUCUUGGGAGAAGAA 
                 NGG 
               
               
                   
                   
                   
                   
                 C 
                   
               
               
                   
               
               
                 F5 
                 R506Q 
                 ABE 
                 179 
                 GGCAAGGAAUACAGGUAUU 
                 NGT 
               
               
                   
                   
                   
                   
                 U 
                   
               
               
                   
               
               
                 F5 
                 R534Q 
                 CBE 
                 180 
                 UCCUCGCCUGUCCAGGGAUC 
                 NGC 
               
               
                   
               
               
                 F7 
                 A294V 
                 ABE 
                 181 
                 GAGCUCCAGGACCGUGGCGC 
                 NNNRR 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                   
               
               
                 F7 
                 C310F 
                 ABE 
                 182 
                 GCAGGAAGUCCUGGGUCAUC 
                 NGC 
               
               
                   
               
               
                 F7 
                 R304Q 
                 ABE 
                 183 
                 CGUGCCCCAGCUGAUGACCC 
                 NGG 
               
               
                   
               
               
                 F7 
                 Q100R 
                 CBE 
                 184 
                 GCAGUACCGCUCACAGCCGC 
                 NGT 
               
               
                   
               
               
                 F8 
                 R2178C 
                 ABE 
                 185 
                 GUGCAAAUGCUAUAAUGAG 
                 NGG 
               
               
                   
                   
                   
                   
                 U 
                   
               
               
                   
               
               
                 F8 
                 R550C 
                 ABE 
                 186 
                 UAAUAGCAGGUCAGGCACCG 
                 NGG 
               
               
                   
               
               
                 F9 
                 T342M 
                 ABE 
                 187 
                 AUGUUCAUGUAUUCCUUGU 
                 NNNRR 
               
               
                   
                   
                   
                   
                 C 
                 T 
               
               
                   
               
               
                 F9 
                 R294Q 
                 ABE 
                 188 
                 AGCAAAAGCAAAAUGUGAU 
                 NGA 
               
               
                   
                   
                   
                   
                 U 
                   
               
               
                   
               
               
                 F9 
                 G106S 
                 ABE 
                 189 
                 AAUGGCAGCAGUUGCAAGG 
                 NGA 
               
               
                   
                   
                   
                   
                 A 
                   
               
               
                   
               
               
                 F9 
                 A279T 
                 ABE 
                 190 
                 GUUGUCACAGGUAAAUACA 
                 NGA 
               
               
                   
                   
                   
                   
                 C 
                   
               
               
                   
               
               
                 F9 
                 R294* 
                 ABE 
                 191 
                 CAUUUCACUUUUGCUCUGUA 
                 NGT 
               
               
                   
               
               
                 F9 
                 R379Q 
                 ABE 
                 192 
                 UUGACCAAGCCACAUGUCUU 
                 NGA 
               
               
                   
               
               
                 FAH 
                 P261L 
                 ABE 
                 193 
                 CACCACCCACAGAGAGACAG 
                 NGG 
               
               
                   
               
               
                 FGF23 
                 R176Q 
                 ABE 
                 194 
                 CGGCAGCACACCCGGAGCGC 
                 NGA 
               
               
                   
               
               
                 G6PC 
                 Q347* 
                 ABE 
                 195 
                 GACCUAGGCGAGGCAGUAG 
                 NGA 
               
               
                   
                   
                   
                   
                 G 
                   
               
               
                   
               
               
                 G6PC 
                 Q347* 
                 ABE 
                 196 
                 GGACCUAGGCGAGGCAGUA 
                 NGG 
               
               
                   
                   
                   
                   
                 G 
                   
               
               
                   
               
               
                 G6PC 
                 Q347* 
                 ABE 
                 197 
                 AGGACCUAGGCGAGGCAGU 
                 NNGRR 
               
               
                   
                   
                   
                   
                 A 
                 T 
               
               
                   
               
               
                 G6PC 
                 R83C 
                 ABE 
                 198 
                 CAGUAUGGACACUGUCCAAA 
                 NNGRR 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                   
               
               
                 G6PD 
                 S188F 
                 ABE 
                 199 
                 GGAGAAGAUGUGGUUGGAC 
                 NNNRR 
               
               
                   
                   
                   
                   
                 A 
                 T 
               
               
                   
               
               
                 GALNS 
                 R386C 
                 ABE 
                 200 
                 UCGCCACAGUAAUAGAAGA 
                 NGG 
               
               
                   
                   
                   
                   
                 U 
                   
               
               
                   
               
               
                 GALT 
                 Q188R 
                 CBE 
                 201 
                 UUACCCGGCAGUGGGGGUG 
                 NGG 
               
               
                   
                   
                   
                   
                 G 
                   
               
               
                   
               
               
                 GBA 
                 N370S 
                 CBE 
                 202 
                 UACAGGAGGCUCUAGGGUA 
                 NGA 
               
               
                   
                   
                   
                   
                 A 
                   
               
               
                   
               
               
                 GBA 
                 N370S 
                 CBE 
                 203 
                 AGGCUCUAGGGUAAGGACA 
                 NGG 
               
               
                   
                   
                   
                   
                 A 
                   
               
               
                   
               
               
                 GBA 
                 L444P 
                 CBE 
                 204 
                 AACGACCCGGACGCAGUGGC 
                 NNNRR 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                   
               
               
                 GBA 
                 L444P 
                 CBE 
                 205 
                 CGACCCGGACGCAGUGGCAC 
                 NGA 
               
               
                   
               
               
                 GCDH 
                 M263V 
                 ABE 
                 206 
                 GAUGAUCACGCCUGUGGCUG 
                 NGG 
               
               
                   
               
               
                 GCDH 
                 R402W 
                 ABE 
                 207 
                 GUGCCAGAUCACGUGAUACU 
                 NGT 
               
               
                   
               
               
                 GLDC 
                 A389V 
                 ABE 
                 208 
                 AUUCACCAAGAGGGCCUAAA 
                 NGA 
               
               
                   
               
               
                 GLDC 
                 G771R 
                 ABE 
                 209 
                 CCCAUCAGAGUGUAAGUUCU 
                 NGG 
               
               
                   
               
               
                 GLDC 
                 T269M 
                 ABE 
                 210 
                 CCCCUCCAUGUCUGGGUACU 
                 NGA 
               
               
                   
               
               
                 GUCY2D 
                 R838C 
                 ABE 
                 211 
                 GCACACGGAGGAGCUGGAGC 
                 NGG 
               
               
                   
               
               
                 GUSB 
                 L175F 
                 ABE 
                 212 
                 GUGAAUGUGUUGUUGAUGG 
                 NNNRR 
               
               
                   
                   
                   
                   
                 C 
                 T 
               
               
                   
               
               
                 HBB 
                 E26K 
                 ABE 
                 213 
                 UUGGUGGUAAGGCCCUGGG 
                 NGG 
               
               
                   
                   
                   
                   
                 C 
                   
               
               
                   
               
               
                 HBB 
                 E26K 
                 ABE 
                 214 
                 UGGUAAGGCCCUGGGCAGG 
                 NGG 
               
               
                   
                   
                   
                   
                 U 
                   
               
               
                   
               
               
                 HBB 
                 E7K 
                 ABE 
                 215 
                 ACUCCUAAGGAGAAGUCUGC 
                 NGT 
               
               
                   
               
               
                 HMBS 
                 R173W 
                 ABE 
                 216 
                 GCCAGGUGUUGAGGUUUCCC 
                 NGC 
               
               
                   
               
               
                 HPRT1 
                 R51* 
                 ABE 
                 217 
                 AUCACAUCUCAAGCAAGACG 
                 NNNRR 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                   
               
               
                 HPRT1 
                 R170* 
                 ABE 
                 218 
                 UUCAUGGGGUCCUUUUCACC 
                 NGC 
               
               
                   
               
               
                 IDS 
                 G374G 
                 ABE 
                 219 
                 UUCUCACCUGCCUCCGGAAG 
                 NGA 
               
               
                   
               
               
                 IDUA 
                 Q70* 
                 ABE 
                 220 
                 CUGCUAGUCCCAGCUGAGGA 
                 NGT 
               
               
                   
               
               
                 IMPDH1 
                 D226N 
                 ABE 
                 221 
                 ACCAACCUGAAGAAGAACCG 
                 NGA 
               
               
                   
               
               
                 KCNJ2 
                 R218W 
                 ABE 
                 222 
                 UUUUCCAAAGAUUGCCCACU 
                 NGC 
               
               
                   
               
               
                 KRT12 
                 L132P 
                 CBE 
                 223 
                 CAAAAUCCUAAUGAUAGAU 
                 NGC 
               
               
                   
                   
                   
                   
                 U 
                   
               
               
                   
               
               
                 LRRK2 
                 G2019S 
                 ABE 
                 224 
                 ACUACAGCAUUGCUCAGUAC 
                 NGC 
               
               
                   
               
               
                 MECP2 
                 R106W 
                 ABE 
                   
                   
                   
               
               
                   
               
               
                 MECP2 
                 R133C 
                 ABE 
                   
                   
                   
               
               
                   
               
               
                 MECP2 
                 R306C 
                 ABE 
                   
                   
                   
               
               
                   
               
               
                 MECP2 
                 R168* 
                 ABE 
                   
                   
                   
               
               
                   
               
               
                 MECP2 
                 R255* 
                 ABE 
                   
                   
                   
               
               
                   
               
               
                 NAGLU 
                 R297* 
                 ABE 
                 225 
                 CUCUCACAGGAAGAGGCUCC 
                 NGA 
               
               
                   
               
               
                 NAGLU 
                 Y140C 
                 CBE 
                 226 
                 CACGAAAGAGCAGCUUUGCG 
                 NGC 
               
               
                   
               
               
                 OPN1LW 
                 C203R 
                 CBE 
                 227 
                 GACUUCACGCGGCCCAGACG 
                 NGT 
               
               
                   
               
               
                 PAH 
                 R408W 
                 ABE 
                 228 
                 AAGGGCCAAGGUAUUGUGG 
                 NGC 
               
               
                   
                   
                   
                   
                 C 
                   
               
               
                   
               
               
                 PAH 
                 I65T 
                 CBE 
                 229 
                 ACACUGAAUCUAGACCUUCU 
                 NGT 
               
               
                   
               
               
                 PAH 
                 R261Q 
                 ABE 
                 230 
                 CUUCCAAGUCUUCCACUGCA 
                 NNNRR 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                   
               
               
                 PCDH15 
                 R245* 
                 ABE 
                 231 
                 UGGUGGUUCACCUCUCAUUC 
                 AGAT 
               
               
                   
               
               
                 PCDH15 
                 R245* 
                 ABE 
                 232 
                 UGGUGGUGGUUCACCUCUCA 
                 TCAGA 
               
               
                   
                   
                   
                   
                 U 
                 T 
               
               
                   
               
               
                 PCDH15 
                 R245* 
                 ABE 
                 233 
                 UUCACCUCUCAUUCAGAUUU 
                 NGG 
               
               
                   
               
               
                 PDE6A 
                 V685M 
                 ABE 
                 234 
                 AAAGAUCAUGGAUCAGUCU 
                 NGA 
               
               
                   
                   
                   
                   
                 A 
                   
               
               
                   
               
               
                 PDS 
                 L236P 
                 CBE 
                 235 
                 CAGCCAAAGAUUGUCCUCAA 
                 NGT 
               
               
                   
               
               
                 PPOX 
                 R59W 
                 ABE 
                 236 
                 UCCCCAAGGUCCAAGCUCAA 
                 NGA 
               
               
                   
               
               
                 PRNP 
                 E200K 
                 ABE 
                 237 
                 CACCAAGACCGACGUUAAGA 
                 NGA 
               
               
                   
               
               
                 PRNP 
                 M129V 
                 CBE 
                 238 
                 UUCCCAGCACGUAGCCGCCA 
                 NGG 
               
               
                   
               
               
                 PRNP 
                 P102L 
                 ABE 
                 239 
                 CUCAGCUUGUUCCACUGACU 
                 NNGRR 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                   
               
               
                 PRNP 
                 D178N 
                 CBE 
                 240 
                 GUGCACAACUGCGUCAAUAU 
                 NNNRR 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                   
               
               
                 PRPF3 
                 T494M 
                 ABE 
                 241 
                 ACCUUCAUGGGGUCUUGAAC 
                 NGC 
               
               
                   
               
               
                 PRPF8 
                 H2309R 
                 CBE 
                 242 
                 GGGCCUGCGCACCUCGUGGU 
                 NGA 
               
               
                   
               
               
                 RHO 
                 P347L 
                 ABE 
                 243 
                 GUCUUAGGCCAGGGCCACCU 
                 NGC 
               
               
                   
               
               
                 RHO 
                 P347L 
                 ABE 
                 244 
                 UAGGCCAGGGCCACCUGGCU 
                 NGT 
               
               
                   
               
               
                 RHO 
                 D190N 
                 ABE 
                 245 
                 AAUCAACUACUACACGCUCA 
                 NGC 
               
               
                   
               
               
                 RP1 
                 R667* 
                 ABE 
                 246 
                 UCAAGAUUUUUUCUUCUUU 
                 NGC 
               
               
                   
                   
                   
                   
                 U 
                   
               
               
                   
               
               
                 RPE65 
                 R44* 
                 ABE 
                 247 
                 ACAUCAAAGGAGACUGCCGG 
                 NGA 
               
               
                   
               
               
                 RPS19 
                 R62Q 
                 ABE 
                 248 
                 GCGCAGCACCUGUACCUCCG 
                 NGG 
               
               
                   
               
               
                 RS1 
                 R102W 
                 ABE 
                 249 
                 CUGUUGAGCCAGGCCUUGUU 
                 NNNRR 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                   
               
               
                 RS1 
                 R141C 
                 ABE 
                 250 
                 AUGUCACAGCACCCCUGGGU 
                 NNGRR 
               
               
                   
                   
                   
                   
                   
                 T 
               
               
                   
               
               
                 SERPINA1 
                 E342K 
                 ABE 
                 251 
                 GACAAGAAAGGGACUGAAG 
                 NGC 
               
               
                   
                   
                   
                   
                 C 
                   
               
               
                   
               
               
                 SERPINA1 
                 E342K 
                 ABE 
                 252 
                 AUCGACAAGAAAGGGACUG 
                 NGC 
               
               
                   
                   
                   
                   
                 A 
                   
               
               
                   
               
               
                 SERPINC1 
                 R48C 
                 ABE 
                 253 
                 ACACACCGGUUGGUGGCCUC 
                 NGG 
               
               
                   
                 (R79C) 
                   
                   
                   
                   
               
               
                   
               
               
                 SGSH 
                 R74C 
                 ABE 
                 254 
                 GGCGCAGCUGGGAGAGCAGC 
                 NGC 
               
               
                   
               
               
                 SMPD1 
                 L302P 
                 CBE 
                 255 
                 CACCUGUGAGGAAGUUCCUG 
                 NGG 
               
               
                   
               
               
                 SNCA 
                 A53T 
                 ABE 
                 256 
                 UGACAACAGGUAAGCUCCAU 
                 NGT 
               
               
                   
               
               
                 SOD1 
                 A4V 
                 ABE 
                 257 
                 CGACCUUCGUCGCCAUAACU 
                 NGC 
               
               
                   
               
               
                 SOD1 
                 H46R 
                 CBE 
                 258 
                 CAUGAACACGGAAUCCAUGC 
                 NGG 
               
               
                   
               
               
                 SOD1 
                 G37R 
                 ABE 
                 259 
                 UAAAAGACUGACUGAAGGC 
                 NGC 
               
               
                   
                   
                   
                   
                 C 
                   
               
               
                   
               
               
                 TECTA 
                 Y1870C 
                 ABE 
                 260 
                 UCAUGUAUAAAAACACACUC 
                 NGG 
               
               
                   
               
               
                 TTR 
                 V50M/V30M 
                 ABE 
                 261 
                 GGCCAUGCAUGUGUUCAGA 
                 NGG 
               
               
                   
                   
                   
                   
                 A 
                   
               
               
                   
               
               
                 USH1C 
                 V72V 
                 ABE 
                 262 
                 CCAGGUAGAAUAUGAUCAG 
                 NGA 
               
               
                   
                   
                   
                   
                 C 
                   
               
               
                   
               
               
                 USH2a 
                 C759F 
                 ABE 
                 263 
                 GGAUUGAAGAAUUUGUUCA 
                 NGA 
               
               
                   
                   
                   
                   
                 C 
                   
               
               
                   
               
               
                 MTM1 
                 c.1261- 
                 CBE 
                 264 
                 AACUGAUGAAGAUAAUUUG 
                 NNNRR 
               
               
                   
                 10A &gt; G 
                   
                   
                 U 
                 T 
               
               
                   
               
               
                 PAH 
                 IVS10- 
                 ABE 
                 265 
                 UCACUUAGGGCCUACAGUAC 
                 NGC 
               
               
                   
                 11G &gt; A 
                   
                   
                   
                   
               
               
                   
               
               
                 PDS 
                 IVS8, +1 
                 ABE 
                 266 
                 GGGAUGAGUGUGGUGUUCC 
                 NNNRR 
               
               
                   
                 G &gt; A 
                   
                   
                 U 
                 T 
               
               
                   
               
               
                 ARSA 
                 C.459 + 1G &gt; A 
                 ABE 
                 267 
                 CGACCAGAUAGGAACCACCC 
                 NGG 
               
               
                   
               
               
                 * is a stop codon. 
               
            
           
         
       
     
     In some embodiments, the disease or disorder is alpha-1 antitrypsin deficiency (A1AD). In some embodiments, the pathogenic mutation is in the SERPINA1 gene which encodes the A1AT protein. In some embodiments, the mutation of the SERPINA1-encoded A1AT protein is E342K (PiZ allele) ( FIG.  3 A ). In some embodiments, the nucleobase “A” at position 7 of the SERPINA1 allele is edited to “G” to restore the PiZ allele to a wild type allele. ( FIGS.  3 B and  3 C ). 
     Delivery System 
     A base editor disclosed herein can be encoded on a nucleic acid that is contained in a viral vector. Viral vectors can include lentivirus, Adenovirus, Retrovirus, and Adeno-associated viruses (AAVs). Viral vectors can be selected based on the application. For example, AAVs are commonly used for gene delivery in vivo due to their mild immunogenicity. 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. For example, the packaging capacity of the AAVs is ˜4.5 kb including two 145 base inverted terminal repeats (ITRs). 
     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. 
     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 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. 
     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. 
     The disclosed strategies for designing base editors can be useful for generating base editors capable of being packaged into a 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. 
     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 cases, 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). 
     A base editor described herein can therefore be delivered with viral vectors. 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 cases, 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. 
     Non-Viral Delivery of Base Editors 
     Non-viral delivery approaches for base editors are also available. 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 4 (below). 
                     TABLE 4                  Lipids Used for Gene Transfer                         Lipid   Abbreviation   Feature               1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine   DOPC   Helper       1,2-Dioleoyl-sn-glycero-3-   DOPE   Helper       phosphatidylethanolamine               Cholesterol       Helper       N-[1-(2,3-Dioleyloxy)prophyl]N,N,N-   DOTMA   Cationic       trimethylammonium chloride               1,2-Dioleoyloxy-3-trimethylammonium-   DOTAP   Cationic       propane               Dioctadecylamidoglycylspermine   DOGS   Cationic       N-(3-Aminopropyl)-N,N-dimethyl-2,3-   GAP-DLRIE   Cationic       bis(dodecyloxy)-1-propanaminium bromide               Cetyltrimethylammonium bromide   CTAB   Cationic       6-Lauroxyhexyl ornithinate   LHON   Cationic       1-(2,3-Dioleoyloxypropyl)-2,4,6-   2Oc   Cationic       trimethylpyridinium               2,3-Dioleyloxy-N-[2(sperminecarboxamido-   DOSPA   Cationic       ethyl]-N,N-dimethyl-1-propanaminium                trifluoroacetate               1,2-Dioleyl-3-trimethylammonium-propane   DOPA   Cationic       N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-   MDRIE   Cationic       bis(tetradecyloxy)-1-propanaminium bromide               Dimyristooxypropyl dimethyl hydroxyethyl    DMRI   Cationic       ammonium bromide               3β-[N-(N′,N′-Dimethylaminoethane)-   DC-Chol   Cationic       carbamoyl]cholesterol               Bis-guanidium-tren-cholesterol   BGTC   Cationic       1,3-Diodeoxy-2-(6-carboxy-spermyl)-   DOSPER   Cationic       propylamide               Dimethyloctadecylammonium bromide   DDAB   Cationic       Dioctadecylamidoglicylspermidin   DSL   Cationic       rac-[(2,3-Dioctadecyloxypropyl)(2-   CLIP-1   Cationic       hydroxyethyl)]-dimethylammonium chloride               rac-[2(2,3-Dihexadecyloxypropyl-   CLIP-6   Cationic       oxymethyloxy)ethyl]trimethylammoniun                bromide               Ethyldimyristoylphosphatidylcholine   EDMPC   Cationic       1,2-Distearyloxy-N,N-dimethyl-3-   DSDMA   Cationic       aminopropane               1,2-Dimyristoyl-trimethylammonium propane   DMTAP   Cationic       O,O′-Dimyristyl-N-lysyl aspartate   DMKE   Cationic       1,2-Distearoyl-sn-glycero-3-   DSEPC   Cationic       ethylphosphocholine               N-Palmitoyl D-erythro-sphingosyl carbamoyl-   CCS   Cationic       spermine               N-t-Butyl-N0-tetradecyl-3-   diC14-   Cationic       tetradecylaminopropionamidine   amidine           Octadecenolyoxy[ethyl-2-heptadecenyl-3    DOTIM   Cationic       hydroxyethyl] imidazolinium chloride               N1-Cholesteryloxycarbonyl-3,7-diazanonane-   CDAN   Cationic       1,9-diamine               2-(3-[Bis(3-amino-propyl)-   RPR209120   Cationic       amino]propylamino)-N-               ditetradecylcarbamoylme-ethyl-acetamide               1,2-dilinoleyloxy-3-dimethylaminopropane   DLinDMA   Cationic       2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-   DLin-KC2-   Cationic       dioxolane   DMA           dilinoleyl-methyl-4-dimethylaminobutyrate   DLin-MC3-   Cationic           DMA                    
Table 5 lists exemplary polymers for use in gene transfer and/or nanoparticle formulations.
 
                     TABLE 5                  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 6 summarizes delivery methods for a polynucleotide encoding a fusion protein described herein.
 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                   
                   
                 Delivery  
                   
                   
                   
               
               
                   
                   
                 into 
                   
                   
                   
               
               
                   
                   
                 Non- 
                 Duration  
                   
                 Type of 
               
               
                   
                   
                 Dividing 
                 of 
                 Genome 
                 Molecule 
               
               
                 Delivery 
                 Vector/Mode 
                 Cells 
                 Expression 
                 Integration 
                 Delivered 
               
               
                   
               
             
            
               
                 Physical 
                 (e.g.. 
                 YES 
                 Transient 
                 NO 
                 Nucleic  
               
               
                   
                 electroporation, 
                   
                   
                   
                 Acids 
               
               
                   
                 particle gun, 
                   
                   
                   
                 and  
               
               
                   
                 Calcium 
                   
                   
                   
                 Proteins 
               
               
                   
                 Phosphate 
                   
                   
                   
                   
               
               
                   
                 transfection 
                   
                   
                   
                   
               
               
                 Viral 
                 Retrovirus 
                 NO 
                 Stable 
                 YES 
                 RNA 
               
               
                   
                 Lentivirus 
                 YES 
                 Stable 
                 YES/NO  
                 RNA 
               
               
                   
                   
                   
                   
                 with 
                   
               
               
                   
                   
                   
                   
                 modification 
                   
               
               
                   
                 Adenovirus 
                 YES 
                 Transient 
                 NO 
                 DNA 
               
               
                   
                 Adeno- 
                 YES 
                 Stable 
                 NO 
                 DNA 
               
               
                   
                 Associated 
                   
                   
                   
                   
               
               
                   
                 Virus (AAV) 
                 YES 
                 Very 
                 NO 
                 DNA 
               
               
                   
                 Vaccinia Virus 
                   
                 Transient 
                   
                   
               
               
                   
                 Herpes Simplex 
                 YES 
                 Stable 
                 NO 
                 DNA 
               
               
                   
                 Virus 
                   
                   
                   
                   
               
               
                 Non-Viral 
                 Cationic 
                 YES 
                 Transient 
                 Depends on 
                 Nucleic  
               
               
                   
                 Liposomes 
                   
                   
                 what is 
                 Acids 
               
               
                   
                   
                   
                   
                 delivered 
                 and  
               
               
                   
                   
                   
                   
                   
                 Proteins 
               
               
                   
                 Polymeric 
                 YES 
                 Transient 
                 Depends on 
                 Nucleic  
               
               
                   
                 Nanoparticles 
                   
                   
                 what is 
                 Acids 
               
               
                   
                   
                   
                   
                 delivered 
                 and  
               
               
                   
                   
                   
                   
                   
                 Proteins 
               
               
                 Biological 
                 Attenuated 
                 YES 
                 Transient 
                 NO 
                 Nucleic  
               
               
                 Non-Viral 
                 Bacteria 
                   
                   
                   
                 Acids 
               
               
                 Delivery 
                 Engineered 
                 YES 
                 Transient 
                 NO 
                 Nucleic  
               
               
                 Vehicles 
                   
                   
                   
                   
                 Acids 
               
               
                   
                 Bacteriophages 
                 YES 
                 Transient 
                 NO 
                 Nucleic  
               
               
                   
                 Mammalian 
                   
                   
                   
                 Acids 
               
               
                   
                 Virus-like 
                   
                   
                   
                   
               
               
                   
                 Particles 
                   
                   
                   
                   
               
               
                   
                 Biological 
                 YES 
                 Transient 
                 NO 
                 Nucleic  
               
               
                   
                 liposomes: 
                   
                   
                   
                 Acids 
               
               
                   
                 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 cases, 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). 
     A base editor described herein with or without one or more guide nucleic can be delivered using adeno associated virus (AAV), lentivirus, adenovirus 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. 
     For in vivo delivery, AAV can be advantageous over other viral vectors. In some cases, 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 cases, AAV allows low probability of causing insertional mutagenesis because it doesn&#39;t integrate into the host genome. 
     AAV has a packaging limit of 4.5 or 4.75 Kb. 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 cases, 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 self-inactivating lentiviral vectors 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 disclosure in some embodiments comprehends a method of modifying a cell or organism. The cell can be a prokaryotic cell or a eukaryotic cell. The cell can be a mammalian cell. The mammalian cell many be a non-human primate, bovine, porcine, rodent or mouse cell. The modification introduced to the cell by the base editors, compositions and methods of the present disclosure can be such that the cell and progeny of the cell are altered for improved production of biologic products such as an antibody, starch, alcohol or other desired cellular output. The modification introduced to the cell by the methods of the present disclosure can be such that the cell and progeny of the cell include an alteration that changes the biologic product produced. 
     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. 
     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. The term “pharmaceutical composition”, as used herein, refers to a composition formulated for pharmaceutical use. 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). 
     As used here, 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.). 
     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. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as “excipient,” “carrier,” “pharmaceutically acceptable carrier,” “vehicle,” or the like are used interchangeably herein. 
     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. 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. 
     Methods of Treating A1AD 
     Provided also are methods of treating A1AD and/or the genetic mutations in SERPINA1 that cause A1AD 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 CG to UA alteration (if the cell is transduced with a cytidine deaminase domain) of a nucleic acid sequence containing mutations in the SERPINA1 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 SERPINA1 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 A1AD. The compositions herein may be also used in the treatment of any other disorders in which A1AD may be implicated. 
     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 MAD) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with A1AD 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. 
     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, including but not limited to one or more of the following: autoimmune disorders (e.g., diabetes, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis); inflammatory disorders (e.g., arthritis, pelvic inflammatory disease); infectious diseases (e.g., viral infections (e.g., HIV, HCV, RSV), bacterial infections, fungal infections, sepsis); neurological disorders (e.g., Alzheimer&#39;s disease, Huntington&#39;s disease; autism; Duchenne muscular dystrophy); cardiovascular disorders (e.g., atherosclerosis, hypercholesterolemia, thrombosis, clotting disorders, angiogenic disorders such as macular degeneration); proliferative disorders (e.g., cancer, benign neoplasms); respiratory disorders (e.g., chronic obstructive pulmonary disease); digestive disorders (e.g., inflammatory bowel disease, ulcers); musculoskeletal disorders (e.g., fibromyalgia, arthritis); endocrine, metabolic, and nutritional disorders (e.g., diabetes, osteoporosis); urological disorders (e.g., renal disease); psychological disorders (e.g., depression, schizophrenia); skin disorders (e.g., wounds, eczema); blood and lymphatic disorders (e.g., anemia, hemophilia); etc. 
     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, e.g., A1AD-associated mutations. 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 A1AD-associated 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. 
     In certain embodiments, the kit is useful for the treatment of a subject having Alpha-1 antitrypsin deficiency (A1AD). 
     The following numbered additional embodiments encompassing the methods and compositions of the base editor systems and uses are envisioned herein:
         1. A method of treating Alpha-1 antitrypsin deficiency (A1AD) in a subject in need thereof, comprising administering to the subject a base editor system comprising   a guide polynucleotide or a nucleic acids encoding the guide polynucleotide;   a polynucleotide programmable DNA binding domain or a nucleic acid encoding the polynucleotide programmable DNA binding domain, and   an adenosine deaminase domain or a nucleic acid encoding the adenosine deaminase domain,   wherein the guide polynucleotide is capable of targeting the base editor system to effect an A⋅T to G⋅C alteration of a single nucleotide polymorphism (SNP) in a SERPINA1 polynucleotide of a cell in the subject, thereby treating A1AD;   wherein the SNP is causative of A1AD.   2. A method of treating Alpha-1 antitrypsin deficiency (A1AD) in a subject in need thereof, comprising   (a) introducing into a cell a base editor system comprising   a guide polynucleotides or a nucleic acid encoding the guide polynucleotide;   a polynucleotide programmable DNA binding domain or a nucleic acid encoding the polynucleotide programmable DNA binding domain, and   an adenosine deaminase domain or a nucleic acid encoding the adenosine deaminase domain, and   (b) administering the cell to the subject,   wherein the guide polynucleotide is capable of targeting the base editor system to effect an A⋅T to G⋅C alteration of a single nucleotide polymorphism (SNP) in a SERPINA1 polynucleotide in the cell, thereby treating A1AD;   wherein the SNP is causative of A1AD.   3. The method of embodiment 2, wherein the cell is a hepatocyte or a progenitor thereof   4. The method of any one of embodiment 2 or 3, wherein the cell is autologous, allogenic, or xenogenic to the subject.   5. A method of correcting a single nucleotide polymorphism (SNP) causative of Alpha-1 antitrypsin deficiency (A1AD) in a SERPINA1 polynucleotide, comprising contacting the SERPINA1 polynucleotide with a base editor system comprising   a guide polynucleotides;   a polynucleotide programmable DNA binding domain, and   an adenosine deaminase domain,   wherein the guide polynucleotides is capable of targeting the base editor system to effect an A⋅T to G⋅C of the SNP in the SERPINA1 polynucleotide, thereby correcting the SNP.   6. A method of producing a modified cell for treatment of Alpha-1 antitrypsin deficiency (A1AD), comprising introducing into a cell a base editor system comprising   a guide polynucleotides or a nucleic acid encoding the guide polynucleotide;   a polynucleotide programmable DNA binding domain or a nucleic acid encoding the polynucleotide programmable DNA binding domain, and   an adenosine deaminase domain or a nucleic acid encoding the adenosine deaminase domain,   wherein the guide polynucleotide is capable of targeting the base editor system to effect an A⋅T to G⋅C alteration of a single nucleotide polymorphism (SNP) causative of A1AD in a SERPINA1 polynucleotide in the cell.   7. The method of embodiment 6, wherein the introduction is in vivo.   8. The method of embodiment 6, wherein the introduction is ex vivo.   9. The method of any one of embodiments 6-8, wherein the cell is a hepatocyte of a progenitor thereof   10. The method of any one of embodiments 6-9, wherein the cell is obtained from a subject having A1AD.   11. The method of any one of the preceding embodiments, wherein SERPINA1 polynucleotide encodes an A1AT protein comprising a lysine at position 342 resulted from the SNP.   12. The method of embodiment 11, wherein the A⋅T to G⋅C alteration substitutes the lysine with a wild type amino acid.   13. A method of treating Alpha-1 antitrypsin deficiency (A1AD) in a subject in need thereof, comprising administering to the subject a base editor system comprising   a guide polynucleotide or a nucleic acid encoding the guide polynucleotide;   a polynucleotide programmable DNA binding domain or a nucleic acid encoding the polynucleotide programmable DNA binding domain, and   an adenosine deaminase domain or a nucleic acid encoding the adenosine deaminase domain,   wherein the guide polynucleotide is capable of targeting the base editor system to effect an A⋅T to G⋅C alteration of a single nucleotide polymorphism (SNP) causative of A1AD in a SERPINA1 polynucleotide of a cell in the subject,   wherein the SERPINA1 polynucleotide encodes a A1AT protein comprising an lysine acid at position 342 resulted from the SNP,   wherein the A⋅T to G⋅C alteration substitutes the lysine with a wild type amino acid, thereby treating A1AD.   14. A method of treating Alpha-1 antitrypsin deficiency (A1AD) in a subject in need thereof, comprising   (a) contacting a cell with a base editor system comprising   a guide polynucleotide or a nucleic acid encoding the guide polynucleotide;   a polynucleotide programmable DNA binding domain or a nucleic acid encoding the polynucleotide programmable DNA binding domain, and   an adenosine deaminase domain or a nucleic acid encoding the adenosine deaminase domain,   (b) administering the cell to the subject,   wherein the guide polynucleotide is capable of targeting the base editor system to effect an A⋅T to G⋅C alteration of a single nucleotide polymorphism (SNP) causative of A1AD in a SERPINA1 polynucleotide in the cell,   wherein the SERPINA1 polynucleotide encodes a A1AT protein comprising a lysine at position 342 resulted from the SNP,   wherein the A⋅T to G⋅C alteration substitutes the lysine with a wild type amino acid, thereby treating A1AD.   15. The method of embodiment 14, wherein the cell is a hepatocyte or a progenitor thereof   16. The method of embodiment 14 or 15, wherein the cell is autologous, allogenic, or xenogenic to the subject.   17. A method of correcting a single nucleotide polymorphism (SNP) causative of Alpha-1 antitrypsin deficiency (A1AD) in a SERPINA1 polynucleotide, comprising contacting the SERPINA1 polynucleotide with a base editor system comprising   a guide polynucleotide;   a polynucleotide programmable DNA binding domain, and   an adenosine deaminase domain,   wherein the guide polynucleotides is capable of targeting the base editor system to effect an A⋅T to G⋅C of the SNP,   wherein the SERPINA1 polynucleotide encodes a A1AT protein comprising a lysine at position 342 resulted from the SNP,   wherein the A⋅T to G⋅C alteration substitutes the lysine with a wild type amino acid, thereby correcting the SNP.   18. A method of producing a modified cell for treatment of A1AD, comprising introducing into a cell a base editor system comprising   a guide polynucleotides or a nucleic acid encoding the guide polynucleotide;   a polynucleotide programmable DNA binding domain or a nucleic acid encoding the polynucleotide programmable DNA binding domain, and   an adenosine deaminase domain or a nucleic acid encoding the adenosine deaminase domain,   wherein the guide polynucleotide is capable of targeting the base editor system to effect an A⋅T to G⋅C alteration of a single nucleotide polymorphism (SNP) causative of A1AD in a SERPINA1 polynucleotide in the cell,   wherein the SERPINA1 polynucleotide encodes a A1AT protein comprising a lysine at position 342 resulted from the SNP,   wherein the A⋅T to G⋅C alteration substitutes the lysine with a wild type amino acid.   19. The method of embodiment 18, wherein the introduction is in vivo.   20. The method of embodiment 18, wherein the introduction is ex vivo.   21. The method of any one of embodiments 18-20, wherein the cell is a hepatocyte or a progenitor thereof   22. The method of any one of embodiments 18-21, wherein the cell is obtained from a subject having A1AD.   23. The method of any one of embodiments 12-22, wherein the wild type amino acid is a glutamic acid.   24. The method of any one of the preceding embodiments, wherein the polynucleotide programmable DNA binding domain is a Cas9 domain.   25. The method of embodiment 24, wherein the Cas9 domain is a nuclease inactive Cas9 domain.   26. The method of embodiment 24, wherein the Cas9 domain is a Cas9 nickase domain.   27. The method of any one of embodiments 24-26, wherein the Cas9 domain comprises a SpCas9 domain.   28. The method of embodiment 27, wherein the SpCas9 domain comprises a D10A and/or a H840A amino acid substitution or corresponding amino acid substitutions thereof   29. The method of embodiment 27 or 28, wherein the SpCas9 domain has specificity for a NGG PAM.   30. The method of any one of embodiments 27-29, wherein the SpCas9 domain has specificity for a NGA PAM, a NGT PAM, or a NGC PAM.   31. The method of any one of embodiments 27-30, wherein the SpCas9 domain comprises amino acid substitutions L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, T1337R and one or more of L1111, D1135L, S1136R, G1218S, E1219V, D1332A, R1335Q, T13371, T1337V, T1337F, and T1337M or corresponding amino acid substitutions thereof   32. The method of any one of embodiments 27-31, wherein the SpCas9 domain comprises amino acid substitutions L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, T1337R and one or more of L1111, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, R1335Q, T13371, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, and T1337M or corresponding amino acid substitutions thereof   33. The method of any one of embodiments 27-32, wherein the SpCas9 domain comprises amino acid substitutions D1135L, S1136R, G1218S, E1219V, A1322R, R1335Q, T1337, and A1322R, and one or more of L1111, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, R1335Q, T13371, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, and T1337M or or corresponding amino acid substitutions thereof   34. The method of any one of embodiments 27-33, wherein the SpCas9 domain comprises amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R, or corresponding amino acid substitutions thereof   35. The method of any one of embodiments 27-34, wherein the SpCas9 domain has specificity for a NG PAM, a NNG PAM, a GAA PAM, a GAT PAM, or a CAA PAM.   36. The method of embodiment 35, wherein the SpCas9 domain comprises amino acid substitutions E480K, E543K, and E1219V or corresponding amino acid substitutions thereof   37. The method of any one of embodiments 27-29, wherein the Cas9 domain comprises a SaCas9 domain.   38. The method of embodiment 27, wherein the SaCas9 domain has specificity for a NNNRRT PAM.   39. The method of embodiment 38, wherein the SaCas9 domain has specificity for a NNGRRT PAM.   40. The method of any one of embodiments 37-39, wherein the SaCas9 domain comprises an amino acid substitution N579A or a corresponding amino acid substitution thereof   41. The method of any one of embodiments 37-40, wherein the SaCas9 domain comprises amino acid substitutions E782K, N968K, and R1015H, or corresponding amino acid substitutions thereof   42. The method of any one of embodiments 27-29, wherein the Cas9 domain comprises a St1Cas9 domain:   43. The method of embodiment 40, wherein the St1Cas9 domain has specificity for a NNACCA PAM.   44. The method of any one of the preceding embodiments, wherein the adenosine deaminase domain is a modified adenosine deaminase domain that does not occur in nature.   45. The method of embodiment 44, wherein the adenosine deaminase domain comprises a TadA domain.   46. The method of embodiment 45, wherein the TadA domain comprises the amino acid sequence of TadA 7.10.   47. The method of any one of the preceding embodiments, wherein the base editor system further comprises a zinc finger domain.   48. The method of embodiment 47, wherein the zinc finger domain comprises recognition helix sequences RNEHLEV (SEQ ID NO: 268), QSTTLKR (SEQ ID NO: 269), and RTEHLAR (SEQ ID NO: 270) or recognition helix sequences RGEHLRQ (SEQ ID NO: 271), QSGTLKR (SEQ ID NO: 272), and RNDKLVP (SEQ ID NO: 273).   49. The method of embodiment 47 or 48, wherein the zinc finger domain is zf1ra or zf1rb.   50. The method of any one of the preceding embodiments, wherein the base editor system further comprises a nuclear localization signal (NLS).   51. The method of any one of the preceding embodiments, wherein the base editor system further comprises one or more linkers.   52. The method of embodiment 51, wherein two or more of the polynucleotide programmable DNA binding domain, the adenosine deaminase domain, the zinc finger domain, and the NLS are connected via a linker.   53. The method of embodiment 52, wherein the linker is a peptide linker, thereby forming a base editing fusion protein.   54. The method of embodiment 53, wherein the peptide linker comprises an amino acid sequence selected from the group consisting of SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 32), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 33), GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS EGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 34), SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 35), SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 36), SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSG GS (SEQ ID NO: 37), PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 38), (SGGS)n (SEQ ID NO: 297), (GGGS)n (SEQ ID NO: 298), (GGGGS)n (SEQ ID NO: 299), (G)n, (EAAAK)n (SEQ ID NO: 300), (GGS)n, SGSETPGTSESATPES (SEQ ID NO: 16), and (XP)n.   55. The method of embodiment 53 or 54, wherein the base editing fusion protein comprises the amino acid sequence selected from the group consisting of       

     
       
         
           
               
               
            
               
                 (SEQ ID NO: 274) 
                   
               
               
                 MPKKKRKVSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPI 
                   
               
               
                   
               
               
                 GRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGAR 
               
               
                   
               
               
                 DAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSS 
               
               
                   
               
               
                 TDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDER 
               
               
                   
               
               
                 EVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTF 
               
               
                   
               
               
                 EPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECA 
               
               
                   
               
               
                 ALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESDLVLGLAIGIG 
               
               
                   
               
               
                 SVGVGILNKVTGEIIHKNSRIFPAAQAENNLVRRTNRQGRRLARRKKHRRVRLNRLFEE 
               
               
                   
               
               
                 SGLITDFTKISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISYLDDASDDGNSSV 
               
               
                   
               
               
                 GDYAQIVKENSKQLETKTPGQIQLERYQTYGQLRGDFTVEKDGKKHRLINVFPTSAYRS 
               
               
                   
               
               
                 EALRILQTQQEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGRYRTSGETLDNIF 
               
               
                   
               
               
                 GILIGKCTFYPDEFRAAKASYTAQEFNLLNDLNNLTVPTETKKLSKEQKNQIINYVKNEK 
               
               
                   
               
               
                 AMGPAKLFKYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDRET 
               
               
                   
               
               
                 LDKLAYVLTLNTEREGIQEALEHEFADGSFSQKQVDELVQFRKANSSIFGKGWHNFSVK 
               
               
                   
               
               
                 LMMELIPELYETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNPVVAKSVRQAI 
               
               
                   
               
               
                 KIVNAAIKEYGDFDNIVIEMARETNEDDEKKAIQKIQKANKDEKDAAMLKAANQYNGK 
               
               
                   
               
               
                 AELPHSVFHGHKQLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHILPLSITFDD 
               
               
                   
               
               
                 SLANKVLVYATANQEKGQRTPYQALDSMDDAWSFRELKAFVRESKTLSNKKKEYLLT 
               
               
                   
               
               
                 EEDISKFDVRKKFIERNLVDTLYASRVVLNALQEHFRAHKIDTKVSVVRGQFTSQLRRH 
               
               
                   
               
               
                 WGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQLLDIETGELISDDEYK 
               
               
                   
               
               
                 ESVFKAPYQHFVDTLKSKEFEDSILFSYQVDSKFNRKISDATIYATRQAKVGKDKADET 
               
               
                   
               
               
                 YVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQTFEKVIEPILENYPNKQINDKG 
               
               
                   
               
               
                 KEVPCNPFLKYKEEHGYIRKYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNKVVLQS 
               
               
                   
               
               
                 VSPWRADVYFNKTTGKYEILGLKYADLQFDKGTGTYKISQEKYNDIKKKEGVDSDSEF 
               
               
                   
               
               
                 KFTLYKNDLLLVKDTETKEQQLFRFLSRTMPKQKHYVELKPYDKQKFEGGEALIKVLG 
               
               
                   
               
               
                 NVANSGQCKKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKLDFPKKKRKVEGADKRT 
               
               
                   
               
               
                 ADGSEFESPKKKRKV, 
               
               
                   
               
               
                 (SEQ ID NO: 275) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTA 
                   
               
               
                   
               
               
                 HAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGA 
               
               
                   
               
               
                 AGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGSS 
               
               
                   
               
               
                 GGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAV 
               
               
                   
               
               
                 LVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCA 
               
               
                   
               
               
                 GAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFF 
               
               
                   
               
               
                 RMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSKRNYILGLAI 
               
               
                   
               
               
                 GITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKL 
               
               
                   
               
               
                 LFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTG 
               
               
                   
               
               
                 NELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQK 
               
               
                   
               
               
                 AYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSV 
               
               
                   
               
               
                 KYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVN 
               
               
                   
               
               
                 EEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEEL 
               
               
                   
               
               
                 TNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKV 
               
               
                   
               
               
                 DLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMI 
               
               
                   
               
               
                 NEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF 
               
               
                   
               
               
                 NYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLA 
               
               
                   
               
               
                 KGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDV 
               
               
                   
               
               
                 KVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVME 
               
               
                   
               
               
                 NQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLY 
               
               
                   
               
               
                 STRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQY 
               
               
                   
               
               
                 GDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVV 
               
               
                   
               
               
                 KLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFI 
               
               
                   
               
               
                 ASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKT 
               
               
                   
               
               
                 QSIKKYSTDILGNLYEVKSKKHPQIIKKGEGADKRTADGSEFESPKKKRKV, 
               
               
                   
               
               
                 (SEQ ID NO: 276) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRH 
                   
               
               
                   
               
               
                 DPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDA 
               
               
                   
               
               
                 KTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD 
               
               
                   
               
               
                 SGGSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVP 
               
               
                   
               
               
                 VGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPC 
               
               
                   
               
               
                 VMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAAL 
               
               
                   
               
               
                 LCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSKRNYI 
               
               
                   
               
               
                 LGLAIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQR 
               
               
                   
               
               
                 VKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVE 
               
               
                   
               
               
                 EDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLL 
               
               
                   
               
               
                 KVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPE 
               
               
                   
               
               
                 ELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK 
               
               
                   
               
               
                 EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDI 
               
               
                   
               
               
                 QEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVP 
               
               
                   
               
               
                 KKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQ 
               
               
                   
               
               
                 KMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLN 
               
               
                   
               
               
                 NPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHIL 
               
               
                   
               
               
                 NLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNN 
               
               
                   
               
               
                 LDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKK 
               
               
                   
               
               
                 VMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLIND 
               
               
                   
               
               
                 TLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIM 
               
               
                   
               
               
                 EQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRN 
               
               
                   
               
               
                 KVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQ 
               
               
                   
               
               
                 AEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTI 
               
               
                   
               
               
                 ASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGEGADKRTADGSEFESPKKKRKVSSGNS 
               
               
                   
               
               
                 NANSRGPSFSSGLVPLSLRGSHSRPGERPFQCRICMRNFSRNEHLEVHTRTHTGEKPFQC 
               
               
                   
               
               
                 RICMRNFSQSTTLKRHLRTHTGEKPFQCRICMRNFSRTEHLARHLKTHLRGSSAQ, 
               
               
                 or 
               
               
                   
               
               
                 (SEQ ID NO: 277) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRH 
                   
               
               
                   
               
               
                 DPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDA 
               
               
                   
               
               
                 KTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD 
               
               
                   
               
               
                 SGGSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVP 
               
               
                   
               
               
                 VGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPC 
               
               
                   
               
               
                 VMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAAL 
               
               
                   
               
               
                 LCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSKRNYI 
               
               
                   
               
               
                 LGLAIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQR 
               
               
                   
               
               
                 VKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVE 
               
               
                   
               
               
                 EDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLL 
               
               
                   
               
               
                 KVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPE 
               
               
                   
               
               
                 ELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK 
               
               
                   
               
               
                 EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDI 
               
               
                   
               
               
                 QEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVP 
               
               
                   
               
               
                 KKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQ 
               
               
                   
               
               
                 KMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLN 
               
               
                   
               
               
                 NPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHIL 
               
               
                   
               
               
                 NLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNN 
               
               
                   
               
               
                 LDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKK 
               
               
                   
               
               
                 VMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLIND 
               
               
                   
               
               
                 TLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIM 
               
               
                   
               
               
                 EQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRN 
               
               
                   
               
               
                 KVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQ 
               
               
                   
               
               
                 AEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTI 
               
               
                   
               
               
                 ASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGEGADKRTADGSEFESPKKKRKVSSGNS 
               
               
                   
               
               
                 NANSRGPSFSSGLVPLSLRGSHSRPGERPFQCRICMRNFSRGEHLRQHTRTHTGEKPFQC 
               
               
                   
               
               
                 RICMRNFSQSGTLKRHLRTHTGEKPFQCRICMRNFSRNDKLVPHLKTHLRGSSAQ. 
               
            
           
         
       
         
         
           
             56. The method of any one of the preceding embodiments, wherein the guide polynucleotide comprises two individual polynucleotides, wherein the two individual polynucleotides are two DNAs, two RNAs or a DNA and a RNA. 
             57. The method of any one of the preceding embodiments, wherein the guide polynucleotides comprise a crRNA and a tracrRNA, wherein the crRNA comprises a nucleic acid sequence complementary to a target sequence in the SERPINA1 polynucleotide. 
             58. The method of embodiment 57, wherein the target sequence comprises a sequence selected from the group consisting of GACAAGAAAGGGACTGAAGC (SEQ ID NO: 278), ATCGACAAGAAAGGGACTGA (SEQ ID NO: 279), and ACACACCGGTTGGTGGCCTC (SEQ ID NO: 280), or a complementary thereof 
             59. The method of embodiment 57 or 58, wherein the base editor system comprises a single guide RNA (sgRNA). 
             60. The method of embodiment 59, wherein the sgRNA comprises a sequence selected from the group consisting of ACTCTaGGCAGAGGTCTCAAAGG (SEQ ID NO: 9) and GCTCTaGGCCGAAGTGTCGCAGG (SEQ ID NO: 10). 
             61. The method of any one of the preceding embodiments, wherein the base editor system comprises a vector comprising one or more of the guide polynucleotide, the polynucleotide programmable DNA binding domain, and the deaminase domain. 
             62. The method of embodiment 61, wherein the vector is an adenovirus vector, an AAV vector, a lentivirus vector, or a retrovirus vector. 
             63. A modified cell comprising a base editor system comprising 
             a guide polynucleotide or a nucleic acid encoding the guide polynucleotide; 
             a polynucleotide programmable DNA binding domain or a nucleic acid encoding the polynucleotide programmable DNA binding domain, and 
             an adenosine deaminase domain or a nucleic acid encoding the adenosine deaminase domain, 
             wherein the guide polynucleotide is capable of targeting the base editor system to effect an A⋅T to G⋅C alteration of a single nucleotide polymorphism (SNP) causative of Alpha-lantitrypsin deficiency (A1AD) in a SERPINA1 polynucleotide in the cell. 
             64. A modified cell comprising a base editor system comprising 
             a guide polynucleotide or a nucleic acid encoding the guide polynucleotide; 
             a polynucleotide programmable DNA binding domain or a nucleic acid encoding the polynucleotide programmable DNA binding domain, and 
             an adenosine deaminase domain or a nucleic acid encoding the adenosine deaminase domain, 
             wherein the guide polynucleotide is capable of targeting the base editor system to effect an A⋅T to G⋅C alteration of a single nucleotide polymorphism (SNP) causative of Alpha-lantitrypsin deficiency (A1AD) in a SERPINA1 polynucleotide in the cell, 
             wherein the SERPINA1 polynucleotide encodes a A1AT protein comprising a lysine at position 324 resulted from the SNP, 
             wherein the A⋅T to G⋅C alteration substitutes the lysine with a wild type amino acid. 
             65. The modified cell of embodiment 63, wherein the SERPINA1 polynucleotide encodes an A1AT protein comprising a lysine at position 342 resulted from the SNP. 
             66. The modified cell of embodiment 65, wherein the A⋅T to G⋅C alteration substitutes the lysine with a wild type amino acid. 
             67. The modified cell of any one of embodiment 63-66, wherein the cell is a hepatocyte or a progenitor thereof 
             68. The modified cell of embodiments 67, wherein the cell is obtained from a subject having A1AD. 
             69. The modified cell of any one of embodiments 66-68, wherein the wild type amino acid is a glutamic acid. 
             70. The modified cell of any one of embodiments 63-66, wherein the polynucleotide programmable DNA binding domain is a Cas9 domain. 
             71. The modified cell of embodiment 70, wherein the Cas9 domain is a nuclease inactive Cas9 domain. 
             72. The modified cell of embodiment 71, wherein the Cas9 domain is a Cas9 nickase domain. 
             73. The modified cell of any one of embodiments 70-72, wherein the Cas9 domain comprises a SpCas9 domain. 
             74. The modified cell of embodiment 73, wherein the SpCas9 domain comprises a D10A and/or a H840A amino acid substitution or corresponding amino acid substitutions thereof 
             75. The modified cell of embodiment 73 or 74, wherein the SpCas9 domain has specificity for a NGG PAM. 
             76. The modified cell of any one of embodiments 73-75, wherein the SpCas9 domain has specificity for a NGA PAM, a NGT PAM, or a NGC PAM. 
             77. The modified cell of any one of embodiments 73-76, wherein the SpCas9 domain comprises amino acid substitutions L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, T1337R and one or more of L1111, D1135L, S1136R, G1218S, E1219V, D1332A, R1335Q, T13371, T1337V, T1337F, and T1337M or corresponding amino acid substitutions thereof 
             78. The modified cell of any one of embodiments 73-76, wherein the SpCas9 domain comprises amino acid substitutions L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, T1337R and one or more of L1111, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, R1335Q, T13371, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, and T1337M or corresponding amino acid substitutions thereof 
             79. The modified cell of any one of embodiments 73-76, wherein the SpCas9 domain comprises amino acid substitutions D1135L, S1136R, G1218S, E1219V, A1322R, R1335Q, T1337, and A1322R, and one or more of L1111, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, R1335Q, T13371, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, and T1337M or or corresponding amino acid substitutions thereof 
             80. The modified cell of any one of embodiments 73-76, wherein the SpCas9 domain comprises amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R, or corresponding amino acid substitutions thereof 
             81. The modified cell of any one of embodiments 73-75, wherein the SpCas9 domain has specificity for a NG PAM, a NNG PAM, a GAA PAM, a GAT PAM, or a CAA PAM. 
             82. The modified cell of embodiment 81, wherein the SpCas9 domain comprises amino acid substitutions E480K, E543K, and E1219V or corresponding amino acid substitutions thereof 
             83. The modified cell of any one of embodiments 70-72, wherein the Cas9 domain comprises a SaCas9 domain. 
             84. The modified cell of embodiment 83, wherein the SaCas9 domain has specificity for a NNNRRT PAM. 
             85. The modified cell of embodiment 84, wherein the SaCas9 domain has specificity for a NNGRRT PAM. 
             86. The modified cell of any one of embodiments 83-85, wherein the SaCas9 domain comprises an amino acid substitution N579A or a corresponding amino acid substitution thereof 
             87. The modified cell of any one of embodiments 83-86, wherein the SaCas9 domain comprises amino acid substitutions E782K, N968K, and R1015H, or corresponding amino acid substitutions thereof 
             88. The modified cell of any one of embodiments 70-72, wherein the Cas9 domain comprises a St1Cas9 domain: 
             89. The modified cell of embodiment 88, wherein the St1Cas9 domain has specificity for a NNACCA PAM. 
             90. The modified cell of any one of the preceding embodiments, wherein the adenosine deaminase domain is a modified adenosine deaminase domain that does not occur in nature. 
             91. The modified cell of embodiment 90, wherein the adenosine deaminase domain comprises a TadA domain. 
             92. The modified cell of embodiment 91, wherein the TadA domain comprises the amino acid sequence of TadA 7.10. 
             93. The modified cell of any one embodiments 63-92, wherein the base editor system further comprises a zinc finger domain. 
             94. The modified cell of embodiment 93, wherein the zinc finger domain comprises recognition helix sequences RNEHLEV (SEQ ID NO: 268), QSTTLKR (SEQ ID NO: 269), and RTEHLAR (SEQ ID NO: 270) or recognition helix sequences RGEHLRQ (SEQ ID NO: 271), QSGTLKR (SEQ ID NO: 272), and RNDKLVP (SEQ ID NO: 273). 
             95. The modified cell of embodiment 93 or 94, wherein the zinc finger domain is zf1ra or zf1rb. 
             96. The modified cell of any one of the preceding embodiments, wherein the base editor system further comprises a nuclear localization signal (NLS). 
             97. The modified cell of any one embodiments 63-96, wherein the base editor system further comprises one or more linkers. 
             98. The modified cell of embodiment 97, wherein two or more of the polynucleotide programmable DNA binding domain, the adenosine deaminase domain, the zinc finger domain, and the NLS are connected via a linker. 
             99. The modified cell of embodiment 98, wherein the linker is a peptide linker, thereby forming a base editing fusion protein. 
             100. The modified cell of embodiment 99, wherein the peptide linker comprises an amino acid sequence selected from the group consisting of SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 32), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 33), GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS EGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 34), SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 35), SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 36), SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSG GS (SEQ ID NO: 37), PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 38), (SGGS)n (SEQ ID NO: 297), (GGGS)n (SEQ ID NO: 298), (GGGGS)n (SEQ ID NO: 299), (G)n, (EAAAK)n (SEQ ID NO: 300), (GGS)n, SGSETPGTSESATPES (SEQ ID NO: 16), and (XP)n. 
             101. The modified cell of embodiment 99 or 100, wherein the base editing fusion protein comprises the amino acid sequence selected from the group consisting of 
           
         
       
    
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 274) 
                   
               
               
                 MPKKKRKVSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPI 
                   
               
               
                   
               
               
                 GRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGAR 
               
               
                   
               
               
                 DAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSS 
               
               
                   
               
               
                 TDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDER 
               
               
                   
               
               
                 EVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTF 
               
               
                   
               
               
                 EPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECA 
               
               
                   
               
               
                 ALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESDLVLGLAIGIG 
               
               
                   
               
               
                 SVGVGILNKVTGEIIHKNSRIFPAAQAENNLVRRTNRQGRRLARRKKHRRVRLNRLFEE 
               
               
                   
               
               
                 SGLITDFTKISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISYLDDASDDGNSSV 
               
               
                   
               
               
                 GDYAQIVKENSKQLETKTPGQIQLERYQTYGQLRGDFTVEKDGKKHRLINVFPTSAYRS 
               
               
                   
               
               
                 EALRILQTQQEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGRYRTSGETLDNIF 
               
               
                   
               
               
                 GILIGKCTFYPDEFRAAKASYTAQEFNLLNDLNNLTVPTETKKLSKEQKNQIINYVKNEK 
               
               
                   
               
               
                 AMGPAKLFKYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDRET 
               
               
                   
               
               
                 LDKLAYVLTLNTEREGIQEALEHEFADGSFSQKQVDELVQFRKANSSIFGKGWHNFSVK 
               
               
                   
               
               
                 LMMELIPELYETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNPVVAKSVRQAI 
               
               
                   
               
               
                 KIVNAAIKEYGDFDNIVIEMARETNEDDEKKAIQKIQKANKDEKDAAMLKAANQYNGK 
               
               
                   
               
               
                 AELPHSVFHGHKQLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHILPLSITFDD 
               
               
                   
               
               
                 SLANKVLVYATANQEKGQRTPYQALDSMDDAWSFRELKAFVRESKTLSNKKKEYLLT 
               
               
                   
               
               
                 EEDISKFDVRKKFIERNLVDTLYASRVVLNALQEHFRAHKIDTKVSVVRGQFTSQLRRH 
               
               
                   
               
               
                 WGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQLLDIETGELISDDEYK 
               
               
                   
               
               
                 ESVFKAPYQHFVDTLKSKEFEDSILFSYQVDSKFNRKISDATIYATRQAKVGKDKADET 
               
               
                   
               
               
                 YVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQTFEKVIEPILENYPNKQINDKG 
               
               
                   
               
               
                 KEVPCNPFLKYKEEHGYIRKYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNKVVLQS 
               
               
                   
               
               
                 VSPWRADVYFNKTTGKYEILGLKYADLQFDKGTGTYKISQEKYNDIKKKEGVDSDSEF 
               
               
                   
               
               
                 KFTLYKNDLLLVKDTETKEQQLFRFLSRTMPKQKHYVELKPYDKQKFEGGEALIKVLG 
               
               
                   
               
               
                 NVANSGQCKKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKLDFPKKKRKVEGADKRT 
               
               
                   
               
               
                 ADGSEFESPKKKRKV, 
               
               
                   
               
               
                 (SEQ ID NO: 275) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTA 
                   
               
               
                   
               
               
                 HAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGA 
               
               
                   
               
               
                 AGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGSS 
               
               
                   
               
               
                 GGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAV 
               
               
                   
               
               
                 LVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCA 
               
               
                   
               
               
                 GAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFF 
               
               
                   
               
               
                 RMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSKRNYILGLAI 
               
               
                   
               
               
                 GITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKL 
               
               
                   
               
               
                 LFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTG 
               
               
                   
               
               
                 NELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQK 
               
               
                   
               
               
                 AYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSV 
               
               
                   
               
               
                 KYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVN 
               
               
                   
               
               
                 EEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEEL 
               
               
                   
               
               
                 TNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKV 
               
               
                   
               
               
                 DLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMI 
               
               
                   
               
               
                 NEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF 
               
               
                   
               
               
                 NYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLA 
               
               
                   
               
               
                 KGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDV 
               
               
                   
               
               
                 KVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVME 
               
               
                   
               
               
                 NQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLY 
               
               
                   
               
               
                 STRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQY 
               
               
                   
               
               
                 GDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVV 
               
               
                   
               
               
                 KLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFI 
               
               
                   
               
               
                 ASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKT 
               
               
                   
               
               
                 QSIKKYSTDILGNLYEVKSKKHPQIIKKGEGADKRTADGSEFESPKKKRKV, 
               
               
                   
               
               
                 (SEQ ID NO: 276) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRH 
                   
               
               
                   
               
               
                 DPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDA 
               
               
                   
               
               
                 KTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD 
               
               
                   
               
               
                 SGGSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVP 
               
               
                   
               
               
                 VGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPC 
               
               
                   
               
               
                 VMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAAL 
               
               
                   
               
               
                 LCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSKRNYI 
               
               
                   
               
               
                 LGLAIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQR 
               
               
                   
               
               
                 VKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVE 
               
               
                   
               
               
                 EDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLL 
               
               
                   
               
               
                 KVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPE 
               
               
                   
               
               
                 ELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK 
               
               
                   
               
               
                 EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDI 
               
               
                   
               
               
                 QEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVP 
               
               
                   
               
               
                 KKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQ 
               
               
                   
               
               
                 KMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLN 
               
               
                   
               
               
                 NPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHIL 
               
               
                   
               
               
                 NLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNN 
               
               
                   
               
               
                 LDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKK 
               
               
                   
               
               
                 VMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLIND 
               
               
                   
               
               
                 TLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIM 
               
               
                   
               
               
                 EQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRN 
               
               
                   
               
               
                 KVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQ 
               
               
                   
               
               
                 AEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTI 
               
               
                   
               
               
                 ASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGEGADKRTADGSEFESPKKKRKVSSGNS 
               
               
                   
               
               
                 NANSRGPSFSSGLVPLSLRGSHSRPGERPFQCRICMRNFSRNEHLEVHTRTHTGEKPFQC 
               
               
                   
               
               
                 RICMRNFSQSTTLKRHLRTHTGEKPFQCRICMRNFSRTEHLARHLKTHLRGSSAQ, 
               
               
                 or 
               
               
                   
               
               
                 (SEQ ID NO: 277) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRH 
                   
               
               
                   
               
               
                 DPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDA 
               
               
                   
               
               
                 KTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD 
               
               
                   
               
               
                 SGGSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVP 
               
               
                   
               
               
                 VGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPC 
               
               
                   
               
               
                 VMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAAL 
               
               
                   
               
               
                 LCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSKRNYI 
               
               
                   
               
               
                 LGLAIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQR 
               
               
                   
               
               
                 VKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVE 
               
               
                   
               
               
                 EDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLL 
               
               
                   
               
               
                 KVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPE 
               
               
                   
               
               
                 ELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK 
               
               
                   
               
               
                 EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDI 
               
               
                   
               
               
                 QEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVP 
               
               
                   
               
               
                 KKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQ 
               
               
                   
               
               
                 KMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLN 
               
               
                   
               
               
                 NPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHIL 
               
               
                   
               
               
                 NLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNN 
               
               
                   
               
               
                 LDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKK 
               
               
                   
               
               
                 VMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLIND 
               
               
                   
               
               
                 TLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIM 
               
               
                   
               
               
                 EQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRN 
               
               
                   
               
               
                 KVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQ 
               
               
                   
               
               
                 AEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTI 
               
               
                   
               
               
                 ASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGEGADKRTADGSEFESPKKKRKVSSGNS 
               
               
                   
               
               
                 NANSRGPSFSSGLVPLSLRGSHSRPGERPFQCRICMRNFSRGEHLRQHTRTHTGEKPFQC 
               
               
                   
               
               
                 RICMRNFSQSGTLKRHLRTHTGEKPFQCRICMRNFSRNDKLVPHLKTHLRGSSAQ. 
               
            
           
         
       
         
         
           
             102. The modified cell of any one of embodiments 63-101, wherein the guide polynucleotide comprises two individual polynucleotides, wherein the two individual polynucleotides are two DNAs, two RNAs or a DNA and a RNA. 
             103. The modified cell of any one of the preceding embodiments, wherein the guide polynucleotides comprise a crRNA and a tracrRNA, wherein the crRNA comprises a nucleic acid sequence complementary to a target sequence in the SERPINA1 polynucleotide. 
             104. The modified cell of embodiment 103, wherein the target sequence comprises a sequence selected from the group consisting of GACAAGAAAGGGACTGAAGC (SEQ ID NO: 278), ATCGACAAGAAAGGGACTGA (SEQ ID NO: 279), and ACACACCGGTTGGTGGCCTC (SEQ ID NO: 280), or a complementary thereof 
             105. The modified cell of embodiment 102 or 103, wherein the base editor system comprises a single guide RNA (sgRNA). 
             106. The modified cell of embodiment 105, wherein the sgRNA comprises a sequence selected from the group consisting of ACTCTaGGCAGAGGTCTCAAAGG (SEQ ID NO: 9) and GCTCTaGGCCGAAGTGTCGCAGG (SEQ ID NO: 10). 
             107. The modified cell of any one of embodiments 63-106, wherein the base editor system comprises a vector comprising one or more of the guide polynucleotide, the polynucleotide programmable DNA binding domain, and the deaminase domain. 
             108. The modified cell of embodiment 107, wherein the vector is an adenovirus vector, an AAV vector, a lentivirus vector, or a retrovirus vector. 
             109. A base editor system comprising 
             a guide polynucleotide or a nucleic acid encoding the guide polynucleotide; 
             a polynucleotide programmable DNA binding domain or a nucleic acid encoding the polynucleotide programmable DNA binding domain, and 
             an adenosine deaminase domain or a nucleic acid encoding the adenosine deaminase domain, 
             wherein the guide polynucleotide is capable of targeting the base editor system to effect an A⋅T to G⋅C alteration of a single nucleotide polymorphism (SNP) causative of Alpha-lantitrypsin deficiency (A1AD) in a SERPINA1 polynucleotide. 
             110. A base editor system comprising 
             a guide polynucleotide or a nucleic acid encoding the guide polynucleotide; 
             a polynucleotide programmable DNA binding domain or a nucleic acid encoding the polynucleotide programmable DNA binding domain, and 
             an adenosine deaminase domain or a nucleic acid encoding the adenosine deaminase domain, 
             wherein the guide polynucleotide is capable of targeting the base editor system to effect an A⋅T to G⋅C alteration of a single nucleotide polymorphism (SNP) causative of Alpha-lantitrypsin deficiency (A1AD) in a SERPINA1 polynucleotide, 
             wherein the SERPINA1 polynucleotide encodes a A1AT protein comprising a lysine at position 324 resulted from the SNP, 
             wherein the A⋅T to G⋅C alteration substitutes the lysine with a wild type amino acid. 
             111. The base editor system of embodiment 109, wherein the SERPINA1 polynucleotide encodes an A1AT protein comprising a lysine at position 342 resulted from the SNP. 
             112. The base editor system of embodiment 111, wherein the A⋅T to G⋅C alteration substitutes the lysine with a wild type amino acid. 
             113. The base editor system of embodiment 110 or 112, wherein the wild type amino acid is a glutamic acid. 
             114. The base editor system of any one of embodiments 109-113, wherein the polynucleotide programmable DNA binding domain is a Cas9 domain. 
             115. The base editor system of embodiment 114, wherein the Cas9 domain is a nuclease inactive Cas9 domain. 
             116. The base editor system of embodiment 114, wherein the Cas9 domain is a Cas9 nickase domain. 
             117. The base editor system of any one of embodiments 114-116, wherein the Cas9 domain comprises a SpCas9 domain. 
             118. The base editor system of embodiment 117, wherein the SpCas9 domain comprises a D10A and/or a H840A amino acid substitution or corresponding amino acid substitutions thereof 
             119. The base editor system of embodiment 117 or 118, wherein the SpCas9 domain has specificity for a NGG PAM. 
             120. The base editor system of any one of embodiments 117-119, wherein the SpCas9 domain has specificity for a NGA PAM, a NGT PAM, or a NGC PAM. 
             121. The base editor system of any one of embodiments 117-119, wherein the SpCas9 domain comprises amino acid substitutions L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, T1337R and one or more of L1111, D1135L, S1136R, G1218S, E1219V, D1332A, R1335Q, T13371, T1337V, T1337F, and T1337M or corresponding amino acid substitutions thereof 
             122. The base editor system of any one of embodiments 117-119, wherein the SpCas9 domain comprises amino acid substitutions L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, T1337R and one or more of L1111, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, R1335Q, T13371, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, and T1337M or corresponding amino acid substitutions thereof 
             123. The base editor system of any one of embodiments 117-119, wherein the SpCas9 domain comprises amino acid substitutions D1135L, S1136R, G1218S, E1219V, A1322R, R1335Q, T1337, and A1322R, and one or more of L1111, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, R1335Q, T13371, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, and T1337M or or corresponding amino acid substitutions thereof 
             124. The base editor system of any one of embodiments 117-119, wherein the SpCas9 domain comprises amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R, or corresponding amino acid substitutions thereof 
             125. The base editor system of any one of embodiments 117-119, wherein the SpCas9 domain has specificity for a NG PAM, a NNG PAM, a GAA PAM, a GAT PAM, or a CAA PAM. 
             126. The base editor system of embodiment 125, wherein the SpCas9 domain comprises amino acid substitutions E480K, E543K, and E1219V or corresponding amino acid substitutions thereof 127. The base editor system of any one of embodiments 114-116, wherein the Cas9 domain comprises a SaCas9 domain. 
             128. The base editor system of embodiment 127, wherein the SaCas9 domain has specificity for a NNNRRT PAM. 
             129. The base editor system of embodiment 128, wherein the SaCas9 domain has specificity for a NNGRRT PAM. 
             130. The base editor system of any one of embodiments 127-129, wherein the SaCas9 domain comprises an amino acid substitution N579A or a corresponding amino acid substitution thereof 
             131. The base editor system of any one of embodiments 127-130, wherein the SaCas9 domain comprises amino acid substitutions E782K, N968K, and R1015H, or corresponding amino acid substitutions thereof 
             132. The base editor system of any one of embodiments 117-119, wherein the Cas9 domain comprises a St1Cas9 domain: 
             133. The base editor system of embodiment 132, wherein the St1Cas9 domain has specificity for a NNACCA PAM. 
             134. The base editor system of any one of embodiments, wherein the adenosine deaminase domain is a modified adenosine deaminase domain that does not occur in nature. 
             135. The base editor system of embodiment 90, wherein the adenosine deaminase domain comprises a TadA domain. 
             136. The base editor system of embodiment 91, wherein the TadA domain comprises the amino acid sequence of TadA 7.10. 
             137. The base editor system of any one of embodiments 109-136, wherein the base editor system further comprises a zinc finger domain. 
             138. The base editor system of embodiment 137, wherein the zinc finger domain comprises recognition helix sequences RNEHLEV (SEQ ID NO: 268), QSTTLKR (SEQ ID NO: 269), and RTEHLAR (SEQ ID NO: 270) or recognition helix sequences RGEHLRQ (SEQ ID NO: 271), QSGTLKR (SEQ ID NO: 272), and RNDKLVP (SEQ ID NO: 273). 
             139. The base editor system of embodiment 136 or 137, wherein the zinc finger domain is zf1ra or zf1rb. 
             140. The base editor system of any one of the preceding embodiments, wherein the base editor system further comprises a nuclear localization signal (NLS). 
             141. The base editor system of any one of embodiments 109-140, wherein the base editor system further comprises one or more linkers. 
             142. The base editor system of embodiment 141, wherein two or more of the polynucleotide programmable DNA binding domain, the adenosine deaminase domain, the zinc finger domain, and the NLS are connected via a linker. 
             143. The base editor system of embodiment 142, wherein the linker is a peptide linker, thereby forming a base editing fusion protein. 
             144. The base editor system of embodiment 143, wherein the peptide linker comprises an amino acid sequence selected from the group consisting of 
           
         
       
    
     
       
         
           
               
            
               
                 (SEQ ID NO: 32) 
               
               
                 SGGSSGSETPGTSESATPESSGGS, 
               
               
                   
               
               
                 (SEQ ID NO: 33) 
               
               
                 SGGSSGGSSGSETPGTSESATPESSGGSSGGS, 
               
               
                   
               
               
                 (SEQ ID NO: 34) 
               
               
                 GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP 
               
               
                   
               
               
                 TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGG 
               
               
                   
               
               
                 SGGS, 
               
               
                   
               
               
                 (SEQ ID NO: 35) 
               
               
                 SGGSSGGSSGSETPGTSESATPES, 
               
               
                   
               
               
                 (SEQ ID NO: 36) 
               
               
                 SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS, 
               
               
                   
               
               
                 (SEQ ID NO: 37) 
               
               
                 SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSE 
               
               
                   
               
               
                 SATPESSGGSSGGS, 
               
               
                   
               
               
                 (SEQ ID NO: 38) 
               
               
                 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG 
               
               
                   
               
               
                 TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS, 
               
               
                   
               
               
                 (SEQ ID NO: 297) 
               
               
                 (SGGS)n, 
               
               
                   
               
               
                 (SEQ ID NO: 298) 
               
               
                 (GGGS)n, 
               
               
                   
               
               
                 (SEQ ID NO: 299) 
               
               
                 (GGGGS)n, 
               
               
                   
               
               
                 (SEQ ID NO: 300) 
               
               
                 (G)n, 
               
               
                   
               
               
                 (EAAAK)n, 
               
               
                   
               
               
                 (SEQ ID NO: 16) 
               
               
                 (GGS)n, 
               
               
                   
               
               
                 SGSETPGTSESATPES, 
               
               
                 and 
               
               
                   
               
               
                 (XP)n. 
               
            
           
         
       
         
         
           
             145. The base editor system of embodiment 143 or 144, wherein the base editing fusion protein comprises the amino acid sequence selected from the group consisting of 
           
         
       
    
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 274) 
                   
               
               
                 MPKKKRKVSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPI 
                   
               
               
                   
               
               
                 GRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGAR 
               
               
                   
               
               
                 DAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSS 
               
               
                   
               
               
                 TDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDER 
               
               
                   
               
               
                 EVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTF 
               
               
                   
               
               
                 EPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECA 
               
               
                   
               
               
                 ALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESDLVLGLAIGIG 
               
               
                   
               
               
                 SVGVGILNKVTGEIIHKNSRIFPAAQAENNLVRRTNRQGRRLARRKKHRRVRLNRLFEE 
               
               
                   
               
               
                 SGLITDFTKISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISYLDDASDDGNSSV 
               
               
                   
               
               
                 GDYAQIVKENSKQLETKTPGQIQLERYQTYGQLRGDFTVEKDGKKHRLINVFPTSAYRS 
               
               
                   
               
               
                 EALRILQTQQEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGRYRTSGETLDNIF 
               
               
                   
               
               
                 GILIGKCTFYPDEFRAAKASYTAQEFNLLNDLNNLTVPTETKKLSKEQKNQIINYVKNEK 
               
               
                   
               
               
                 AMGPAKLFKYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDRET 
               
               
                   
               
               
                 LDKLAYVLTLNTEREGIQEALEHEFADGSFSQKQVDELVQFRKANSSIFGKGWHNFSVK 
               
               
                   
               
               
                 LMMELIPELYETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNPVVAKSVRQAI 
               
               
                   
               
               
                 KIVNAAIKEYGDFDNIVIEMARETNEDDEKKAIQKIQKANKDEKDAAMLKAANQYNGK 
               
               
                   
               
               
                 AELPHSVFHGHKQLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHILPLSITFDD 
               
               
                   
               
               
                 SLANKVLVYATANQEKGQRTPYQALDSMDDAWSFRELKAFVRESKTLSNKKKEYLLT 
               
               
                   
               
               
                 EEDISKFDVRKKFIERNLVDTLYASRVVLNALQEHFRAHKIDTKVSVVRGQFTSQLRRH 
               
               
                   
               
               
                 WGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQLLDIETGELISDDEYK 
               
               
                   
               
               
                 ESVFKAPYQHFVDTLKSKEFEDSILFSYQVDSKFNRKISDATIYATRQAKVGKDKADET 
               
               
                   
               
               
                 YVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQTFEKVIEPILENYPNKQINDKG 
               
               
                   
               
               
                 KEVPCNPFLKYKEEHGYIRKYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNKVVLQS 
               
               
                   
               
               
                 VSPWRADVYFNKTTGKYEILGLKYADLQFDKGTGTYKISQEKYNDIKKKEGVDSDSEF 
               
               
                   
               
               
                 KFTLYKNDLLLVKDTETKEQQLFRFLSRTMPKQKHYVELKPYDKQKFEGGEALIKVLG 
               
               
                   
               
               
                 NVANSGQCKKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKLDFPKKKRKVEGADKRT 
               
               
                   
               
               
                 ADGSEFESPKKKRKV, 
               
               
                   
               
               
                 (SEQ ID NO: 275) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTA 
                   
               
               
                   
               
               
                 HAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGA 
               
               
                   
               
               
                 AGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGSS 
               
               
                   
               
               
                 GGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAV 
               
               
                   
               
               
                 LVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCA 
               
               
                   
               
               
                 GAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFF 
               
               
                   
               
               
                 RMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSKRNYILGLAI 
               
               
                   
               
               
                 GITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKL 
               
               
                   
               
               
                 LFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTG 
               
               
                   
               
               
                 NELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQK 
               
               
                   
               
               
                 AYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSV 
               
               
                   
               
               
                 KYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVN 
               
               
                   
               
               
                 EEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEEL 
               
               
                   
               
               
                 TNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKV 
               
               
                   
               
               
                 DLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMI 
               
               
                   
               
               
                 NEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF 
               
               
                   
               
               
                 NYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLA 
               
               
                   
               
               
                 KGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDV 
               
               
                   
               
               
                 KVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVME 
               
               
                   
               
               
                 NQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLY 
               
               
                   
               
               
                 STRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQY 
               
               
                   
               
               
                 GDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVV 
               
               
                   
               
               
                 KLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFI 
               
               
                   
               
               
                 ASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKT 
               
               
                   
               
               
                 QSIKKYSTDILGNLYEVKSKKHPQIIKKGEGADKRTADGSEFESPKKKRKV, 
               
               
                   
               
               
                 (SEQ ID NO: 276) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRH 
                   
               
               
                   
               
               
                 DPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDA 
               
               
                   
               
               
                 KTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD 
               
               
                   
               
               
                 SGGSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVP 
               
               
                   
               
               
                 VGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPC 
               
               
                   
               
               
                 VMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAAL 
               
               
                   
               
               
                 LCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSKRNYI 
               
               
                   
               
               
                 LGLAIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQR 
               
               
                   
               
               
                 VKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVE 
               
               
                   
               
               
                 EDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLL 
               
               
                   
               
               
                 KVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPE 
               
               
                   
               
               
                 ELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK 
               
               
                   
               
               
                 EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDI 
               
               
                   
               
               
                 QEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVP 
               
               
                   
               
               
                 KKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQ 
               
               
                   
               
               
                 KMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLN 
               
               
                   
               
               
                 NPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHIL 
               
               
                   
               
               
                 NLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNN 
               
               
                   
               
               
                 LDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKK 
               
               
                   
               
               
                 VMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLIND 
               
               
                   
               
               
                 TLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIM 
               
               
                   
               
               
                 EQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRN 
               
               
                   
               
               
                 KVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQ 
               
               
                   
               
               
                 AEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTI 
               
               
                   
               
               
                 ASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGEGADKRTADGSEFESPKKKRKVSSGNS 
               
               
                   
               
               
                 NANSRGPSFSSGLVPLSLRGSHSRPGERPFQCRICMRNFSRNEHLEVHTRTHTGEKPFQC 
               
               
                   
               
               
                 RICMRNFSQSTTLKRHLRTHTGEKPFQCRICMRNFSRTEHLARHLKTHLRGSSAQ, 
               
               
                 or 
               
               
                   
               
               
                 (SEQ ID NO: 277) 
                   
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRH 
                   
               
               
                   
               
               
                 DPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDA 
               
               
                   
               
               
                 KTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD 
               
               
                   
               
               
                 SGGSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVP 
               
               
                   
               
               
                 VGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPC 
               
               
                   
               
               
                 VMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAAL 
               
               
                   
               
               
                 LCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSKRNYI 
               
               
                   
               
               
                 LGLAIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQR 
               
               
                   
               
               
                 VKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVE 
               
               
                   
               
               
                 EDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLL 
               
               
                   
               
               
                 KVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPE 
               
               
                   
               
               
                 ELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK 
               
               
                   
               
               
                 EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDI 
               
               
                   
               
               
                 QEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVP 
               
               
                   
               
               
                 KKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQ 
               
               
                   
               
               
                 KMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLN 
               
               
                   
               
               
                 NPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHIL 
               
               
                   
               
               
                 NLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNN 
               
               
                   
               
               
                 LDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKK 
               
               
                   
               
               
                 VMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLIND 
               
               
                   
               
               
                 TLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIM 
               
               
                   
               
               
                 EQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRN 
               
               
                   
               
               
                 KVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQ 
               
               
                   
               
               
                 AEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTI 
               
               
                   
               
               
                 ASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKGEGADKRTADGSEFESPKKKRKVSSGNS 
               
               
                   
               
               
                 NANSRGPSFSSGLVPLSLRGSHSRPGERPFQCRICMRNFSRGEHLRQHTRTHTGEKPFQC 
               
               
                   
               
               
                 RICMRNFSQSGTLKRHLRTHTGEKPFQCRICMRNFSRNDKLVPHLKTHLRGSSAQ. 
               
            
           
         
       
         
         
           
             146. The base editor system of any one of the preceding embodiments, wherein the guide polynucleotide comprises two individual polynucleotides, wherein the two individual polynucleotides are two DNAs, two RNAs or a DNA and a RNA. 
             147. The base editor system of any one of the embodiments 109-146, wherein the guide polynucleotides comprise a crRNA and a tracrRNA, wherein the crRNA comprises a nucleic acid sequence complementary to a target sequence in the SERPINA1 polynucleotide. 
             148. The base editor system of embodiment 147, wherein the target sequence comprises a sequence selected from the group consisting of: GACAAGAAAGGGACUGAAGC (SEQ ID NO: 306), AUCGACAAGAAAGGGACUGA (SEQ ID NO: 307), and ACACACCGGUUGGUGGCCUC (SEQ ID NO: 308) or a complementary thereof 
             149. The base editor system of embodiment 147 or 148, wherein the base editor system comprises a single guide RNA (sgRNA). 
             150. The base editor system of embodiment 149, wherein the sgRNA comprises a sequence selected from the group consisting of ACTCTaGGCAGAGGTCTCAAAGG (SEQ ID NO: 9) and GCTCTaGGCCGAAGTGTCGCAGG (SEQ ID NO: 10). 
             151. The base editor system of any one embodiments 109-150, wherein the base editor system comprises a vector comprising one or more of the guide polynucleotide, the polynucleotide programmable DNA binding domain, and the deaminase domain. 
             152. The base editor system of embodiment 151, wherein the vector is an adenovirus vector, an AAV vector, a lentivirus vector, or a retrovirus vector. 
             153. A method of treating a disease in a subject in need thereof, comprising administering to the subject a base editor system comprising 
             a guide polynucleotide or a nucleic acid encoding the guide polynucleotide; 
             a polynucleotide programmable DNA binding domain or a nucleic acid encoding the polynucleotide programmable DNA binding domain, and 
             an deaminase domain or a nucleic acid encoding the adenosine deaminase domain, 
             wherein the guide polynucleotide is capable of targeting the base editor system to effect deamination of a pathogenic single nucleotide polymorphism (SNP) in a target polynucleotide of a cell in the subject, 
             wherein the pathogenic SNP is causative of a pathogenic amino acid mutation in Table 3A or Table 3B, wherein the deamination of the pathogenic SNP results in a conversion of the pathogenic SNP to its wild-type allele, thereby treating the disease. 
             154. A method of treating a disease in a subject in need thereof, comprising 
             (a) introducing into a cell a base editor system comprising 
             a guide polynucleotides or a nucleic acid encoding the guide polynucleotide; 
             a polynucleotide programmable DNA binding domain or a nucleic acid encoding the polynucleotide programmable DNA binding domain, and 
             an deaminase domain or a nucleic acid encoding the deaminase domain, and 
             (b) administering the cell to the subject, 
             wherein the guide polynucleotide is capable of targeting the base editor system to effect deamination of a pathogenic single nucleotide polymorphism (SNP) in a target polynucleotide of a cell in the subject, 
             wherein the pathogenic SNP is causative of a pathogenic amino acid mutation in Table 3A or Table 3B, wherein the deamination of the pathogenic SNP results in a conversion of the pathogenic SNP to its wild-type allele, thereby treating the disease. 
             155. A method of correcting a SNP causative of a disease in a target polynucleotide, comprising contacting the target polynucleotide with a base editor system comprising 
             a guide polynucleotides; 
             a polynucleotide programmable DNA binding domain, and 
             an deaminase domain, 
             wherein the guide polynucleotide is capable of targeting the base editor system to effect deamination of a pathogenic single nucleotide polymorphism (SNP) in a target polynucleotide of a cell in the subject, 
             wherein the pathogenic SNP is causative of a pathogenic amino acid mutation in Table 3A or Table 3B, wherein the deamination of the pathogenic SNP results in a conversion of the pathogenic SNP to its wild-type allele, thereby correcting the pathogenic SNP in the target polynucleotide. 
             156. A method of producing a modified cell for treatment of a disease, comprising introducing into a cell a base editor system comprising 
             a guide polynucleotides or a nucleic acid encoding the guide polynucleotide; 
             a polynucleotide programmable DNA binding domain or a nucleic acid encoding the polynucleotide programmable DNA binding domain, and 
             an deaminase domain or a nucleic acid encoding the deaminase domain, 
             wherein the guide polynucleotide is capable of targeting the base editor system to effect deamination of a pathogenic single nucleotide polymorphism (SNP) in a target polynucleotide of a cell in the subject, 
             wherein the pathogenic SNP is causative of a pathogenic amino acid mutation in Table 3A or Table 3B, wherein the deamination of the SNP results in a conversion of the pathogenic SNP to its wild-type allele, thereby correcting the pathogenic SNP in the target polynucleotide. 
             157. The method of embodiment 156, wherein the introduction is in vivo or ex vivo. 
             158. The method of embodiment 156 or 157, wherein the cell is obtained from a subject having the disease. 
             159. The method of any one of embodiments 156-158, wherein the disease is selected from the group consisting of Stargardt disease, pseudoxanthoma elasticum, medium-chain acyl-CoA dehydrogenase deficiency, severe combined immunodeficiency, primary hypoxaluria, autosomal recessive hypercholesterolemia, metachromatic leukodystrophy, Marteauz-Lamy Syndrome (MSPVI), Citrullinemia Type I, Darier disease classic homocysteinuria, cystic fibrosis, choroideremia, Neuronal ceroid lipofuscinosis, autosomal dominant deafness carnitine palmitoyltransferase II deficiency, cystinosis, autosomal recessive deafness, agammaglobulinemia, congenital factor XI deficiency, congenital factor V deficiency, congenital factor VII deficiency, hemophilia A, hemophilia B, tyrosinemia type 1, autosomal dominant hypophosphatemic rickets von Gierke disease, Mediterranean G6PD deficiency Morquio Syndrome (MPSIVA classic galactosemia, Gaucher diesease, glutaryl-CoA dehydrogenase deficiency glycine encephalopathy, cone-rod dystrophy, Sly Syndrome (MPSVII), sickle cell disease, intermitent porphyria, Lesch-Nyhan syndrome, Hunter syndrome, Hurler syndrome (MSPII), retinitis pigmentosa Andersen-Tawil syndrome, Meesmann epithelial corneal dystrophy, Parkinson&#39;s disease, Sanfilippo syndrome B (MPSIIIB), CADASIL syndromeblue-cone monochromatismphenylketonuriaPendred syndrome variegate porphyria neuronal ceroid lipofuscinosis 1Creutzfeldt-Jakob disease (CJD), hereditary chronic pancreatitis, Leber congenital amaurosis 2, Blackfan-Diamond anemia, Sanfilippo syndrome A (MPSIIIA), Neimann-Pick disease type A ATTR amyloidosis, retinitis pigmentosa/Usher syndrome type 1C, and myotubular myopathy. 
             160. The method of any one of embodiments 156-159, wherein the target polynucleotide comprises a gene in Table 3A or Table 3B. 
             161. The method of any one of embodiments 156-160, wherein the pathogenic amino acid mutation comprises a pathogenic mutation in Table 3A or Table 3B. 
             162. The method of any one of embodiments 156-161, wherein the polynucleotide programmable DNA binding domain is a Cas9 domain. 
             163. The method of embodiment 162, wherein the Cas9 domain is a nuclease inactive Cas9 domain or a Cas9 nickase domain. 
             164. The method of embodiment 162 or 163, wherein the Cas9 domain comprises a SpCas9 domain. 
             165. The method of embodiment 164, wherein the SpCas9 domain comprises a D10A and/or a H840A amino acid substitution or corresponding amino acid substitutions thereof 166. The method of embodiment 164 or 165, wherein the SpCas9 domain has specificity for a NGN PAM. 
             167. The method of any one of embodiments 164-166, wherein the Cas9 domain comprises amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R, or corresponding amino acid substitutions thereof 168. The method of embodiment 164 or 165, wherein the SpCas9 domain has specificity for a NG PAM, a NNG PAM, a GAA PAM, a GAT PAM, or a CAA PAM. 
             169. The method of embodiment 168, wherein the SpCas9 domain comprises amino acid substitutions E480K, E543K, and E1219V or corresponding amino acid substitutions thereof 
             170. The method of embodiment 162 or 163, wherein the Cas9 domain comprises a SaCas9. 
             171. The method of embodiment 170, wherein the SaCas9 domain has specificity for a NNNRRT PAM. 
             172. The method of embodiment 170 or 171, wherein the SaCas9 domain comprises an amino acid substitution N579A or a corresponding amino acid substitution thereof 
             173. The method of any one of embodiments 170-172, wherein the SaCas9 domain comprises amino acid substitutions E782K, N968K, and R1015H, or corresponding amino acid substitutions thereof 
             174. The method of embodiment 162 or 163, wherein the Cas9 domain comprises a St1Cas9 domain. 
             175. The method of embodiment 174, wherein the St1Cas9 domain has specificity for a NNACCA PAM. 
             176. The method of any one of embodiments 156-175, wherein the deaminase domain comprises a cytidine deaminase domain. 
             177. The method of embodiment 176, wherein the cytidine deaminase domain comprises an APOBEC1 domain. 
             178. The method of any one of embodiments 156-175, wherein the deaminase domain comprises an adenosine deaminase domain. 
             179. The method of embodiment 178, wherein the adenosine deaminase domain comprises a TadA 7.10 domain. 
             180. The method of any one of embodiments 156-179, wherein the base editor system further comprises a UGI domain. 
             181. The method of any one of embodiments 156-180, wherein the base editor system further comprises a zinc finger domain. 
             182. The method of any one of embodiments 156-181, wherein the base editor system further comprises one or more linkers. 
             183. The method of embodiment 182, wherein two or more of the polynucleotide programmable DNA binding domain, the deaminase domain, the UGI domain and the zinc finger domain are connected via a linker, 
             184. The method of embodiment 183, wherein the linker is a peptide linker, thereby forming a base editing fusion protein. 
             185. The method of embodiment 184, wherein the base editing fusion protein comprises the amino acid sequence of BE4. 
             186. The method of embodiment 184, wherein the base editing fusion protein comprises the amino acid sequence of 
           
         
       
    
     
       
         
           
               
            
               
                 (SEQ ID NO: 281) 
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG 
               
               
                   
               
               
                 RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG 
               
               
                   
               
               
                 RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR 
               
               
                   
               
               
                 MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS 
               
               
                   
               
               
                 EVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLH 
               
               
                   
               
               
                 DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV 
               
               
                   
               
               
                 VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMP 
               
               
                   
               
               
                 RQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK 
               
               
                   
               
               
                 YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD 
               
               
                   
               
               
                 SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES 
               
               
                   
               
               
                 FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI 
               
               
                   
               
               
                 YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS 
               
               
                   
               
               
                 GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS 
               
               
                   
               
               
                 NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD 
               
               
                   
               
               
                 ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ 
               
               
                   
               
               
                 SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT 
               
               
                   
               
               
                 FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP 
               
               
                   
               
               
                 LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN 
               
               
                   
               
               
                 EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK 
               
               
                   
               
               
                 TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK 
               
               
                   
               
               
                 DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR 
               
               
                   
               
               
                 RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF 
               
               
                   
               
               
                 KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH 
               
               
                   
               
               
                 KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT 
               
               
                   
               
               
                 QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN 
               
               
                   
               
               
                 KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE 
               
               
                   
               
               
                 RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL 
               
               
                   
               
               
                 ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN 
               
               
                   
               
               
                 GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG 
               
               
                   
               
               
                 FSKESILPKRNSDKLIARKKDWDPKKYGGFmqPTVAYSVLVVAKVEKGKS 
               
               
                   
               
               
                 KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF 
               
               
                   
               
               
                 ELENGRKRMLASAkfLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ 
               
               
                   
               
               
                 KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE 
               
               
                   
               
               
                 QAENIIHLFTLTNLGAPrAFKYFDTTIaRKeYrSTKEVLDATLIHQSITG 
               
               
                   
               
               
                 LYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV. 
               
            
           
         
       
         
         
           
             187. The method of any one of embodiments 156-186, wherein the deamination results in less than 10% indel formation. 
             188. A base editor system comprising 
             a guide polynucleotide or a nucleic acid encoding the guide polynucleotide; 
             a polynucleotide programmable DNA binding domain or a nucleic acid encoding the polynucleotide programmable DNA binding domain, and 
             a deaminase domain or a nucleic acid encoding the adenosine deaminase domain, 
             wherein the guide polynucleotide is capable of targeting the base editor system to effect deamination of a pathogenic single nucleotide polymorphism (SNP) in a target polynucleotide, wherein the pathogenic SNP is causative of a pathogenic amino acid mutation in Table 3A or Table 3B, wherein the deamination of the pathogenic SNP results in a conversion of the pathogenic SNP to its wild-type allele, wherein the target polynucleotide comprises a targeting sequence in Table 3A or Table 3B. 
           
         
       
    
     EXAMPLES 
     The following examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein. 
     Example 1. PAM Variant Validation in Base Editors 
     Novel CRISPR systems and PAM variants enable the base editors to make precise corrections at a target SNP. 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) and PCT/US2016/058344 (WO2017/070632), 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. 
     Example 2. Gene Editing to Correct Alpha-1 Antitrypsin Deficiency (A1AD) 
     Alpha-1 antitrypsin (A1A or A1AT) is a protease inhibitor encoded by the SERPINA1 gene on chromosome 14. This glycoprotein is synthesized mainly in the liver and is secreted into the blood, with serum concentrations of 1.5-3.0 g/L (20-52 μmon) in healthy adults ( FIG.  1   ). A1AT diffuses into the lung interstitium and alveolar lining fluid, where it inactivates neutrophil elastase, thereby protecting the lung tissue from protease-mediated damage. Alpha-1 antitrypsin deficiency (A1AD) is inherited in an autosomal codominant fashion. 
     Over 100 genetic variants of the SERPINA1 gene have been described, but not all are associated with disease. The alphabetic designation of these variants is based on their speed of migration on gel electrophoresis. The most common variant is the M (medium mobility) allele, and the two most frequent deficiency alleles are PiS and PiZ (the latter having the slowest rate of migration). Several mutations have been described that produce no measurable serum protein; these are referred to as “null” alleles. The most common genotype is MM, which produces normal serum levels of alpha-1 antitrypsin. Most people with severe deficiency are homozygous for the Z allele (ZZ). The Z protein misfolds and polymerizes during its production in the endoplasmic reticulum of hepatocytes; these abnormal polymers are trapped in the liver, greatly reducing the serum levels of alpha-1 antitrypsin. The liver disease seen in patients with alpha-1 antitrypsin deficiency is caused by the accumulation of abnormal alpha-1 antitrypsin protein in hepatocytes and the consequent cellular responses, including autophagy, the endoplasmic reticulum stress response and apoptosis.  FIG.  2    shows the most common genotypes (MM, MZ, SS, SZ and ZZ) and the respective serum levels of alpha-1 antitrypsin. Reduced circulating levels of alpha-1 antitrypsin lead to increased neutrophil elastase activity in the lungs; this imbalance of protease and antiprotease activities results in the lung disease associated with this condition ( FIG.  1   ). 
     Alpha-1 antitrypsin deficiency (A1AD) is most common in caucasians, and it most frequently affects the lungs and liver. In the lungs, the most common manifestation is early-onset (patients in their 30s and 40s) panacinar emphysema most pronounced in the lung bases. However, diffuse or upper lobe emphysema can occur, as can bronchiectasis. The most frequently described symptoms include dyspnea, wheezing and cough. Pulmonary function testing of affected individuals shows findings consistent with COPD; however, bronchodilator responsiveness may be observed and may be misdiagnosed as asthma. 
     Liver disease caused by the ZZ genotype manifests in various ways. Affected infants can present in the newborn period with cholestatic jaundice, sometimes with acholic stools (pale or clay-coloured) and hepatomegaly. Conjugated bilirubin, transaminases and gamma-glutamyl transferase levels in blood are elevated. Liver disease in older children and adults can present with an incidental finding of elevated transaminases or with signs of established cirrhosis, including variceal hemorrhage or ascites. Alpha-1 antitrypsin deficiency also predisposes patients to hepatocellular carcinoma. Although the homozygous ZZ genotype is necessary for liver disease to develop, a heterozygous Z mutation can act as a genetic modifier for other diseases by conferring a greater risk of more severe liver disease, such as in hepatitis C infection and cystic fibrosis liver disease. 
     The two most common clinical variants of A1AD are the E264V (PiS) and E342K (PiZ) alleles. More than half of A1AD patients harbor at least one copy of the mutation E342K. Nuclease genome editing via homology directed repair (HDR) is inefficient, and the abundant indels will lower circulating levels and worsen lung symptoms. Gene therapy via AAV to liver worsens liver pathology due to additional misfolded protein. AAVs encoding both wild-type A1AT and siRNA that knocks down E342K A1AT show promise for addressing both pathologies. 
       FIG.  3 A  shows a precise correction base editing strategy for a mutation in the SERPINA1 gene. The “A” nucleobase at position 7 in the sequence (A7), “Target A,” can be edited to restore wild-type.  FIG.  3 B  shows a characterization of A1AT protein secretion as a function of alternate alleles generated by a DNA editor (E342K, D341G, E342G). HEK293T were transfected with vectors encoding A1AT variants, and supernatants were assessed by ELISA for A1AT content. This assay characterized the diminished secretion of an E342K-containing A1AT relative to WT A1AT. The results indicated that the PAM option is AGCT, which is expected to result in editing of “A” at position 5 and/or position 7 (A5 and/or A7) of the SERPINA1 sequence. A1AT function was found to be restored when A7 editing resulted in wild-type protein; when A5 and A7 editing resulted in glutamic acid (E) at position 342 and D341G; and when A7 and A8 were converted to WT and E342G. The assessed phenotypes (activities/function) of the recombinant mutant A1AT included both secretion of protein from cells and inhibition of neutrophil elastase. The functional activity of the A1AT variants is shown in  FIG.  3 C  (inhibition of elastase). Interestingly, the D341G mutation had significant elastase activity, which confirms a restorative outcome for the A5 and A7 editing.  FIG.  4    shows a strategy for generating polypeptide variants (e.g., adenosine deaminase variants). 
     Example 3. Base Editing in HEK298T Cells 
     Base editing efficiency of various SNPs in different genes was tested in HEK298T cells (Table 7). For plasmid transfections, HEK293T cells were transiently transfected with Mirus TransIT293, which is a high efficiency, low toxicity DNA transfection reagent optimized for HEK293 cells, in a 3 μl:1 μg ratio using 250 ng of gRNA plasmid and 750 ng of base editor plasmid. For mRNA transfections, HEK293T cells were electroporated with 3 μg of total RNA using the Neon System at 1150V using two 20 ms pulses. After four days for plasmid transfections and two days for RNA electroporation, genomic DNA was extracted from the cells with a simple lysis buffer containing 0.05% SDS, 25 μg/ml proteinase K, 10 mM Tris pH 8.0, followed by heat inactivation at 85° C. Genomic sites were PCR amplified and sequenced on a MiSeq. Results were analyzed as has been described for base frequencies at each position and for percent indels. For example, the details of indel calculations 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. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Liver target editing of SNP correction 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                 US 
                   
                   
                   
                   
                 Percent 
               
               
                   
                   
                 Patient 
                 Base 
                 SEQ ID 
                   
                   
                 Precise 
               
               
                 Disease 
                 Target 
                 Estimate 
                 Editor 
                 NO: 
                 Protospacer 
                 PAM 
                 Correction 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   Mucopoly - 
                 IDUA 
                 600 
                 ABE 
                 282 
                 GCTCTAGGCCGA 
                 NG 
                 12.53 
               
               
                   saccharidosis  1 
                 W402* 
                   
                   
                   
                 AGTGTCGC 
                 G 
                   
               
               
                   
               
               
                 
                   Homocystinuria 
                 
                 CBS 
                 900 
                 CBE 
                 283 
                 TCACTGGGGTGG 
                 NG 
                 26.1 
               
               
                   
                 I278T 
                   
                   
                   
                 ATCCCGAA 
                 G 
                   
               
               
                   
               
               
                 
                   Homocystinuria 
                 
                 CBS 
                 unknown 
                 ABE 
                 284 
                 GTGGGCATCCTC 
                 NG 
                 1.11 
               
               
                   
                 T191M 
                   
                   
                   
                 ACAATCTC 
                 C 
                   
               
               
                   
               
               
                 Glycogen 
                 G6PC 
                 500 
                 ABE 
                 285 
                 GGACCTAGGCGA 
                 NG 
                 37.22 
               
               
                 Storage 
                 Q347* 
                   
                   
                   
                 GGCAGTAG 
                 G 
                   
               
               
                 Disorder 1a 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glycogen 
                 G6PC 
                 500 
                 ABE 
                 286 
                 GACCTAGGCGAG 
                 NG 
                 43.89 
               
               
                 Storage 
                 Q347* 
                   
                   
                   
                 GCAGTAGG 
                 A 
                   
               
               
                 Disorder 1a 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Glycogen 
                 G6PC 
                 900 
                 ABE 
                 287 
                 CAGTATGGACAC 
                 NN 
                 1.41 
               
               
                 Storage 
                 R83C 
                   
                   
                   
                 TGTCCAAA 
                 GR 
                   
               
               
                 Disorder 1a 
                   
                   
                   
                   
                   
                 RT 
                   
               
               
                   
               
               
                 Alpha-1 
                 SERPINA1 
                 30000 
                 ABE 
                 288 
                 ATCGACAAGAAA 
                 NG 
                 0.39 
               
               
                 Antitrypsin 
                 E342K 
                   
                   
                   
                 GGGACTGA 
                 C 
                   
               
               
                 Deficiency 
                   
                   
                   
                   
                   
                   
                   
               
               
                 (A1AD) 
               
               
                   
               
            
           
         
       
     
     Example 4. Improved NGC-PAM ABE Generated by Mutation Shuffling 
     HEK293T cells that contained an integrated lentiviral cassette of the PiZ ORF expressing a A1AT variant containing E342K (HEK293T-E342K) were transfected with plasmids expressing variant ABEs using Lipo2000. Following transfection, base editing in A1AT was characterized. This approach identified a number of improved ABEs, including Var-3, which has the following amino acid sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 289) 
               
               
                 MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIG 
               
               
                   
               
               
                 RHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG 
               
               
                   
               
               
                 RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFR 
               
               
                   
               
               
                 MRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSS 
               
               
                   
               
               
                 EVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLH 
               
               
                   
               
               
                 DPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRV 
               
               
                   
               
               
                 VFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMP 
               
               
                   
               
               
                 RQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKK 
               
               
                   
               
               
                 YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD 
               
               
                   
               
               
                 SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES 
               
               
                   
               
               
                 FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI 
               
               
                   
               
               
                 YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS 
               
               
                   
               
               
                 GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS 
               
               
                   
               
               
                 NFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD 
               
               
                   
               
               
                 ILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ 
               
               
                   
               
               
                 SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT 
               
               
                   
               
               
                 FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGP 
               
               
                   
               
               
                 LARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPN 
               
               
                   
               
               
                 EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK 
               
               
                   
               
               
                 TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK 
               
               
                   
               
               
                 DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRR 
               
               
                   
               
               
                 RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF 
               
               
                   
               
               
                 KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH 
               
               
                   
               
               
                 KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT 
               
               
                   
               
               
                 QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDN 
               
               
                   
               
               
                 KVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE 
               
               
                   
               
               
                 RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKL 
               
               
                   
               
               
                 ESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN 
               
               
                   
               
               
                 GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG 
               
               
                   
               
               
                 FSKESILPKRNSDKLIARKKDWDPKKYGGFmqPTVAYSVLVVAKVEKGKS 
               
               
                   
               
               
                 KKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLF 
               
               
                   
               
               
                 ELENGRKRMLASAkfLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ 
               
               
                   
               
               
                 KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIRE 
               
               
                   
               
               
                 QAENIIHLFTLTNLGAPrAFKYFDTTIaRKeYrSTKEVLDATLIHQSITG 
               
               
                   
               
               
                 LYETRIDLSQLGGDEGADKRTADGSEFESPKKKRKV 
               
            
           
         
       
     
     HEK293T-E342K were transfected by Neon electroporation using 2.5 μg Var-3 ABE mRNA and 1000 ng gRNA 191 length 20 nt. 
     The gRNA backbone provided as sgRNA for spCas9 base editors is as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 2) 
               
               
                 5′- GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAU 
               
               
                   
               
               
                 CAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3′ 
               
            
           
         
       
     
     The gRNAs useful in the methods of the invention include the following: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 3) 
               
               
                 5′-ACCAUCGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGU 
               
               
                   
               
               
                 UAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU 
               
               
                   
               
               
                 GCUUUU-3′; 
               
               
                   
               
               
                 (SEQ ID NO: 4) 
               
               
                 5′-CCAUCGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGUU 
               
               
                   
               
               
                 AAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG 
               
               
                   
               
               
                 CUUUU-3′; 
               
               
                   
               
               
                 (SEQ ID NO: 5) 
               
               
                 5′-CAUCGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGUUA 
               
               
                   
               
               
                 AAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC 
               
               
                   
               
               
                 UUUU-3′; 
               
               
                   
               
               
                 (SEQ ID NO: 6) 
               
               
                 5′-AUCGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGUUAA 
               
               
                   
               
               
                 AAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU 
               
               
                   
               
               
                 UUU-3′; 
               
               
                   
               
               
                 (SEQ ID NO: 7) 
               
               
                 5′-UCGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAA 
               
               
                   
               
               
                 AUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUU 
               
               
                   
               
               
                 UU-3′; 
               
               
                 and 
               
               
                   
               
               
                 (SEQ ID NO: 8) 
               
               
                 5′-CGACAAGAAAGGGACUGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAA 
               
               
                   
               
               
                 UAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU 
               
               
                   
               
               
                 U-3′. 
               
            
           
         
       
     
     Editing with mRNA and sgRNA was primarily unlinked at A5 and A7. The Var-3 ABE vastly outperformed other variant ABEs and resulted in a total beneficial correction of more than 35% (A7 only=WT)+(A5+A7=WT+D341G—no change in secretion). 
     Example 5. Optimizing gRNA Length for Correction of E342K in HEK293T Cells 
     Synthego gRNAs and mRNA for ABE Var-3 were transfected into HEK293T-E342K using the Neon System in duplicate. gRNAs having target complementary lengths of 18 and 19 nucleotides yielded a better ratio of editing at the desired A7 position compared with the A5 position ( FIG.  5   ). This pattern is consistent with a mechanism by which truncated gRNAs shorten the accessible region of the R-loop and thus lower editing of positions close to the end of the gRNA. 
     The 18 and 19 nucleotide gRNAs and ABE Var-3 are tested for their ability to correct E342K mutations in induced pluripotent stem cell-derived E342 hepatocytes and in a PiZZ mouse model. SpCas9 mutants having altered PAM binding specificities were generated using NGC PAM evolution ( FIGS.  6 A,  6 B ). These SpCas9 mutants were selected to enrich for mutations within the PAM-interacting (PI) domain of Cas9. The library is screened for SpCas9s having altered PAM specificities. 
     Example 6. Gene Editing to Correct Glycogen Storage Disorder Type 1a (Von Gierke Disease) 
     Glycogen Storage Disorder Type 1 (also known as GSD1 or Von Gierke Disease) is an inherited disorder that results in a deficiency in glycogenolysis and gluconeogenesis, with accumulation of glycogen in tissues, causing severe hypoglycemia and lactic acidosis with potential CNS damage. About one in 100,000 newborns in the United States are born with GSD1. 
     There are two types of GSD1, Type 1a (GSD1a) and Type 1b (GSD1b), which are caused by different genetic mutations. GSD1a is caused by a mutation in glucose-6-phosphatase (G6PC) and affects about 80% of patients with GSD1. About 25% of Caucasian patients carry the recessive mutation Q347*. Current treatment regimen involves regular or continuous cornstarch feeding between meals (amylase converts starch directly to glucose). 
     Base Editing Strategy for the Correction of the Q347X Mutation 
     There is a direct correction of Q347* to restore expression of G6PC and normalize glucose metabolism. Base editors may be used for the correction of Q347X by efficiently converting A&gt;G at a targeted site. A representative target site (highlighted) is shown in  FIG.  7   . A precise correction at this site would yield the following conversion: TAG&gt;CAG (stop codon&gt;Glutamine). The base editor may utilize either the NGG PAM recognition sequence or the NGA PAM recognition sequence. The tissue and delivery strategy may include liver lipid nanoparticle (LNP) delivery. 
     In Vitro Transfection of iPSc-Derived Hepatocytes Harboring the Q347X Mutation 
     Base editing was tested using an in vitro transfection method in iPSc-derived hepatocytes (Definigen, Lot 00419 F 002). The GSD1a iPSc-derived hepatocytes are compound heterozygous (Q347X/G222R) and harbor the Q347X mutation. GSD1a cells were plated and allowed to mature. 
     As shown in  FIGS.  8 A and  8 B , a transfection schedule was selected based on the known maturation cycle for GSD1a iPSc-derived hepatocytes.  FIG.  8 A  provides a timeline of the transfection schedule showing representative time points for plating, transfection, and cell harvest.  FIG.  8 B  shows representative images of maturing GSD1a iPSc-derived hepatocytes on Day 5 and Day 7. After maturation (e.g. Day 12), the GSD1a cells were transfected with the base editor ABE7.10 VRQR/gRNA 272. 48-72 hrs post transfection (e.g. Day 14), the GSD1a transfected cells were harvested for gDNA. 
     Representative base editing precise correction data of G6PC Q347X for GSD1a is shown in  FIGS.  9 A and  9 B . In  FIG.  9 A , base editing efficiency of G6PC Q347X in HEK293T cells for ABE-On target, ABE-Bystander, Indels, and Nuclease-Indels was examined using either NGA PAM or NGG PAM sequences. The targeted/insert sequence for G6PC using NGG PAM is shown below:
         gg a  cct  a gg cga ggc agt ag ggg       

     The above target/insert sequence contains two “a” nucleobases corresponding to bystander (shown in italic and underlining) and on target (shown in bold and underlining). 
     The targeted/insert sequence for G6PC using NGA PAM is shown below: 
     
       
         
           
               
               
            
               
                   
                 g   a    cct    a   gg cga ggc agt ag gga 
               
            
           
         
       
     
     The above target/insert sequence contains two “a” nucleobases corresponding to bystander (shown in italic and underlining) and on target (shown in bold and underlining). 
     The NGA PAM gRNA 272 yielded &gt;40% precise correction of Q347X in HEK293T cells with low indels and no detectable bystander V384A ( FIG.  9 A ). Thus, high base editing efficiency was achieved using HEK293 cells. 
     In  FIG.  9 B , the base editing efficiency in G6PC Q347X in patient iPSc-derived hepatocytes for ABE-On target, ABE-Bystander, Indels, and Nuclease-Indels was examined using either NGA PAM or NGG PAM sequences. Similar editing of G6PC Q347X in patient iPSc-derived hepatocytes was observed with both NGA (n=4) and NGG (n=2), with negligible Indels, bystander V384A. As shown in  FIG.  9 B , the precise correction in heterozygous patient iPS-derived Q347X hepatocytes resulted in about 8%-15% A&gt;G conversion efficiency. While lower than the HEK293 cells, the base editing in Q347X iPSc hepaotcytes yielded cleaner results as compared to bystander. 
     A-to-G Conversion Efficiency for ABE Variants in Patient iPSc-Derived Hepatocytes 
     The A-to-G conversion efficiency was tested for ABE variants in G6PC Q347X patient iPSc-derived hepatocytes. mRNA variants were generated (TriLink pBxt464, MSP464, MSP465, MSP471) and transfected into GSD1a patient iPSc-derived hepatocytes on Day 12 of culture. On Day 14, the cells were harvested for gDNA isolation and PAS staining was conducted. 
     The targeted/insert sequence for G6PC using GGA PAM is shown below: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 290) 
               
               
                   
                 G   a   cct   a   ggcgaggcagtagg 
               
            
           
         
       
     
     The above target/insert sequence contains two “a” nucleobases corresponding to bystander (shown in italic and underlining) and on target (shown in bold and underlining). 
     The percent of base editing correction efficiency of Q347X was similar among the mRNA variants with about 10% A-to-G conversion efficiency for the heterozygous sequence ( FIG.  10   ). 
     Example 7. Gene Editing to Correct Mucopolysaccharidosis Type 1 (Hurler Syndrome) 
     Mucopolysaccharidosis type 1 (also known as MPS1 or Hurler Syndrome) is 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 gene (IDUA), leading to deficiency of alpha-L-iduronidase, which is essential for the breakdown of glycosaminoglycans in lysosomes. 
     The amino acid sequence of a representative mouse alpha-L-iduronidase (IDUA) protein, found under NCBI Reference Sequence No. NP_032351.2, is provided below: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 291) 
               
            
           
           
               
               
            
               
                 1 
                 MRPPRPSSAM LTFFAAFLAA PLALAESPYL VRVDAARPLR 
               
               
                   
               
               
                   
                 PLLPFWRSTG FCPPLPHDQA 
               
               
                   
               
               
                 61 
                 DQYDLSWDQQ LNLAYIGAVP HSGIEQVRIH WLLDLITARK 
               
               
                   
               
               
                   
                 SPGQGLMYNF THLDAFLDLL 
               
               
                   
               
               
                 121 
                 MENQLLPGFE LMGSPSGYFT DFDDKQQVFE WKDLVSLLAR 
               
               
                   
               
               
                   
                 RYIGRYGLTH VSKWNFETWN 
               
               
                   
               
               
                 181 
                 EPDHHDFDNV SMTTQGFLNY YDACSEGLRI ASPTLKLGGP 
               
               
                   
               
               
                   
                 GDSFHPLPRS PMCWSLLGHC 
               
               
                   
               
               
                 241 
                 ANGTNFFTGE VGVRLDYISL HKKGAGSSIA ILEQEMAVVE 
               
               
                   
               
               
                   
                 QVQQLFPEFK DTPIYNDEAD 
               
               
                   
               
               
                 301 
                 PLVGWSLPQP WRADVTYAAL VVKVIAQHQN LLFANSSSSM 
               
               
                   
               
               
                   
                 RYVLLSNDNA FLSYHPYPFS 
               
               
                   
               
               
                 361 
                 QRTLTARFQV NNTHPPHVQL LRKPVLTVMG LMALLDGEQL 
               
               
                   
               
               
                   
                 WAEVSKAGAV LDSNHTVGVL 
               
               
                   
               
               
                 421 
                 ASTHHPEGSA AAWSTTVLIY TSDDTHAHPN HSIPVTLRLR 
               
               
                   
               
               
                   
                 GVPPGLDLVY IVLYLDNQLS 
               
               
                   
               
               
                 481 
                 SPYSAWQHMG QPVFPSAEQF RRMRMVEDPV AEAPRPFPAR 
               
               
                   
               
               
                   
                 GRLTLHRKLP VPSLLLVHVC 
               
               
                   
               
               
                 541 
                 TRPLKPPGQV SRLRALPLTH GQLILVWSDE RVGSKCLWTY 
               
               
                   
               
               
                   
                 EIQFSQKGEE YAPINRRPST 
               
               
                   
               
               
                 601 
                 FNLFVFSPDT AVVSGSYRVR ALDYWARPGP FSDPVTYLDV 
               
               
                   
               
               
                   
                 PAS 
               
            
           
         
       
     
     A representative mouse alpha-L-iduronidase (IDUA) nucleic acid sequence, found under NCBI Reference Sequence No. NM_008325.4 is provided below: 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 292) 
                   
               
            
           
           
               
               
               
            
               
                 1 
                 ctctgtgccc acccactgcc aagagggaca ggtctcaaag gtcagggcag tgtcccggga 
                   
               
               
                   
               
               
                 61 
                 aggagggcat cggctcctgg gagcggcctt aggacgcggg gtggactctc accatcgcac 
               
               
                   
               
               
                 121 
                 aggaagccag ccagtcccca gatgaagtcc gagcagaggt ggcagaagag cacctacagg 
               
               
                   
               
               
                 181 
                 cctccagcga gaccgagaca gccgcaagaa taatggccgc tctgagacac ccaagcactg 
               
               
                   
               
               
                 241 
                 ctaatgttgg ttccattttt ggagcgcctg ggacgcagcg gaactcgcca gcacggggcg 
               
               
                   
               
               
                 301 
                 gcgcgtgact gggttccttt ttgtcccggc ctggcgagag gtcacgtggg gcgttacgca 
               
               
                   
               
               
                 361 
                 gaggcggaac actgcgaccg ccgcctaaaa agcttgctgt ttaggggcac ctggatatcc 
               
               
                   
               
               
                 421 
                 caaccatgcg acccccgcgt ccctcctcag ctatgctgac gttttttgct gcgttcttgg 
               
               
                   
               
               
                 481 
                 ccgcgccctt ggcgctggct gagtcaccgt acctggtgcg tgtggacgca gcccgcccgc 
               
               
                   
               
               
                 541 
                 tgaggcctct gttgcccttc tggaggagca ccggcttctg ccccccactg cctcacgacc 
               
               
                   
               
               
                 601 
                 aggctgacca gtacgacctt agttgggacc agcaactgaa ccttgcctac ataggtgccg 
               
               
                   
               
               
                 661 
                 tacctcacag tggcattgag caggtccgga tacactggct gctggatctc atcacagcca 
               
               
                   
               
               
                 721 
                 ggaagtcacc tgggcaggga cttatgtaca acttcaccca cttggatgca ttcttggacc 
               
               
                   
               
               
                 781 
                 ttctcatgga gaaccagctt ctccctggat ttgagctcat gggcagtcct tctgggtact 
               
               
                   
               
               
                 841 
                 tcacggactt tgatgacaag cagcaggtgt ttgaatggaa ggacctggtt tctctcttgg 
               
               
                   
               
               
                 901 
                 ccaggagata cattggtagg tatgggctga cacacgtttc caagtggaac tttgagactt 
               
               
                   
               
               
                 961 
                 ggaatgaacc agaccaccat gactttgaca acgtgtccat gaccacacaa ggcttcctga 
               
               
                   
               
               
                 1021 
                 attactatga tgcctgctct gaggggctgc gcattgccag ccccactttg aagttgggtg 
               
               
                   
               
               
                 1081 
                 gtcctgggga ttccttccac cccctgccaa ggtcaccaat gtgctggagc ctcctgggtc 
               
               
                   
               
               
                 1141 
                 actgtgccaa tggaaccaac ttcttcactg gcgaggtggg cgtgcgtctg gattacatct 
               
               
                   
               
               
                 1201 
                 ccctgcacaa gaagggtgca ggtagctcca tcgccatcct ggagcaggag atggcagttg 
               
               
                   
               
               
                 1261 
                 tggagcaggt ccagcagctc ttccctgagt tcaaggatac ccctatttac aatgacgagg 
               
               
                   
               
               
                 1321 
                 cagaccctct ggtgggctgg tccctgccac aaccttggag agctgatgtg acttatgcgg 
               
               
                   
               
               
                 1381 
                 ccctggtggt gaaggtcatt gcacagcacc agaacctgct gtttgccaac agcagttcct 
               
               
                   
               
               
                 1441 
                 ccatgcgcta tgtgctcctc agcaatgaca atgccttcct gagctaccac ccgtaccctt 
               
               
                   
               
               
                 1501 
                 tctcccagcg cacacttact gctcgattcc aggtcaacaa tactcaccca ccccacgtgc 
               
               
                   
               
               
                 1561 
                 agttgctgcg aaagccagta ctcacagtca tggggctcat ggccctgttg gatggagaac 
               
               
                   
               
               
                 1621 
                 aactctgggc agaggtctca aaggctgggg ctgtgttgga cagcaatcat acagtgggtg 
               
               
                   
               
               
                 1681 
                 tcctggccag cacccatcac cctgaaggct ccgcagcggc ctggagtacc acagtcctca 
               
               
                   
               
               
                 1741 
                 tctacactag tgatgacacc cacgcacacc ccaaccacag tatccctgtg actcttcgcc 
               
               
                   
               
               
                 1801 
                 tgcgtggggt acctcctggc ttggatcttg tctacatagt actctactta gacaatcaac 
               
               
                   
               
               
                 1861 
                 tcagcagccc ctacagtgcg tggcagcaca tgggccagcc agtcttcccc tctgcagagc 
               
               
                   
               
               
                 1921 
                 agttccgacg tatgcgcatg gtggaggacc ccgtggctga ggcaccacgc ccctttcctg 
               
               
                   
               
               
                 1981 
                 ctaggggccg cctgacccta caccggaagc ttccggtgcc atcactcctg ctggtgcatg 
               
               
                   
               
               
                 2041 
                 tatgcacacg ccccttgaag ccacctgggc aggtcagccg gctccgtgca ctgcccctga 
               
               
                   
               
               
                 2101 
                 cacatggaca gctgattttg gtctggtcag atgagcgtgt gggctccaag tgcctgtgga 
               
               
                   
               
               
                 2161 
                 catatgagat ccagttttcc cagaaaggtg aagagtatgc cccaatcaac aggaggccgt 
               
               
                   
               
               
                 2221 
                 ctacttttaa cctctttgtg ttcagcccag acacagctgt ggtctctggc tcctaccgag 
               
               
                   
               
               
                 2281 
                 ttcgagcatt ggattactgg gcccggccag gccccttctc cgaccctgtg acttacctgg 
               
               
                   
               
               
                 2341 
                 atgtccctgc ctcatgagag ccactggctc ctagtgactt gtgaatctgt gctgactggt 
               
               
                   
               
               
                 2401 
                 gaatggagtc aaccagtatg agctagactg ccattagcta ggcagctgac tgtcagcttc 
               
               
                   
               
               
                 2461 
                 tattgttctt cccctatttc cctttaaagt gtctttctct acctcagact tagggtcagt 
               
               
                   
               
               
                 2521 
                 ctttgtggct aagcacttta taggcccagt tggagtgacc tttgcccacc ttcctcccca 
               
               
                   
               
               
                 2581 
                 tgcccagctg ttcaaaaagt ttaaatgtgg gactggaaag atggctcagt agataaagtg 
               
               
                   
               
               
                 2641 
                 cttgctgtgc aggcccaggg acttgtgttc agatatctag cactcatgta taggctgggc 
               
               
                   
               
               
                 2701 
                 atggcaatat atgcctattg tcctagtgtt ggtggaaggg acagagacag gccagggttc 
               
               
                   
               
               
                 2761 
                 cctggccttc cagtctacct gaaactgcaa gctccaggtt cagtaagaaa ccctgtttta 
               
               
                   
               
               
                 2821 
                 gaaaaatcaa gtagagtgct tggtacacac acacacacac acacacagag 
               
               
                   
               
               
                 2881 
                 tctaaattta gtttcttgag cttctgtaat atcaaaaatg aagttatgta cttctgaaat 
               
               
                   
               
               
                 2941 
                 acaatactgc acagagtaag catcttcatt ccaacaggaa aaagaaatga cagggaagga 
               
               
                   
               
               
                 3001 
                 tttaagtgaa acaagaccaa agcatagcaa gacaaacgtt aaatcctgca gctccattct 
               
               
                   
               
               
                 3061 
                 cagcatcggg gcccatgatc ctgtgatgtg ctggacagtc tgtgtctgtg gtgttgccat 
               
               
                   
               
               
                 3121 
                 ttccagccgc catgaccttt ctcctaggct ggtgtcttgg gcttcctatt gattcgataa 
               
               
                   
               
               
                 3181 
                 accgcgatga atgagcagaa gcatctgggt agggaagcgt tgacttcact tgtattccta 
               
               
                   
               
               
                 3241 
                 cattacagtc tatcatcgaa ggcagtcagg caggcacctg gaggcaggaa gtcatggaga 
               
               
                   
               
               
                 3301 
                 ggccatggag gggtgctgct tactcagact atgatcttac acatcccggg atcaccagcc 
               
               
                   
               
               
                 3361 
                 aaagggtggg cccccaccca caacggtctg gaccctccca catcaatcac tagtttaaga 
               
               
                   
               
               
                 3421 
                 aaacaggctt atctataggt caatcttgtt ggggcatttt tctcaattga ggttccttct 
               
               
                   
               
               
                 3481 
                 tcccaaatga ctctagcttg taataaactg aaataaagcc accccaatct tgccacacat 
               
               
                   
               
               
                 3541 
                 tacctggcct cccaagtctt cctttgaaat ctgggtggaa gccaacataa ccctgtcact 
               
               
                   
               
               
                 3601 
                 gtaactctga tattctacct tctaagccag catcctgtgg atcacagcct actatcagct 
               
               
                   
               
               
                 3661 
                 tgagtggtag ttgaggactc ctgggtcatc catggctaca ataagcatga agtgcctgag 
               
               
                   
               
               
                 3721 
                 gcttagtccc atgcatttgt ggagaatatt atgatgatga tatctagtag aggggggagg 
               
               
                   
               
               
                 3781 
                 ctgttcacct caaagggaac aggaagtaga gtggggatag aattaagcta aatacttctt 
               
               
                   
               
               
                 3841 
                 cactgacccg ctttgtttaa ctcagccctg catcctaaag tttctagaat ctccccaaac 
               
               
                   
               
               
                 3901 
                 agacctatta cctggaaact acctttaagg tgtaagcctg ggctgatctt aataggctga 
               
               
                   
               
               
                 3961 
                 tgttcctacc ttggttctgg ccacgggaag aaccctctct cctttagtac aaacccctgg 
               
               
                   
               
               
                 4021 
                 tgtgccccac cagagagcct gttggacaca cgtgtcctta attcattctg cacatttttc 
               
               
                   
               
               
                 4081 
                 ttctctccat cagcacacag aagttcagca tacagaggtt tgtgctgaaa tgtaggcgta 
               
               
                   
               
               
                 4141 
                 ctcccaagct ctccccagag tactatcacc tactgtccag gaaatgagtc tgagtgcagt 
               
               
                   
               
               
                 4201 
                 gctcatatac actgcatggc tacatccaaa gtcagagttc ctctgccctc atgcctcttg 
               
               
                   
               
               
                 4261 
                 aagtgaacga aatgtgatga ccttctgcag ggtgtttttt agtcctctgt ggaccctagg 
               
               
                   
               
               
                 4321 
                 ctggccttgg catcttggct cacctgtccc agagttacta ctattaagat tacaggtgtg 
               
               
                   
               
               
                 4381 
                 taccaccatg cctgccaatt acctctcact ttaaataaaa tatgacattt 
               
            
           
         
       
     
     The amino acid sequence of a representative human alpha-L-iduronidase (IDUA) protein, found under NCBI Reference Sequence No. NP_000194.2, is provided below: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 293) 
               
            
           
           
               
               
            
               
                 1 
                 MRPLRPRAAL LALLASLLAA PPVAPAEAPH LVHVDAARAL 
               
               
                   
               
               
                   
                 WPLRRFWRST GFCPPLPHSQ 
               
               
                   
               
               
                 61 
                 ADQYVLSWDQ QLNLAYVGAV PHRGIKQVRT HWLLELVTTR 
               
               
                   
               
               
                   
                 GSTGRGLSYN FTHLDGYLDL 
               
               
                   
               
               
                 121 
                 LRENQLLPGF ELMGSASGHF TDFEDKQQVF EWKDLVSSLA 
               
               
                   
               
               
                   
                 RRYIGRYGLA HVSKWNFETW 
               
               
                   
               
               
                 181 
                 NEPDHHDFDN VSMTMQGFLN YYDACSEGLR AASPALRLGG 
               
               
                   
               
               
                   
                 PGDSFHTPPR SPLSWGLLRH 
               
               
                   
               
               
                 241 
                 CHDGTNFFTG EAGVRLDYIS LHRKGARSSI SILEQEKVVA 
               
               
                   
               
               
                   
                 QQIRQLFPKF ADTPIYNDEA 
               
               
                   
               
               
                 301 
                 DPLVGWSLPQ PWRADVTYAA MVVKVIAQHQ NLLLANTTSA 
               
               
                   
               
               
                   
                 FPYALLSNDN AFLSYHPHPF 
               
               
                   
               
               
                 361 
                 AQRTLTARFQ VNNTRPPHVQ LLRKPVLTAM GLLALLDEEQ 
               
               
                   
               
               
                   
                 LWAEVSQAGT VLDSNHTVGV 
               
               
                   
               
               
                 421 
                 LASAHRPQGP ADAWRAAVLI YASDDTRAHP NRSVAVTLRL 
               
               
                   
               
               
                   
                 RGVPPGPGLV YVTRYLDNGL 
               
               
                   
               
               
                 481 
                 CSPDGEWRRL GRPVFPTAEQ FRRMRAAEDP VAAAPRPLPA 
               
               
                   
               
               
                   
                 GGRLTLRPAL RLPSLLLVHV 
               
               
                   
               
               
                 541 
                 CARPEKPPGQ VTRLRALPLT QGQLVLVWSD EHVGSKCLWT 
               
               
                   
               
               
                   
                 YEIQFSQDGK AYTPVSRKPS 
               
               
                   
               
               
                 601 
                 TFNLFVFSPD TGAVSGSYRV RALDYWARPG PFSDPVPYLE 
               
               
                   
               
               
                   
                 VPVPRGPPSP GNP 
               
            
           
         
       
     
     A representative human alpha-L-iduronidase (IDUA) nucleic acid sequence, found under NCBI Reference Sequence No. NM_000203, is provided below: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 305) 
               
            
           
           
               
               
            
               
                 1 
                 agtgcagccc gaagccccgc agtccccgag cacgcgtggc 
               
               
                   
               
               
                   
                 catgcgtccc ctgcgccccc 
               
               
                   
               
               
                 61 
                 gcgccgcgct gctggcgctc ctggcctcgc tcctggccgc 
               
               
                   
               
               
                   
                 gcccccggtg gccccggccg 
               
               
                   
               
               
                 121 
                 aggccccgca cctggtgcat gtggacgcgg cccgcgcgct 
               
               
                   
               
               
                   
                 gtggcccctg cggcgcttct 
               
               
                   
               
               
                 181 
                 ggaggagcac aggcttctgc cccccgctgc cacacagcca 
               
               
                   
               
               
                   
                 ggctgaccag tacgtcctca 
               
               
                   
               
               
                 241 
                 gctgggacca gcagctcaac ctcgcctatg tgggcgccgt 
               
               
                   
               
               
                   
                 ccctcaccgc ggcatcaagc 
               
               
                   
               
               
                 301 
                 aggtccggac ccactggctg ctggagcttg tcaccaccag 
               
               
                   
               
               
                   
                 ggggtccact ggacggggcc 
               
               
                   
               
               
                 361 
                 tgagctacaa cttcacccac ctggacgggt acctggacct 
               
               
                   
               
               
                   
                 tctcagggag aaccagctcc 
               
               
                   
               
               
                 421 
                 tcccagggtt tgagctgatg ggcagcgcct cgggccactt 
               
               
                   
               
               
                   
                 cactgacttt gaggacaagc 
               
               
                   
               
               
                 481 
                 agcaggtgtt tgagtggaag gacttggtct ccagcctggc 
               
               
                   
               
               
                   
                 caggagatac atcggtaggt 
               
               
                   
               
               
                 541 
                 acggactggc gcatgtttcc aagtggaact tcgagacgtg 
               
               
                   
               
               
                   
                 gaatgagcca gaccaccacg 
               
               
                   
               
               
                 601 
                 actttgacaa cgtctccatg accatgcaag gcttcctgaa 
               
               
                   
               
               
                   
                 ctactacgat gcctgctcgg 
               
               
                   
               
               
                 661 
                 agggtctgcg cgccgccagc cccgccctgc ggctgggagg 
               
               
                   
               
               
                   
                 ccccggcgac tccttccaca 
               
               
                   
               
               
                 721 
                 ccccaccgcg atccccgctg agctggggcc tcctgcgcca 
               
               
                   
               
               
                   
                 ctgccacgac ggtaccaact 
               
               
                   
               
               
                 781 
                 tcttcactgg ggaggcgggc gtgcggctgg actacatctc 
               
               
                   
               
               
                   
                 cctccacagg aagggtgcgc 
               
               
                   
               
               
                 841 
                 gcagctccat ctccatcctg gagcaggaga aggtcgtcgc 
               
               
                   
               
               
                   
                 gcagcagatc cggcagctct 
               
               
                   
               
               
                 901 
                 tccccaagtt cgcggacacc cccatttaca acgacgaggc 
               
               
                   
               
               
                   
                 ggacccgctg gtgggctggt 
               
               
                   
               
               
                 961 
                 ccctgccaca gccgtggagg gcggacgtga cctacgcggc 
               
               
                   
               
               
                   
                 catggtggtg aaggtcatcg 
               
               
                   
               
               
                 1021 
                 cgcagcatca gaacctgcta ctggccaaca ccacctccgc 
               
               
                   
               
               
                   
                 cttcccctac gcgctcctga 
               
               
                   
               
               
                 1081 
                 gcaacgacaa tgccttcctg agctaccacc cgcacccctt 
               
               
                   
               
               
                   
                 cgcgcagcgc acgctcaccg 
               
               
                   
               
               
                 1141 
                 cgcgcttcca ggtcaacaac acccgcccgc cgcacgtgca 
               
               
                   
               
               
                   
                 gctgttgcgc aagccggtgc 
               
               
                   
               
               
                 1201 
                 tcacggccat ggggctgctg gcgctgctgg atgaggagca 
               
               
                   
               
               
                   
                 gctctgggcc gaagtgtcgc 
               
               
                   
               
               
                 1261 
                 aggccgggac cgtcctggac agcaaccaca cggtgggcgt 
               
               
                   
               
               
                   
                 cctggccagc gcccaccgcc 
               
               
                   
               
               
                 1321 
                 cccagggccc ggccgacgcc tggcgcgccg cggtgctgat 
               
               
                   
               
               
                   
                 ctacgcgagc gacgacaccc 
               
               
                   
               
               
                 1381 
                 gcgcccaccc caaccgcagc gtcgcggtga ccctgcggct 
               
               
                   
               
               
                   
                 gcgcggggtg ccccccggcc 
               
               
                   
               
               
                 1441 
                 cgggcctggt ctacgtcacg cgctacctgg acaacgggct 
               
               
                   
               
               
                   
                 ctgcagcccc gacggcgagt 
               
               
                   
               
               
                 1501 
                 ggcggcgcct gggccggccc gtcttcccca cggcagagca 
               
               
                   
               
               
                   
                 gttccggcgc atgcgcgcgg 
               
               
                   
               
               
                 1561 
                 ctgaggaccc ggtggccgcg gcgccccgcc ccttacccgc 
               
               
                   
               
               
                   
                 cggcggccgc ctgaccctgc 
               
               
                   
               
               
                 1621 
                 gccccgcgct gcggctgccg tcgcttttgc tggtgcacgt 
               
               
                   
               
               
                   
                 gtgtgcgcgc cccgagaagc 
               
               
                   
               
               
                 1681 
                 cgcccgggca ggtcacgcgg ctccgcgccc tgcccctgac 
               
               
                   
               
               
                   
                 ccaagggcag ctggttctgg 
               
               
                   
               
               
                 1741 
                 tctggtcgga tgaacacgtg ggctccaagt gcctgtggac 
               
               
                   
               
               
                   
                 atacgagatc cagttctctc 
               
               
                   
               
               
                 1801 
                 aggacggtaa ggcgtacacc ccggtcagca ggaagccatc 
               
               
                   
               
               
                   
                 gaccttcaac ctctttgtgt 
               
               
                   
               
               
                 1861 
                 tcagcccaga cacaggtgct gtctctggct cctaccgagt 
               
               
                   
               
               
                   
                 tcgagccctg gactactggg 
               
               
                   
               
               
                 1921 
                 cccgaccagg ccccttctcg gaccctgtgc cgtacctgga 
               
               
                   
               
               
                   
                 ggtccctgtg ccaagagggc 
               
               
                   
               
               
                 1981 
                 ccccatcccc gggcaatcca tgagcctgtg ctgagcccca 
               
               
                   
               
               
                   
                 gtgggttgca cctccaccgg 
               
               
                   
               
               
                 2041 
                 cagtcagcga gctggggctg cactgtgccc atgctgccct 
               
               
                   
               
               
                   
                 cccatcaccc cctttgcaat 
               
               
                   
               
               
                 2101 
                 atatttttat attttattat tttcttttat atcttggtac 
               
               
                   
               
               
                   
                 caacgccccc tttaaagcgg 
               
               
                   
               
               
                 2161 
                 ctttgcacag gtca 
               
            
           
         
       
     
     Base editing was tested in the HEK293T cell line with mouse and human IDUA target sequences ( FIG.  11   ). The targeted/insert sequence for mouse IDUA is shown below and corresponds to nucleic acids 1612-1824 of the mouse IDUA gene sequence shown above. 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 294) 
               
               
                   
                 atggagaaca actct   a   ggca gaggtctcaa aggctggggc 
               
               
                   
                   
               
               
                   
                 tgtgttggac agcaatcata cagtgggtgt cctggccagc 
               
               
                   
                   
               
               
                   
                 acccatcacc ctgaaggctc cgcagcggcc tggagtacca 
               
               
                   
                   
               
               
                   
                 cagtcctcat ctacactagt gatgacaccc acgcacaccc 
               
               
                   
                   
               
               
                   
                 caaccacagt atccctgtga ctcttcgcct gcgtggggta 
               
               
                   
                   
               
               
                   
                 cctcctggct tgg 
               
            
           
         
       
     
     The above mouse target/insert sequence contains an “a” nucleobase (shown in bold and underlining), while the mouse IDUA gene sequence contains a “g” nucleobase at position 1627 of the IDUA sequence. 
     The targeted/insert sequence in the human IDUA polynucleotide sequence is shown below and corresponds to nucleic acids 1231-1324-of the human IDUA gene sequence shown above. 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 295) 
               
               
                   
                 atgaggagca gctct   a   ggcc gaagtgtcgc aggccgggac 
               
               
                   
                   
               
               
                   
                 cgtcctggac agcaaccaca cggtgggcgt cctggccagc 
               
               
                   
                   
               
               
                   
                 gcccaccgcc ccca 
               
            
           
         
       
     
     The above human target/insert sequence contains an “a” nucleobase (shown in bold and underlining), while the human IDUA gene sequence contains a “g” nucleobase at position 1246 of the IDUA sequence. 
     For plasmid transfections, HEK293T cells were transfected with 250 ng of gRNA plasmid and 750 ng of base editor plasmid at 30,000 cells per well of a 48-well plate. The base editor SpCas9-ABE7.10 was used having the NGG PAM sequence. The mouse gRNA sequence is shown below and contains an “a” nucleobase (shown in bold and underlining): 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 10) 
               
               
                   
                 gctct   a   ggccgaagtgtcgc agg 
               
            
           
         
       
     
     The human gRNA sequence is shown below and contains an “a” nucleobase (shown in bold and underlining): 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 9) 
               
               
                   
                 actct   a   ggcagaggtctcaa agg 
               
            
           
         
       
     
     Example 8. Materials and Methods 
     The results provided in the Examples described herein were obtained using the following materials and methods. 
     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: 2). This scaffold was used for the PAMs shown in the tables herein, e.g., NGG, NGA, NGC, NGT PAMs; the gRNA encompasses the scaffold sequence and the spacer sequence (target sequence) for disease-associated genes (e.g., Tables 3A and 3B) 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). 
     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 listed herein above were 5′ phosphorylated using T4 Polynucleotide Kinase (New England Biolabs) according to the manufacturer&#39;s instructions. 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 DpnI (20 U, New England Biolabs) at 37° C. for 1 hour, purified on a QIAprep spin column (Qiagen), and ligated using QuickLigase (New England Biolabs) according to the manufacturer&#39;s instructions. DNA vector amplification was carried out using Mach1 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-rAPOBEC1-Linker—dCas9 Fusions. 
       E. coli  BL21 STAR (DE3)-competent cells (ThermoFisher Scientific) were transformed with plasmids (e.g. plasmids encoding pET28b-His6-rAPOBEC1-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 4,000 g 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 25,000 g 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, unlabeled 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 MgCl 2 , 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 μl, Qiagen) and isopropanol (25 μl) and purified on a 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 the gene of interest (e.g. SERPINA1, G6PC, IDUA, etc.) (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. 
     SEQUENCES 
     Table 8 below presents a representative list of wild-type and variant (E342K) SERPINA1-encoded amino acid sequences and open reading frame (ORF) nucleic acid sequences of the wild-type and variant (E342K) SERPINA1 polynucleotides as utilized in the described embodiments. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Exemplary Sequences 
               
            
           
           
               
               
               
            
               
                   
                 SEQ ID 
                   
               
               
                   
                 NO 
                 Sequences 
               
               
                   
               
               
                 SERPINA1 
                 296 
                 MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHD 
               
               
                 Amino 
                   
                 QDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF 
               
               
                 acids 
                   
                 AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLN 
               
               
                   
                   
                 QPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVN 
               
               
                   
                   
                 FGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYI 
               
               
                   
                   
                 FFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFN 
               
               
                   
                   
                 IQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHD 
               
               
                   
                   
                 IITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSN 
               
               
                   
                   
                 GADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAI 
               
               
                   
                   
                 PMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK 
               
               
                   
               
               
                 SERPINA1 
                 301 
                 ATGCCGTCTTCTGTCTCGTGGGGCATCCTCCTGCTGGCAGG 
               
               
                 ORF 
                   
                 CCTGTGCTGCCTGGTCCCTGTCTCCCTGGCTGAGGATCCCC 
               
               
                   
                   
                 AGGGAGATGCTGCCCAGAAGACAGATACATCCCACCATG 
               
               
                   
                   
                 ATCAGGATCACCCAACCTTCAACAAGATCACCCCCAACCT 
               
               
                   
                   
                 GGCTGAGTTCGCCTTCAGCCTATACCGCCAGCTGGCACAC 
               
               
                   
                   
                 CAGTCCAACAGCACCAATATCTTCTTCTCCCCAGTGAGCA 
               
               
                   
                   
                 TCGCTACAGCCTTTGCAATGCTCTCCCTGGGGACCAAGGC 
               
               
                   
                   
                 TGACACTCACGATGAAATCCTGGAGGGCCTGAATTTCAAC 
               
               
                   
                   
                 CTCACGGAGATTCCGGAGGCTCAGATCCATGAAGGCTTCC 
               
               
                   
                   
                 AGGAACTCCTCCGTACCCTCAACCAGCCAGACAGCCAGCT 
               
               
                   
                   
                 CCAGCTGACCACCGGCAATGGCCTGTTCCTCAGCGAGGGC 
               
               
                   
                   
                 CTGAAGCTAGTGGATAAGTTTTTGGAGGATGTTAAAAAGT 
               
               
                   
                   
                 TGTACCACTCAGAAGCCTTCACTGTCAACTTCGGGGACAC 
               
               
                   
                   
                 CGAAGAGGCCAAGAAACAGATCAACGATTACGTGGAGAA 
               
               
                   
                   
                 GGGTACTCAAGGGAAAATTGTGGATTTGGTCAAGGAGCTT 
               
               
                   
                   
                 GACAGAGACACAGTTTTTGCTCTGGTGAATTACATCTTCTT 
               
               
                   
                   
                 TAAAGGCAAATGGGAGAGACCCTTTGAAGTCAAGGACAC 
               
               
                   
                   
                 CGAGGAAGAGGACTTCCACGTGGACCAGGTGACCACCGT 
               
               
                   
                   
                 GAAGGTGCCTATGATGAAGCGTTTAGGCATGTTTAACATC 
               
               
                   
                   
                 CAGCACTGTAAGAAGCTGTCCAGCTGGGTGCTGCTGATGA 
               
               
                   
                   
                 AATACCTGGGCAATGCCACCGCCATCTTCTTCCTGCCTGAT 
               
               
                   
                   
                 GAGGGGAAACTACAGCACCTGGAAAATGAACTCACCCAC 
               
               
                   
                   
                 GATATCATCACCAAGTTCCTGGAAAATGAAGACAGAAGGT 
               
               
                   
                   
                 CTGCCAGCTTACATTTACCCAAACTGTCCATTACTGGAACC 
               
               
                   
                   
                 TATGATCTGAAGAGCGTCCTGGGTCAACTGGGCATCACTA 
               
               
                   
                   
                 AGGTCTTCAGCAATGGGGCTGACCTCTCCGGGGTCACAGA 
               
               
                   
                   
                 GGAGGCACCCCTGAAGCTCTCCAAGGCCGTGCATAAGGCT 
               
               
                   
                   
                 GTGCTGACCATCGACGAGAAAGGGACTGAAGCTGCTGGG 
               
               
                   
                   
                 GCCATGTTTTTAGAGGCCATACCCATGTCTATCCCCCCCGA 
               
               
                   
                   
                 GGTCAAGTTCAACAAACCCTTTGTCTTCTTAATGATTGAAC 
               
               
                   
                   
                 AAAATACCAAGTCTCCCCTCTTCATGGGAAAAGTGGTGAA 
               
               
                   
                   
                 TCCCACCCAAAAA 
               
               
                   
               
               
                 SERPINA1 
                 302 
                 MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHD 
               
               
                 E342K 
                   
                 QDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF 
               
               
                 Amino 
                   
                 AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLN 
               
               
                 Acids 
                   
                 QPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVN 
               
               
                   
                   
                 FGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYI 
               
               
                   
                   
                 FFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFN 
               
               
                   
                   
                 IQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHD 
               
               
                   
                   
                 IITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSN 
               
               
                   
                   
                 GADLSGVTEEAPLKLSKAVHKAVLTIDKKGTEAAGAMFLEA 
               
               
                   
                   
                 IPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK 
               
               
                   
               
               
                 SERPINA1 
                 303 
                 ATGCCGTCTTCTGTCTCGTGGGGCATCCTCCTGCTGGCAGG 
               
               
                 E342K 
                   
                 CCTGTGCTGCCTGGTCCCTGTCTCCCTGGCTGAGGATCCCC 
               
               
                 ORF 
                   
                 AGGGAGATGCTGCCCAGAAGACAGATACATCCCACCATG 
               
               
                   
                   
                 ATCAGGATCACCCAACCTTCAACAAGATCACCCCCAACCT 
               
               
                   
                   
                 GGCTGAGTTCGCCTTCAGCCTATACCGCCAGCTGGCACAC 
               
               
                   
                   
                 CAGTCCAACAGCACCAATATCTTCTTCTCCCCAGTGAGCA 
               
               
                   
                   
                 TCGCTACAGCCTTTGCAATGCTCTCCCTGGGGACCAAGGC 
               
               
                   
                   
                 TGACACTCACGATGAAATCCTGGAGGGCCTGAATTTCAAC 
               
               
                   
                   
                 CTCACGGAGATTCCGGAGGCTCAGATCCATGAAGGCTTCC 
               
               
                   
                   
                 AGGAACTCCTCCGTACCCTCAACCAGCCAGACAGCCAGCT 
               
               
                   
                   
                 CCAGCTGACCACCGGCAATGGCCTGTTCCTCAGCGAGGGC 
               
               
                   
                   
                 CTGAAGCTAGTGGATAAGTTTTTGGAGGATGTTAAAAAGT 
               
               
                   
                   
                 TGTACCACTCAGAAGCCTTCACTGTCAACTTCGGGGACAC 
               
               
                   
                   
                 CGAAGAGGCCAAGAAACAGATCAACGATTACGTGGAGAA 
               
               
                   
                   
                 GGGTACTCAAGGGAAAATTGTGGATTTGGTCAAGGAGCTT 
               
               
                   
                   
                 GACAGAGACACAGTTTTTGCTCTGGTGAATTACATCTTCTT 
               
               
                   
                   
                 TAAAGGCAAATGGGAGAGACCCTTTGAAGTCAAGGACAC 
               
               
                   
                   
                 CGAGGAAGAGGACTTCCACGTGGACCAGGTGACCACCGT 
               
               
                   
                   
                 GAAGGTGCCTATGATGAAGCGTTTAGGCATGTTTAACATC 
               
               
                   
                   
                 CAGCACTGTAAGAAGCTGTCCAGCTGGGTGCTGCTGATGA 
               
               
                   
                   
                 AATACCTGGGCAATGCCACCGCCATCTTCTTCCTGCCTGAT 
               
               
                   
                   
                 GAGGGGAAACTACAGCACCTGGAAAATGAACTCACCCAC 
               
               
                   
                   
                 GATATCATCACCAAGTTCCTGGAAAATGAAGACAGAAGGT 
               
               
                   
                   
                 CTGCCAGCTTACATTTACCCAAACTGTCCATTACTGGAACC 
               
               
                   
                   
                 TATGATCTGAAGAGCGTCCTGGGTCAACTGGGCATCACTA 
               
               
                   
                   
                 AGGTCTTCAGCAATGGGGCTGACCTCTCCGGGGTCACAGA 
               
               
                   
                   
                 GGAGGCACCCCTGAAGCTCTCCAAGGCCGTGCATAAGGCT 
               
               
                   
                   
                 GTGCTGACCATCGACaAGAAAGGGACTGAAGCTGCTGGGG 
               
               
                   
                   
                 CCATGTTTTTAGAGGCCATACCCATGTCTATCCCCCCCGAG 
               
               
                   
                   
                 GTCAAGTTCAACAAACCCTTTGTCTTCTTAATGATTGAACA 
               
               
                   
                   
                 AAATACCAAGTCTCCCCTCTTCATGGGAAAAGTGGTGAAT 
               
               
                   
                   
                 CCCACCCAAAAA