Patent Publication Number: US-2022220462-A1

Title: Nucleobase editors and uses thereof

Description:
RELATED APPLICATIONS 
     This application is a continuation of and claims priority under 35 U.S.C. § 120 to U.S. application, U.S. Ser. No. 15/960,171, filed Apr. 23, 2018, which is a continuation of and claims priority under 35 U.S.C. § 120 to international PCT Application, PCT/US2016/058344, filed Oct. 22, 2016, and is a continuation of and claims priority under 35 U.S.C. § 120 to U.S. application, U.S. Ser. No. 15/331,852, filed Oct. 22, 2016, which claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent applications, U.S. Ser. No. 62/245,828 filed Oct. 23, 2015, U.S. Ser. No. 62/279,346 filed Jan. 15, 2016, U.S. Ser. No. 62/311,763 filed Mar. 22, 2016, U.S. Ser. No. 62/322,178 filed Apr. 13, 2016, U.S. Ser. No. 62/357,352 filed Jun. 30, 2016, U.S. Ser. No. 62/370,700 filed Aug. 3, 2016, U.S. Ser. No. 62/398,490 filed Sep. 22, 2016, U.S. Ser. No. 62/408,686 filed Oct. 14, 2016, and U.S. Ser. No. 62/357,332 filed Jun. 30, 2016; each of which is incorporated herein by reference. 
    
    
     GOVERNMENT SUPPORT 
     This invention was made with Government support under grant number EB022376 (formerly GM065400) awarded by the National Institutes of Health. The Government has certain rights in the invention. 
    
    
     REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB 
     This application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 9, 2021, is named H082470213US10-SEQ-EPG and is 4,463,178 bytes in size. 
     BACKGROUND OF THE INVENTION 
     Targeted editing of nucleic acid sequences, for example, the targeted cleavage or the targeted introduction of a specific modification into genomic DNA, is a highly promising approach for the study of gene function and also has the potential to provide new therapies for human genetic diseases. 1  An ideal nucleic acid editing technology possesses three characteristics: (1) high efficiency of installing the desired modification; (2) minimal off-target activity; and (3) the ability to be programmed to edit precisely any site in a given nucleic acid, e.g., any site within the human genome. 2  Current genome engineering tools, including engineered zinc finger nucleases (ZFNs), 3  transcription activator like effector nucleases (TALENs), 4  and most recently, the RNA-guided DNA endonuclease Cas9, 5  effect sequence-specific DNA cleavage in a genome. This programmable cleavage can result in mutation of the DNA at the cleavage site via non-homologous end joining (NHEJ) or replacement of the DNA surrounding the cleavage site via homology-directed repair (HDR). 6,7    
     One drawback to the current technologies is that both NHEJ and HDR are stochastic processes that typically result in modest gene editing efficiencies as well as unwanted gene alterations that can compete with the desired alteration. 8  Since many genetic diseases in principle can be treated by effecting a specific nucleotide change at a specific location in the genome (for example, a C to T change in a specific codon of a gene associated with a disease), 9  the development of a programmable way to achieve such precision gene editing would represent both a powerful new research tool, as well as a potential new approach to gene editing-based human therapeutics. 
     SUMMARY OF THE INVENTION 
     The clustered regularly interspaced short palindromic repeat (CRISPR) system is a recently discovered prokaryotic adaptive immune system 10  that has been modified to enable robust and general genome engineering in a variety of organisms and cell lines. 11  CRISPR-Cas (CRISPR associated) systems are protein-RNA complexes that use an RNA molecule (sgRNA) as a guide to localize the complex to a target DNA sequence via base-pairing. 12  In the natural systems, a Cas protein then acts as an endonuclease to cleave the targeted DNA sequence. 13  The target DNA sequence must be both complementary to the sgRNA, and also contain a “protospacer-adjacent motif” (PAM) at the 3′-end of the complementary region in order for the system to function. 14    
     Among the known Cas proteins,  S. pyogenes  Cas9 has been mostly widely used as a tool for genome engineering. 15  This Cas9 protein is a large, multi-domain protein containing two distinct nuclease domains. Point mutations can be introduced into Cas9 to abolish nuclease activity, resulting in a dead Cas9 (dCas9) that still retains its ability to bind DNA in a sgRNA-programmed manner. 16  In principle, when fused to another protein or domain, dCas9 can target that protein to virtually any DNA sequence simply by co-expression with an appropriate sgRNA. 
     The potential of the dCas9 complex for genome engineering purposes is immense. Its unique ability to bring proteins to specific sites in a genome programmed by the sgRNA in theory can be developed into a variety of site-specific genome engineering tools beyond nucleases, including transcriptional activators, transcriptional repressors, histone-modifying proteins, integrases, and recombinases. 11  Some of these potential applications have recently been implemented through dCas9 fusions with transcriptional activators to afford RNA-guided transcriptional activators, 17,18  transcriptional repressors, 16,19,20  and chromatin modification enzymes. 21  Simple co-expression of these fusions with a variety of sgRNAs results in specific expression of the target genes. These seminal studies have paved the way for the design and construction of readily programmable sequence-specific effectors for the precise manipulation of genomes. 
     Significantly, 80-90% of protein mutations responsible for human disease arise from the substitution, deletion, or insertion of only a single nucleotide. 6  Most current strategies for single-base gene correction include engineered nucleases (which rely on the creation of double-strand breaks, DSBs, followed by stochastic, inefficient homology-directed repair, HDR), and DNA-RNA chimeric oligonucleotides. 22  The latter strategy involves the design of a RNA/DNA sequence to base pair with a specific sequence in genomic DNA except at the nucleotide to be edited. The resulting mismatch is recognized by the cell&#39;s endogenous repair system and fixed, leading to a change in the sequence of either the chimera or the genome. Both of these strategies suffer from low gene editing efficiencies and unwanted gene alterations, as they are subject to both the stochasticity of HDR and the competition between HDR and non-homologous end-joining, NHEJ. 23-25  HDR efficiencies vary according to the location of the target gene within the genome, 26  the state of the cell cycle, 27  and the type of cell/tissue. 28  The development of a direct, programmable way to install a specific type of base modification at a precise location in genomic DNA with enzyme-like efficiency and no stochasticity therefore represents a powerful new approach to gene editing-based research tools and human therapeutics. 
     Some aspects of the disclosure are based on the recognition that certain configurations of a dCas9 domain, and a cytidine deaminase domain fused by a linker are useful for efficiently deaminating target cytidine residues. Other aspects of this disclosure relate to the recognition that a nucleobase editing fusion protein with a cytidine deaminase domain fused to the N-terminus of a nuclease inactive Cas9 (dCas9) via a linker was capable of efficiently deaminating target nucleic acids in a double stranded DNA target molecule. See for example, Examples 3 and 4 below, which demonstrate that the fusion proteins, which are also referred to herein as base editors, generate less indels and more efficiently deaminate target nucleic acids than other base editors, such as base editors without a UGI domain. In some embodiments, the fusion protein comprises a nuclease-inactive Cas9 (dCas9) domain and an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain, where the deaminase domain is fused to the N-terminus of the dCas9 domain via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 7). In some embodiments, the nuclease-inactive Cas9 (dCas9) domain of comprises the amino acid sequence set forth in SEQ ID NO: 263. In some embodiments, the deaminase is rat APOBEC1 (SEQ ID NO: 284). In some embodiments, the deaminase is human APOBEC1 (SEQ ID NO: 282). In some embodiments, the deaminase is pmCDA1 (SEQ ID NO: 5738). In some embodiments, the deaminase is human APOBEC3G (SEQ ID NO: 275). In some embodiments, the deaminase is a human APOBEC3G variant of any one of (SEQ ID NOs: 5739-5741). 
     Some aspects of the disclosure are based on the recognition that certain configurations of a dCas9 domain, and a cytidine deaminase domain fused by a linker are useful for efficiently deaminating target cytidine residues. Other aspects of this disclosure relate to the recognition that a nucleobase editing fusion protein with an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain fused to the N-terminus of a nuclease inactive Cas9 (dCas9) via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 7) was capable of efficiently deaminating target nucleic acids in a double stranded DNA target molecule. In some embodiments, the fusion protein comprises a nuclease-inactive Cas9 (dCas9) domain and an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain, where the deaminase domain is fused to the N-terminus of the dCas9 domain via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 7). 
     In some embodiments, the fusion protein comprises the amino acid residues 11-1629 of the amino acid sequence set forth in SEQ ID NO: 591. In some embodiments, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 591. In some embodiments, the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 5737, 5743, 5745, and 5746. 
     Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within a subject&#39;s genome, e.g., a human&#39;s genome. In some embodiments, fusion proteins of Cas9 (e.g., dCas9, nuclease active Cas9, or Cas9 nickase) and deaminases or deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and deaminases or deaminase domains, are provided. 
     Some aspects of this disclosure provide fusion proteins comprising a Cas9 protein as provided herein that is fused to a second protein (e.g., an enzymatic domain such as a cytidine deaminase domain), thus forming a fusion protein. In some embodiments, the second protein comprises an enzymatic domain, or a binding domain. In some embodiments, the enzymatic domain is 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, the enzymatic domain is a nucleic acid editing domain. In some embodiments, the nucleic acid editing domain is a deaminase domain. In some embodiments, the deaminase is a cytosine deaminase or a cytidine 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 APOBEC3A 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). It should be appreciated that the deaminase may be from any suitable organism (e.g., a human or a rat). In some embodiments, the deaminase is from a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase is rat APOBEC1 (SEQ ID NO: 284). In some embodiments, the deaminase is human APOBEC1 (SEQ ID NO: 282). In some embodiments, the deaminase is pmCDA1. 
     Some aspects of this disclosure provide fusion proteins comprising: (i) a nuclease-inactive Cas9 (dCas9) domain comprising the amino acid sequence of SEQ ID NO: 263; and (ii) an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain, wherein the deaminase domain is fused to the N-terminus of the dCas9 domain via a linker comprising the amino acid sequence of SGSETPGTSESATPES (SEQ ID NO: 7). In some embodiments, the deaminase is rat APOBEC1 (SEQ ID NO: 284). In some embodiments, the deaminase is human APOBEC1 (SEQ ID NO: 282). In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 591. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 5737. In some embodiments, the deaminase is pmCDA1 (SEQ ID NO: 5738). In some embodiments, the deaminase is human APOBEC3G (SEQ ID NO: 275). In some embodiments, the deaminase is a human APOBEC3G variant of any one of SEQ ID NOs: 5739-5741. 
     Some aspects of this disclosure provide fusion proteins comprising: (i) a Cas9 nickase domain and (ii) an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain, wherein the deaminase domain is fused to the N-terminus of the Cas9 nickase domain. In some embodiments, the Cas9 nickase domain comprises a D10X mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid except for D. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises a D10A mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises a histidine at amino acid position 840 of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding amino acid position in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises the amino acid sequence as set forth in SEQ ID NO: 267. In some embodiments, the deaminase is rat APOBEC1 (SEQ ID NO: 284). In some embodiments, the deaminase is human APOBEC1 (SEQ ID NO: 282). In some embodiments, the deaminase is pmCDA1. 
     Some aspects of this disclosure provide fusion proteins comprising: (i) a Cas9 nickase domain and (ii) an apolipoprotein B mRNA-editing complex 1 (APOBEC1) deaminase domain, wherein the deaminase domain is fused to the N-terminus of the Cas9 nickase domain. In some embodiments, the Cas9 nickase domain comprises a D10X mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid except for D. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises a D10A mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises a histidine at amino acid position 840 of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding amino acid position in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises the amino acid sequence as set forth in SEQ ID NO: 267. In some embodiments, the deaminase is rat APOBEC1 (SEQ ID NO: 284). In some embodiments, the deaminase is human APOBEC1 (SEQ ID NO: 282). In some embodiments, the deaminase is pmCDA1. 
     Other aspects of this disclosure relate to the recognition that fusion proteins comprising a deaminase domain, a dCas9 domain and a uracil glycosylase inhibitor (UGI) domain demonstrate improved efficiency for deaminating target nucleotides in a nucleic acid molecule. Without wishing to be bound by any particular theory, cellular DNA-repair response to the presence of U:G heteroduplex DNA may be responsible for a decrease in nucleobase editing efficiency in cells. Uracil DNA glycosylase (UDG) catalyzes removal of U from DNA in cells, which may initiate base excision repair, with reversion of the U:G pair to a C:G pair as the most common outcome. As demonstrated herein, Uracil DNA Glycosylase Inhibitor (UGI) may inhibit human UDG activity. Without wishing to be bound by any particular theory, base excision repair may be inhibited by molecules that bind the single strand, block the edited base, inhibit UGI, inhibit base excision repair, protect the edited base, and/or promote “fixing” of the non-edited strand, etc. Thus, this disclosure contemplates fusion proteins comprising a dCas9-cytidine deaminase domain that is fused to a UGI domain. 
     In some embodiments, the fusion protein comprises a nuclease-inactive Cas9 (dCas9) domain; a nucleic acid editing domain; and a uracil glycosylase inhibitor (UGI) domain. In some embodiments, the dCas9 domain comprises a D10X mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid except for D. In some embodiments, the amino acid sequence of the dCas9 domain comprises a D10A mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the amino acid sequence of the dCas9 domain comprises an H840X mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid except for H. In some embodiments, the amino acid sequence of the dCas9 domain comprises an H840A mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the dCas9 domain comprises the amino acid sequence as set forth in SEQ ID NO: 263. 
     Further aspects of this disclosure relate to the recognition that fusion proteins using a Cas9 nickase as the Cas9 domain demonstrate improved efficiency for editing nucleic acids. For example, aspects of this disclosure relate to the recognition that fusion proteins comprising a Cas9 nickase, a deaminase domain and a UGI domain demonstrate improved efficiency for editing nucleic acids. For example, the improved efficiency for editing nucleotides is described below in the Examples section. 
     Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of modifying a specific nucleotide base without generating a significant proportion of indels. An “indel”, as used herein, refers to the insertion or deletion of a nucleotide base within a nucleic acid. Such insertions or deletions can lead to frame shift mutations within a coding region of a gene. In some embodiments, it is desirable to generate base editors that efficiently modify (e.g. mutate or deaminate) a specific nucleotide within a nucleic acid, without generating a large number of insertions or deletions (i.e., indels) in the nucleic acid. In certain embodiments, any of the base editors provided herein are capable of generating a greater proportion of intended modifications (e.g., point mutations or deaminations) versus indels. 
     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, a fusion protein comprises a Cas9 nickase domain, a nucleic acid editing domain; and a uracil glycosylase inhibitor (UGI) domain. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises a D10X mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid except for D. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises a D10A mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises a histidine at amino acid position 840 of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding amino acid position in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the amino acid sequence of the Cas9 nickase domain comprises the amino acid sequence as set forth in SEQ ID NO: 267. 
     In some embodiments, the deaminase domain of the fusion protein is fused to the N-terminus of the dCas9 domain or the Cas9 nickase. In some embodiments, the UGI domain is fused to the C-terminus of the dCas9 domain or the Cas9 nickase. In some embodiments, the dCas9 domain or the Cas9 nickase and the nucleic acid editing domain are fused via a linker. In some embodiments, the dCas9 domain or the Cas9 nickase and the UGI domain are fused via a linker. 
     In certain embodiments, linkers may be used to link any of the peptides or peptide domains of the invention. The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In certain embodiments, the linker is a polpeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises amino acids. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may included funtionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates. 
     In some embodiments, the linker comprises the amino acid sequence (GGGGS) n  (SEQ ID NO: 5), (G) n , (EAAAK) n  (SEQ ID NO: 6), (GGS) n , (SGGS) n (SEQ ID NO: 4288), SGSETPGTSESATPES (SEQ ID NO: 7), (XP) n , or any combination thereof, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, the linker comprises the amino acid sequence (GGS) n , wherein n is 1, 3, or 7. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 7). 
     In some embodiments, the fusion protein comprises the structure [nucleic acid editing domain]-[optional linker sequence]-[dCas9 or Cas9 nickase]-[optional linker sequence]-[UGI]. In some embodiments, the fusion protein comprises the structure [nucleic acid editing domain]-[optional linker sequence]-[UGI]-[optional linker sequence]-[dCas9 or Cas9 nickase]; [UGI]-[optional linker sequence]-[nucleic acid editing domain]-[optional linker sequence]-[dCas9 or Cas9 nickase]; [UGI]-[optional linker sequence]-[dCas9 or Cas9 nickase]-[optional linker sequence]-[nucleic acid editing domain]; [dCas9 or Cas9 nickase]-[optional linker sequence]-[UGI]-[optional linker sequence]-[nucleic acid editing domain]; or [dCas9 or Cas9 nickase]-[optional linker sequence]-[nucleic acid editing domain]-[optional linker sequence]-[UGI]. 
     In some embodiments, the nucleic acid editing domain comprises a deaminase. In some embodiments, the nucleic acid editing domain comprises a deaminase. In some embodiments, the deaminase is a cytidine 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, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, an APOBEC3H deaminase, or an APOBEC4 deaminase. In some embodiments, the deaminase is an activation-induced deaminase (AID). In some embodiments, the deaminase is a Lamprey CDA1 (pmCDA1) deaminase. 
     In some embodiments, the deaminase is from a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase is from a human. In some embodiments the deaminase is from a rat. In some embodiments, the deaminase is a rat APOBEC1 deaminase comprising the amino acid sequence set forth in (SEQ ID NO: 284). In some embodiments, the deaminase is a human APOBEC1 deaminase comprising the amino acid sequence set forth in (SEQ ID NO: 282). In some embodiments, the deaminase is pmCDA1 (SEQ ID NO: 5738). In some embodiments, the deaminase is human APOBEC3G (SEQ ID NO: 275). In some embodiments, the deaminase is a human APOBEC3G variant of any one of (SEQ ID NOs: 5739-5741). In some embodiments, the deaminase 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 any one of the amino acid sequences set forth in SEQ ID NOs: 266-284 or 5725-5741. 
     In some embodiments, the UGI domain comprises an amino acid sequence that 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 SEQ ID NO: 600. In some embodiments, the UGI domain comprises the amino acid sequence as set forth in SEQ ID NO: 600. 
     Some aspects of this disclosure provide complexes comprising a Cas9 protein or a Cas9 fusion protein as provided herein, and a guide RNA bound to the Cas9 protein or the Cas9 fusion protein. 
     Some aspects of this disclosure provide methods of using the Cas9 proteins, fusion proteins, or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule (a) with a Cas9 protein or a fusion protein as provided herein and with a 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; or (b) with a Cas9 protein, a Cas9 fusion protein, or a Cas9 protein or fusion protein complex with a gRNA as provided herein. 
     Some aspects of this disclosure provide kits comprising a nucleic acid construct, comprising (a) a nucleotide sequence encoding a Cas9 protein or a Cas9 fusion protein as provided herein; and (b) a heterologous promoter that drives expression of the sequence of (a). In some embodiments, the kit further comprises an expression construct encoding a guide RNA backbone, wherein the construct comprises a cloning site positioned to allow the cloning of a nucleic acid sequence identical or complementary to a target sequence into the guide RNA backbone. 
     Some aspects of this disclosure provide polynucleotides encoding a Cas9 protein of a fusion protein as provided herein. Some aspects of this disclosure provide vectors comprising such polynucleotides. In some embodiments, the vector comprises a heterologous promoter driving expression of polynucleotide. 
     Some aspects of this disclosure provide cells comprising a Cas9 protein, a fusion protein, a nucleic acid molecule, and/or a vector as provided herein. 
     The description of exemplary embodiments of the reporter systems above is provided for illustration purposes only and not meant to be limiting. Additional reporter systems, e.g., variations of the exemplary systems described in detail above, are also embraced by this disclosure. 
     The summary above is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the Detailed Description, the Drawings, the Examples, and the Claims. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows the deaminase activity of deaminases on single stranded DNA substrates. Single stranded DNA substrates using randomized PAM sequences (NNN PAM) were used as negative controls. Canonical PAM sequences used (NGG PAM) 
         FIG. 2  shows activity of Cas9:deaminase fusion proteins on single stranded DNA substrates. 
         FIG. 3  illustrates double stranded DNA substrate binding by Cas9:deaminase:sgRNA complexes. 
         FIG. 4  illustrates a double stranded DNA deamination assay. 
         FIG. 5  demonstrates that Cas9 fusions can target positions 3-11 of double-stranded DNA target sequences (numbered according to the schematic in  FIG. 5 ). Upper Gel: 1 μM rAPOBEC1-GGS-dCas9, 125 nM dsDNA, 1 equivalent sgRNA. Mid Gel: 1 μM rAPOBEC1-(GGS) 3  (SEQ ID NO: 596)-dCas9, 125 nM dsDNA, 1 equivalent sgRNA. Lower Gel: 1.85 μM rAPOBEC1-XTEN-dCas9, 125 nM dsDNA, 1 equivalent sgRNA. 
         FIG. 6  demonstrates that the correct guide RNA, e.g., the correct sgRNA, is required for deaminase activity. 
         FIG. 7  illustrates the mechanism of target DNA binding of in vivo target sequences by deaminase-dCas9:sgRNA complexes. 
         FIG. 8  shows successful deamination of exemplary disease-associated target sequences. 
         FIG. 9  shows in vitro C→T editing efficiencies using His6-rAPOBEC1-XTEN-dCas9. 
         FIG. 10  shows C→T editing efficiencies in HEK293T cells is greatly enhanced by fusion with UGI. 
         FIGS. 11A to 11C  show NBE1 mediates specific, guide RNA-programmed C to U conversion in vitro.  FIG. 11A : Nucleobase editing strategy. DNA with a target C (red) at a locus specified by a guide RNA (green) is bound by dCas9 (blue), which mediates the local denaturation of the DNA substrate. Cytidine deamination by a tethered APOBEC1 enzyme (orange) converts the target C to U. The resulting G:U heteroduplex can be permanently converted to an A:T base pair following DNA replication or repair. If the U is in the template DNA strand, it will also result in an RNA transcript containing a G to A mutation following transcription.  FIG. 11B : Deamination assay showing an activity window of approximately five nucleotides. Following incubation of NBE1-sgRNA complexes with dsDNA substrates at 37° C. for 2 h, the 5′ fluorophore-labeled DNA was isolated and incubated with USER enzyme (uracil DNA glycosylase and endonuclease VIII) at 37° C. for 1 h to induce DNA cleavage at the site of any uracils. The resulting DNA was resolved on a denaturing polyacrylamide gel, and any fluorophore-linked strands were visualized. Each lane is labeled according to the position of the target C within the protospacer, or with “-” if no target C is present, counting the base distal from the PAM as position 1.  FIG. 11C : Deaminase assay showing the sequence specificity and sgRNA-dependence of NBE1. The DNA substrate with a target C at position 7 was incubated with NBE1 as in  FIG. 11B  with either the correct sgRNA, a mismatched sgRNA, or no sgRNA. No C to U editing is observed with the mismatched sgRNA or with no sgRNA. The positive control sample contains a DNA sequence with a U synthetically incorporated at position 7. 
         FIGS. 12A to 12B  show effects of sequence context and target C position on nucleobase editing efficiency in vitro.  FIG. 12A : Effect of changing the sequence surrounding the target C on editing efficiency in vitro. The deamination yield of 80% of targeted strands (40% of total sequencing reads from both strands) for C 7  in the protospacer sequence 5′-TTATTTCGTGGATTTATTTA-3′(SEQ ID NO: 264) was defined as 1.0, and the relative deamination efficiencies of substrates containing all possible single-base mutations at positions 1-6 and 8-13 are shown. Values and error bars reflect the mean and standard deviation of two or more independent biological replicates performed on different days.  FIG. 12B : Positional effect of each NC motif on editing efficiency in vitro. Each NC target motif was varied from positions 1 to 8 within the protospacer as indicated in the sequences shown on the right (the PAM shown in red, the protospacer plus one base 5′ to the protospacer are also shown). The percentage of total sequence reads containing T at each of the numbered target C positions following incubation with NBE1 is shown in the graph. Note that the maximum possible deamination yield in vitro is 50% of total sequencing reads (100% of targeted strands). Values and error bars reflect the mean and standard deviation of two or three independent biological replicates performed on different days.  FIG. 12B  depicts SEQ ID NOs: 285 through 292 from top to bottom, respectively. 
         FIGS. 13A to 13C  show nucleobase editing in human cells.  FIG. 13A : Protospacer (black) and PAM (red) sequences of the six mammalian cell genomic loci targeted by nucleobase editors. Target Cs are indicated with subscripted numbers corresponding to their positions within the protospacer.  FIG. 13A  depicts SEQ ID NOs: 293 through 298 from top to bottom, respectively.  FIG. 13B : HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE3 and an appropriate sgRNA. Three days after transfection, genomic DNA was extracted and analyzed by high-throughput DNA sequencing at the six loci. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, are shown for NBE1, NBE2, and NBE3 at all six genomic loci, and for wt Cas9 with a donor HDR template at three of the six sites (EMX1, HEK293 site 3, and HEK293 site 4). Values and error bars reflect the mean and standard deviation of three independent biological replicates performed on different days.  FIG. 13C : Frequency of indel formation, calculated as described in the Methods, is shown following treatment of HEK293T cells with NBE2 and NBE3 for all six genomic loci, or with wt Cas9 and a single-stranded DNA template for HDR at three of the six sites (EMX1, HEK293 site 3, and HEK293 site 4). Values reflect the mean of at least three independent biological replicates performed on different days. 
         FIGS. 14A to 14C  show NBE2- and NBE3-mediated correction of three disease-relevant mutations in mammalian cells. For each site, the sequence of the protospacer is indicated to the right of the name of the mutation, with the PAM highlighted in green and the base responsible for the mutation indicated in bold with a subscripted number corresponding to its position within the protospacer. The amino acid sequence above each disease-associated allele is shown, together with the corrected amino acid sequence following nucleobase editing in red. Underneath each sequence are the percentages of total sequencing reads with the corresponding base. Cells were nucleofected with plasmids encoding NBE2 or NBE3 and an appropriate sgRNA. Two days after nucleofection, genomic DNA was extracted and analyzed by HTS to assess pathogenic mutation correction.  FIG. 14A : The Alzheimer&#39;s disease-associated APOE4 allele is converted to APOE3′ in mouse astrocytes by NBE3 in 11% of total reads (44% of nucleofected astrocytes). Two nearby Cs are also converted to Ts, but with no change to the predicted sequence of the resulting protein (SEQ ID NO: 299).  FIG. 14B  The cancer-associated p53 N239D mutation is corrected by NBE2 in 11% of treated human lymphoma cells (12% of nucleofected cells) that are heterozygous for the mutation (SEQ ID NO: 300).  FIG. 14C  The p53 Y163C mutation is corrected by NBE3 in 7.6% of nucleofected human breast cancer cells (SEQ ID NO: 301). 
         FIGS. 15A to 15D  show effects of deaminase-dCas9 linker length and composition on nucleobase editing. Gel-based deaminase assay showing the deamination window of nucleobase editors with deaminase-Cas9 linkers of GGS ( FIG. 15A ), (GGS) 3  (SEQ ID NO: 596) ( FIG. 15B ), XTEN ( FIG. 15C ), or (GGS) 7  (SEQ ID NO: 597) ( FIG. 15D ). Following incubation of 1.85 μM editor-sgRNA complexes with 125 nM dsDNA substrates at 37° C. for 2 h, the dye-conjugated DNA was isolated and incubated with USER enzyme (uracil DNA glycosylase and endonuclease VIII) at 37° C. for an additional hour to cleave the DNA backbone at the site of any uracils. The resulting DNA was resolved on a denaturing polyacrylamide gel, and the dye-conjugated strand was imaged. Each lane is numbered according to the position of the target C within the protospacer, or with—if no target C is present. 8U is a positive control sequence with a U synthetically incorporated at position 8. 
         FIGS. 16A to 16B  show NBE1 is capable of correcting disease-relevant mutations in vitro.  FIG. 16A : Protospacer and PAM sequences (red) of seven disease-relevant mutations. The disease-associated target C in each case is indicated with a subscripted number reflecting its position within the protospacer. For all mutations except both APOE4 SNPs, the target C resides in the template (non-coding) strand.  FIG. 16A  depicts SEQ ID NOs: 302 through 308 from top to bottom, respectively.  FIG. 16B : Deaminase assay showing each dsDNA oligonucleotide before (−) and after (+) incubation with NBE1, DNA isolation, and incubation with USER enzymes to cleave DNA at positions containing U. Positive control lanes from incubation of synthetic oligonucleotides containing U at various positions within the protospacer with USER enzymes are shown with the corresponding number indicating the position of the U. 
         FIG. 17  shows processivity of NBE1. The protospacer and PAM (red) of a 60-mer DNA oligonucleotide containing eight consecutive Cs is shown at the top. The oligonucleotide (125 nM) was incubated with NBE1 (2 μM) for 2 h at 37° C. The DNA was isolated and analyzed by high-throughput sequencing. Shown are the percent of total reads for the most frequent nine sequences observed. The vast majority of edited strands (&gt;93%) have more than one C converted to T. This figure depicts SEQ ID NO: 309. 
         FIGS. 18A to 18H  show the effect of fusing UGI to NBE1 to generate NBE2.  FIG. 18A : Protospacer and PAM (red) sequences of the six mammalian cell genomic loci targeted with nucleobase editors. Editable Cs are indicated with labels corresponding to their positions within the protospacer.  FIG. 18A  depicts SEQ ID NOs: 293 through 298 from top to bottom, respectively.  FIGS. 18B to 18G : HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE1 and UGI, and an appropriate sgRNA. Three days after transfection, genomic DNA was extracted and analyzed by high-throughput DNA sequencing at the six loci. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, are shown for NBE1, NBE1 and UGI, and NBE2 at all six genomic loci.  FIG. 18H : C to T mutation rates at 510 Cs surrounding the protospacers of interest for NBE1, NBE1 plus UGI on a separate plasmid, NBE2, and untreated cells are shown. The data show the results of 3,000,000 DNA sequencing reads from 1.5×106 cells. Values reflect the mean of at least two biological experiments conducted on different days. 
         FIG. 19  shows nucleobase editing efficiencies of NBE2 in U2OS and HEK293T cells. Cellular C to T conversion percentages by NBE2 are shown for each of the six targeted genomic loci in HEK293T cells and U2OS cells. HEK293T cells were transfected using lipofectamine 2000, and U2OS cells were nucleofected. U2OS nucleofection efficiency was 74%. Three days after plasmid delivery, genomic DNA was extracted and analyzed for nucleobase editing at the six genomic loci by FITS. Values and error bars reflect the mean and standard deviation of at least two biological experiments done on different days. 
         FIG. 20  shows nucleobase editing persists over multiple cell divisions. Cellular C to T conversion percentages by NBE2 are displayed at two genomic loci in HEK293T cells before and after passaging the cells. HEK293T cells were transfected using Lipofectamine 2000. Three days post transfection, the cells were harvested and split in half. One half was subjected to HTS analysis, and the other half was allowed to propagate for approximately five cell divisions, then harvested and subjected to HTS analysis. 
         FIG. 21  shows genetic variants from ClinVar that can be corrected in principle by nucleobase editing. The NCBI ClinVar database of human genetic variations and their corresponding phenotypes 68  was searched for genetic diseases that can be corrected by current nucleobase editing technologies. The results were filtered by imposing the successive restrictions listed on the left. The x-axis shows the number of occurrences satisfying that restriction and all above restrictions on a logarithmic scale. 
         FIG. 22  shows in vitro identification of editable Cs in six genomic loci. Synthetic 80-mers with sequences matching six different genomic sites were incubated with NBE1 then analyzed for nucleobase editing via HTS. For each site, the sequence of the protospacer is indicated to the right of the name of the site, with the PAM highlighted in red. Underneath each sequence are the percentages of total DNA sequencing reads with the corresponding base. A target C was considered as “editable” if the in vitro conversion efficiency is &gt;10%. Note that maximum yields are 50% of total DNA sequencing reads since the non-targeted strand is not a substrate for nucleobase editing. This figure depicts SEQ ID NOs: 293 through 298 from top to bottom, respectively. 
         FIG. 23  shows activities of NBE1, NBE2, and NBE3 at EMX1 off-targets. HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE3 and a sgRNA matching the EMX1 sequence using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus the top ten known Cas9 off-target loci for the EMX1 sgRNA, as previously determined using the GUIDE-seq method 55 . EMX1 off-target 5 locus did not amplify and is not shown. Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for NBE1, NBE2, and NBE3. On the far right are displayed the total number of sequencing reads reported for each sequence. This figure depicts SEQ ID NOs: 293, and 310 through 318 from top to bottom, respectively. 
         FIG. 24  shows activities of NBE1, NBE2, and NBE3 at FANCF off-targets. HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE3 and a sgRNA matching the FANCF sequence using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus all of the known Cas9 off-target loci for the FANCF sgRNA, as previously determined using the GUIDE-seq method 55 . Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for NBE1, NBE2, and NBE3. On the far right are displayed the total number of sequencing reads reported for each sequence. This figure depicts SEQ ID NOs: 294 and 319 through 326 from top to bottom, respectively. 
         FIG. 25  shows activities of NBE1, NBE2, and NBE3 at HEK293 site 2 off-targets. HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE3 and a sgRNA matching the HEK293 site 2 sequence using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus all of the known Cas9 off-target loci for the HEK293 site 2 sgRNA, as previously determined using the GUIDE-seq method 55 . Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for NBE1, NBE2, and NBE3. On the far right are displayed the total number of sequencing reads reported for each sequence. This figure depicts SEQ ID NOs: 295, 327, and 328 from top to bottom, respectively. 
         FIG. 26  shows activities of NBE1, NBE2, and NBE3 at HEK293 site 3 off-targets. HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE3 and a sgRNA matching the HEK293 site 3 sequence using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus all of the known Cas9 off-target loci for the HEK293 site 3 sgRNA, as previously determined using the GUIDE-seq method 55 . Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for NBE1, NBE2, and NBE3. On the far right are displayed the total number of sequencing reads reported for each sequence. This figure depicts SEQ ID NOs: 296 and 659 through 663 from top to bottom, respectively. 
         FIG. 27  shows activities of NBE1, NBE2, and NBE3 at HEK293 site 4 off-targets. HEK293T cells were transfected with plasmids expressing NBE1, NBE2, or NBE3 and a sgRNA matching the HEK293 site 4 sequence using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus the top ten known Cas9 off-target loci for the HEK293 site 4 sgRNA, as previously determined using the GUIDE-seq method 55 . Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for NBE1, NBE2, and NBE3. On the far right are displayed the total number of sequencing reads reported for each sequence. This figure depicts SEQ ID NOs: 297 and 664 through 673 from top to bottom, respectively. 
         FIG. 28  shows non-target C mutation rates. Shown here are the C to T mutation rates at 2,500 distinct cytosines surrounding the six on-target and 34 off-target loci tested, representing a total of 14,700,000 sequence reads derived from approximately 1.8×106 cells. 
         FIGS. 29A to 29C  show base editing in human cells.  FIG. 29A  shows possible base editing outcomes in mammalian cells. Initial editing resulted in a U:G mismatch. Recognition and excision of the U by uracil DNA glycosylase (UDG) initiated base excision repair (BER), which lead to reversion to the C:G starting state. BER was impeded by BE2 and BE3, which inhibited UDG. The U:G mismatch was also processed by mismatch repair (MMR), which preferentially repaired the nicked strand of a mismatch. BE3 nicked the non-edited strand containing the G, favoring resolution of the U:G mismatch to the desired U:A or T:A outcome.  FIG. 29B  shows HEK293T cells treated as described in the Materials and Methods in the Examples below. The percentage of total DNA sequencing read with Ts at the target positions indicated show treatment with BE1, BE2, or BE3, or for treatment with wt Cas9 with a donor HDR template.  FIG. 29C  shows frequency of indel formation following the treatment in  FIG. 29B . Values are listed in  FIG. 34 . For  FIGS. 29B and 29C , values and error bars reflect the mean and s.d. of three independent biological replicates performed on different days. 
         FIGS. 30A to 30B  show BE3-mediated correction of two disease-relevant mutations in mammalian cells. The sequence of the protospacer is shown to the right of the mutation, with the PAM in blue and the target base in red with a subscripted number indicating its position within the protospacer. Underneath each sequence are the percentages of total sequencing reads with the corresponding base. Cells were treated as described in the Materials and Methods.  FIG. 30A  shows the Alzheimer&#39;s disease-associated APOE4 allele converted to APOE3r in mouse astrocytes by BE3 in 74.9% of total reads. Two nearby Cs were also converted to Ts, but with no change to the predicted sequence of the resulting protein. Identical treatment of these cells with wt Cas9 and donor ssDNA results in only 0.3% correction, with 26.1% indel formation.  FIG. 30B  shows the cancer associated p53 Y163C mutation corrected by BE3 in 7.6% of nucleofected human breast cancer cells with 0.7% indel formation. Identical treatment of these cells with wt Cas9 and donor ssDNA results in no mutation correction with 6.1% indel formation. This figure depicts SEQ ID NOs: 675 to 680 from top to bottom, respectively. 
         FIG. 31  shows activities of BE1, BE2, and BE3 at HEK293 site 2 off-targets. HEK293T cells were transfected with plasmids expressing BE1, BE2, or BE3 and a sgRNA matching the HEK293 site 2 sequence using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus all of the known Cas9 and dCas9 off-target loci for the HEK293 site 2 sgRNA, as previously determined by Joung and coworkers using the GUIDE-seq method (63), and Adli and coworkers using chromatin immunoprecipitation high-throughput sequencing (ChIP-seq) experiments (18). Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for BE1, BE2, and BE3. On the far right are displayed the total number of sequencing reads reported, and the ChIP-seq signal intensity reported for each sequence. This figure depicts SEQ ID NOs: 681 to 688 from top to bottom, respectively. 
         FIG. 32  shows activities of BE1, BE2, and BE3 at HEK293 site 3 off-targets. HEK293T cells were transfected with plasmids expressing BE1, BE2, or BE3 and a sgRNA matching the HEK293 site 3 sequence using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus all of the known Cas9 off-target loci and the top five known dCas9 off-target loci for the HEK293 site 3 sgRNA, as previously determined by Joung and coworkers using the GUIDE-seq method 54 , and using chromatin immunoprecipitation high-throughput sequencing (ChIP-seq) experiments 61 . Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for BE1, BE2, and BE3. On the far right are displayed the total number of sequencing reads reported, and the ChIP-seq signal intensity reported for each sequence. This figure depicts SEQ ID NOs: 689 to 699 from top to bottom, respectively. 
         FIG. 33  shows activities of BE1, BE2, and BE3 at HEK293 site 4 off-targets. HEK293T cells were transfected with plasmids expressing BE1, BE2, or BE3 and a sgRNA matching the HEK293 site 4 sequence using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci, plus the top ten known Cas9 off-target loci and the top five known dCas9 off-target loci for the HEK293 site 4 sgRNA, as previously determined using the GUIDE-seq method 54 , and using chromatin immunoprecipitation high-throughput sequencing (ChIP-seq) experiments 61 . Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for BE1, BE2, and BE3. On the far right are displayed the total number of sequencing reads reported, and the ChIP-seq signal intensity reported for each sequence. This figure depicts SEQ ID NOs: 700 to 712 from top to bottom, respectively. 
         FIG. 34  shows mutation rates of non-protospacer bases following BE3-mediated correction of the Alzheimer&#39;s disease-associated APOE4 allele to APOE3r in mouse astrocytes. The DNA sequence of the 50 bases on either side of the protospacer from  FIG. 30A  and  FIG. 34B  is shown with each base&#39;s position relative to the protospacer. The side of the protospacer distal to the PAM is designated with positive numbers, while the side that includes the PAM is designated with negative numbers, with the PAM shown in blue. Underneath each sequence are the percentages of total DNA sequencing reads with the corresponding base for untreated cells, for cells treated with BE3 and an sgRNA targeting the APOE4 C158R mutation, or for cells treated with BE3 and an sgRNA targeting the VEGFA locus. Neither BE3-treated sample resulted in mutation rates above those of untreated controls. This figure depicts SEQ ID NOs: 713 to 716 from top to bottom, respectively. 
         FIG. 35  shows mutation rates of non-protospacer bases following BE3-mediated correction of the cancer-associated p53 Y163C mutation in HCC1954 human cells. The DNA sequence of the 50 bases on either side of the protospacer from  FIG. 30B  and  FIG. 39B  is shown with each base&#39;s position relative to the protospacer. The side of the protospacer distal to the PAM is designated with positive numbers, while the side that includes the PAM is designated with negative numbers, with the PAM shown in blue. Underneath each sequence are the percentages of total sequencing reads with the corresponding base for untreated cells, for cells treated with BE3 and an sgRNA targeting the TP53 Y163C mutation, or for cells treated with BE3 and an sgRNA targeting the VEGFA locus. Neither BE3-treated sample resulted in mutational rates above those of untreated controls. This figure depicts SEQ ID NOs: 717 to 720 from top to bottom, respectively. 
         FIGS. 36A to 36F  show the effects of deaminase, linker length, and linker composition on base editing.  FIG. 36A  shows a gel-based deaminase assay showing activity of rAPOBEC1, pmCDA1, hAID, hAPOBEC3G, rAPOBEC1-GGS-dCas9, rAPOBEC1-(GGS) 3  (SEQ ID NO: 596)-dCas9, and dCas9-(GGS) 3  (SEQ ID NO: 596)-rAPOBEC1 on ssDNA. Enzymes were expressed in a mammalian cell lysate-derived in vitro transcription-translation system and incubated with 1.8 μM dye-conjugated ssDNA and USER enzyme (uracil DNA glycosylase and endonuclease VIII) at 37° C. for 2 hours. The resulting DNA was resolved on a denaturing polyacrylamide gel and imaged. The positive control is a sequence with a U synthetically incorporated at the same position as the target C.  FIG. 36B  shows coomassie-stained denaturing PAGE gel of the expressed and purified proteins used in  FIGS. 36C to 36F .  FIGS. 36C to 36F  show gel-based deaminase assay showing the deamination window of base editors with deaminase-Cas9 linkers of GGS ( FIG. 36C ), (GGS) 3  (SEQ ID NO: 596) ( FIG. 36D ), XTEN ( FIG. 36E ), or (GGS) 7  (SEQ ID NO: 597) ( FIG. 36F ). Following incubation of 1.85 μM deaminase-dCas9 fusions complexed with sgRNA with 125 nM dsDNA substrates at 37° C. for 2 hours, the dye-conjugated DNA was isolated and incubated with USER enzyme at 37° C. for 1 hour to cleave the DNA backbone at the site of any uracils. The resulting DNA was resolved on a denaturing polyacrylamide gel, and the dye-conjugated strand was imaged. Each lane is numbered according to the position of the target C within the protospacer, or with—if no target C is present. 8U is a positive control sequence with a U synthetically incorporated at position 8. 
         FIGS. 37A to 37C  show BE1 base editing efficiencies are dramatically decreased in mammalian cells.  FIG. 37A  Protospacer (black and red) and PAM (blue) sequences of the six mammalian cell genomic loci targeted by base editors. Target Cs are indicated in red with subscripted numbers corresponding to their positions within the protospacer.  FIG. 37B  shows synthetic 80-mers with sequences matching six different genomic sites were incubated with BE1 then analyzed for base editing by HTS. For each site, the sequence of the protospacer is indicated to the right of the name of the site, with the PAM highlighted in blue. Underneath each sequence are the percentages of total DNA sequencing reads with the corresponding base. We considered a target C as “editable” if the in vitro conversion efficiency is &gt;10%. Note that maximum yields are 50% of total DNA sequencing reads since the non-targeted strand is unaffected by BE1. Values are shown from a single experiment.  FIG. 37C  shows HEK293T cells were transfected with plasmids expressing BE1 and an appropriate sgRNA. Three days after transfection, genomic DNA was extracted and analyzed by high-throughput DNA sequencing at the six loci. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, are shown for BE1 at all six genomic loci. Values and error bars of all data from HEK293T cells reflect the mean and standard deviation of three independent biological replicates performed on different days.  FIG. 37A  depicts SEQ ID NOs: 721 to 726 from top to bottom, respectively.  FIG. 37B  depicts SEQ ID NOs: 727 to 732 from top to bottom, respectively. 
         FIG. 38  shows base editing persists over multiple cell divisions. Cellular C to T conversion percentages by BE2 and BE3 are shown for HEK293 sites 3 and 4 in HEK293T cells before and after passaging the cells. HEK293T cells were nucleofected with plasmids expressing BE2 or BE3 and an sgRNA targeting HEK293 site 3 or 4. Three days after nucleofection, the cells were harvested and split in half. One half was subjected to HTS analysis, and the other half was allowed to propagate for approximately five cell divisions, then harvested and subjected to HTS analysis. Values and error bars reflect the mean and standard deviation of at least two biological experiments. 
         FIGS. 39A to 39C  show non-target C/G mutation rates. Shown here are the C to T and G to A mutation rates at 2,500 distinct cytosines and guanines surrounding the six on-target and 34 off-target loci tested, representing a total of 14,700,000 sequence reads derived from approximately 1.8×10 6  cells.  FIGS. 39A and 39B  show cellular non-target C to T and G to A conversion percentages by BE1, BE2, and BE3 are plotted individually against their positions relative to a protospacer for all 2,500 cytosines/guanines. The side of the protospacer distal to the PAM is designated with positive numbers, while the side that includes the PAM is designated with negative numbers.  FIG. 39C  shows average non-target cellular C to T and G to A conversion percentages by BE1, BE2, and BE3 are shown, as well as the highest and lowest individual conversion percentages. 
         FIGS. 40A to 40B  show additional data sets of BE3-mediated correction of two disease-relevant mutations in mammalian cells. For each site, the sequence of the protospacer is indicated to the right of the name of the mutation, with the PAM highlighted in blue and the base responsible for the mutation indicated in red bold with a subscripted number corresponding to its position within the protospacer. The amino acid sequence above each disease-associated allele is shown, together with the corrected amino acid sequence following base editing in green. Underneath each sequence are the percentages of total sequencing reads with the corresponding base. Cells were nucleofected with plasmids encoding BE3 and an appropriate sgRNA. Two days after nucleofection, genomic DNA was extracted from the nucleofected cells and analyzed by HTS to assess pathogenic mutation correction.  FIG. 40A  shows the Alzheimer&#39;s disease-associated APOE4 allele is converted to APOE3r in mouse astrocytes by BE3 in 58.3% of total reads only when treated with the correct sgRNA. Two nearby Cs are also converted to Ts, but with no change to the predicted sequence of the resulting protein. Identical treatment of these cells with wt Cas9 and donor ssDNA results in 0.2% correction, with 26.7% indel formation.  FIG. 40B  shows the cancer-associated p53 Y163C mutation is corrected by BE3 in 3.3% of nucleofected human breast cancer cells only when treated with the correct sgRNA. Identical treatment of these cells with wt Cas9 and donor ssDNA results in no detectable mutation correction with 8.0% indel formation.  FIGS. 40A to 40B  depict SEQ ID NOs: 733 to 740 from top to bottom, respectively. 
         FIG. 41  shows a schematic representation of an exemplary USER (Uracil-Specific Excision Reagent) Enzyme-based assay, which may be used to test the activity of various deaminases on single-stranded DNA (ssDNA) substrates. 
         FIG. 42  is a schematic of the pmCDA-nCas9-UGI-NLS construct and its activity at the HeK-3 site relative to the base editor (rAPOBEC1) and the negative control (untreated). 
         FIG. 43  is a schematic of the pmCDA1-XTEN-nCas9-UGI-NLS construct and its activity at the HeK-3 site relative to the base editor (rAPOBEC1) and the negative control (untreated). 
         FIG. 44  shows the percent of total sequencing reads with target C converted to T using cytidine deaminases (CDA) or APOBEC. 
         FIG. 45  shows the percent of total sequencing reads with target C converted to A using deaminases (CDA) or APOBEC. 
         FIG. 46  shows the percent of total sequencing reads with target C converted to G using deaminases (CDA) or APOBEC. 
         FIG. 47  is a schematic of the huAPOBEC3G-XTEN-nCas9-UGI-NLS construct and its activity at the HeK-2 site relative to a mutated form (huAPOBEC3G*(D316R_D317R)-XTEN-nCas9-UGI-NLS, the base editor (rAPOBEC1) and the negative control (untreated). 
         FIG. 48  shows the schematic of the LacZ construct used in the selection assay of Example 7. 
         FIG. 49  shows reversion data from different plasmids and constructs. 
         FIG. 50  shows the verification of lacZ reversion and the purification of reverted clones. 
         FIG. 51  is a schematic depicting a deamination selection plasmid used in Example 7. 
         FIG. 52  shows the results of a chloramphenicol reversion assay (pmCDA1 fusion). 
         FIGS. 53A to 53B  demonstrated DNA correction induction of two constructs. 
         FIG. 54  shows the results of a chloramphenicol reversion assay (huAPOBEC3G fusion). 
         FIG. 55  shows the activities of BE3 and HF-BE3 at EMX1 off-targets. The sequences, from top to bottom, correspond to SEQ ID NOs: 286-292, 299-301. 
         FIG. 56  shows on-target base editing efficiencies of BE3 and HF-BE3. 
         FIG. 57  is a graph demonstrating that mutations affect cytidine deamination with varying degrees. Combinations of mutations that each slightly impairs catalysis allow selective deamination at one position over others. The FANCF site was GGAATC 6 C 7 C 8 TTC 11 TGCAGCACCTGG (SEQ ID NO: 303). 
         FIG. 58  is a schematic depicting next generation base editors. 
         FIG. 59  is a schematic illustrating new base editors made from Cas9 variants. 
         FIG. 60  shows the base-edited percentage of different NGA PAM sites. 
         FIG. 61  shows the base-edited percentage of cytidines using NGCG PAM EMX (VRER BE3) and the C 1 TC 3 C 4 C 5 ATC 8 AC 10 ATCAACCGGT (SEQ ID NO: 304) spacer. 
         FIG. 62  shows the based-edited percentages resulting from different NNGRRT PAM sites. 
         FIG. 63  shows the based-edited percentages resulting from different NNHRRT PAM sites. 
         FIGS. 64A to 64C  show the base-edited percentages resulting from different TTTN PAM sites using Cpf1 BE2. The spacers used were: TTTCCTC 3 C 4 C 5 C 6 C 7 C 8 C 9 AC 11 AGGTAGAACAT ( FIG. 64A , SEQ ID NO: 305), TTTCC 1 C 2 TC 4 TGTC 8 C 9 AC 11 ACCCTCATCCTG ( FIG. 64B , SEQ ID NO: 306), and TTTCC 1 C 2 C 3 AGTC 7 C 8 TC 10 C 11 AC 13 AC 15 C 16 C 17 TGAAAC ( FIG. 64C , SEQ ID NO: 307). 
         FIG. 65  is a schematic depicting selective deamination as achieved through kinetic modulation of cytidine deaminase point mutagenesis. 
         FIG. 66  is a graph showing the effect of various mutations on the deamination window probed in cell culture with multiple cytidines in the spacer. The spacer used was: TGC 3 C 4 C 5 C 6 TC 8 C 9 C 10 TC 12 C 13 C 14 TGGCCC (SEQ ID NO: 308). 
         FIG. 67  is a graph showing the effect of various mutations on the deamination window probed in cell culture with multiple cytidines in the spacer. The spacer used was: AGAGC 5 C 6 C 7 C 8 C 9 C 10 C 11 TC 13 AAAGAGA (SEQ ID NO: 309). 
         FIG. 68  is a graph showing the effect of various mutations on the FANCF site with a limited number of cytidines. The spacer used was: GGAATC 6 C 7 C 8 TTC 11 TGCAGCACCTGG (SEQ ID NO: 303). Note that the triple mutant (W90Y, R126E, R132E) preferentially edits the cytidine at the sixth position. 
         FIG. 69  is a graph showing the effect of various mutations on the HEK3 site with a limited number of cytidines. The spacer used was: GGCC 4 C 5 AGACTGAGCACGTGATGG (SEQ ID NO: 310). Note that the double and triple mutants preferentially edit the cytidine at the fifth position over the cytidine in the fourth position. 
         FIG. 70  is a graph showing the effect of various mutations on the EMX1 site with a limited number of cytidines. The spacer used was: GAGTC 5 C 6 GAGCAGAAGAAGAAGGG (SEQ ID NO: 311). Note that the triple mutant only edits the cytidine at the fifth position, not the sixth. 
         FIG. 71  is a graph showing the effect of various mutations on the HEK2 site with a limited number of cytidines. The spacer used was: GAAC 4 AC 6 AAAGCATAGACTGCGGG (SEQ ID NO: 312). 
         FIG. 72  shows on-target base editing efficiencies of BE3 and BE3 comprising mutations W90Y R132E in immortalized astrocytes. 
         FIG. 73  depicts a schematic of three Cpf1 fusion constructs. 
         FIG. 74  shows a comparison of plasmid delivery of BE3 and HF-BE3 (EMX1, FANCF, and RNF2). 
         FIG. 75  shows a comparison of plasmid delivery of BE3 and HF-BE3 (HEK3 and HEK 4). 
         FIG. 76  shows off-target editing of EMX-1 at all 10 sites. 
         FIG. 77  shows deaminase protein lipofection to HEK cells using a GAGTCCGAGCAGAAGAAGAAG (SEQ ID NO: 313) spacer. The EMX-1 on-target and EMX-1 off target site 2 were examined. 
         FIG. 78  shows deaminase protein lipofection to HEK cells using a GGAATCCCTTCTGCAGCACCTGG (SEQ ID NO: 314) spacer. The FANCF on target and FANCF off target site 1 were examined. 
         FIG. 79  shows deaminase protein lipofection to HEK cells using a GGCCCAGACTGAGCACGTGA (SEQ ID NO: 315) spacer. The HEK-3 on target site was examined. 
         FIG. 80  shows deaminase protein lipofection to HEK cells using a GGCACTGCGGCTGGAGGTGGGGG (SEQ ID NO: 316) spacer. The HEK-4 on target, off target site 1, site 3, and site 4. 
         FIG. 81  shows the results of an in vitro assay for sgRNA activity for sgHR_13 (GTCAGGTCGAGGGTTCTGTC (SEQ ID NO: 317) spacer; C8 target: G51 to STOP), sgHR_14 (GGGCCGCAGTATCCTCACTC (SEQ ID NO: 318) spacer; C7 target; C7 target: Q68 to STOP), and sgHR_15 (CCGCCAGTCCCAGTACGGGA (SEQ ID NO: 319) spacer; C10 and C11 are targets: W239 or W237 to STOP). 
         FIG. 82  shows the results of an in vitro assay for sgHR_17 (CAACCACTGCTCAAAGATGC (SEQ ID NO: 320) spacer; C4 and C5 are targets: W410 to STOP), and sgHR_16 (CTTCCAGGATGAGAACACAG (SEQ ID NO: 321) spacer; C4 and C5 are targets: W273 to STOP). 
         FIG. 83  shows the direct injection of BE3 protein complexed with sgHR_13 in zebrafish embryos. 
         FIG. 84  shows the direct injection of BE3 protein complexed with sgHR_16 in zebrafish embryos. 
         FIG. 85  shows the direct injection of BE3 protein complexed with sgHR_17 in zebrafish embryos. 
         FIG. 86  shows exemplary nucleic acid changes that may be made using base editors that are capable of making a cytosine to thymine change. 
         FIG. 87  shows an illustration of apolipoprotein E (APOE) isoforms, demonstrating how a base editor (e.g., BE3) may be used to edit one APOE isoform (e.g., APOE4) into another APOE isoform (e.g., APOE3r) that is associated with a decreased risk of Alzheimer&#39;s disease. 
         FIG. 88  shows base editing of APOE4 to APOE3r in mouse astrocytes. 
         FIG. 89  shows base editing of PRNP to cause early truncation of the protein at arginine residue 37. 
         FIG. 90  shows that knocking out UDG (which UGI inhibits) dramatically improves the cleanliness of efficiency of C to T base editing. 
         FIG. 91  shows that use of a base editor with the nickase but without UGI leads to a mixture of outcomes, with very high indel rates. 
         FIGS. 92A to 92G  show that SaBE3, SaKKH-BE3, VQR-BE3, EQR-BE3, and VRER-BE3 mediate efficient base editing at target sites containing non-NGG PAMs in human cells.  FIG. 92A  shows base editor architectures using  S. pyogenes  and  S. aureus  Cas9.  FIG. 92B  shows recently characterized Cas9 variants with alternate or relaxed PAM requirements.  FIGS. 92C and 92D  show HEK293T cells treated with the base editor variants shown as described in Example 12. The percentage of total DNA sequencing reads (with no enrichment for transfected cells) with C converted to T at the target positions indicated are shown. The PAM sequence of each target tested is shown below the X-axis. The charts show the results for SaBE3 and SaKKH-BE3 at genomic loci with NNGRRT PAMs ( FIG. 92C ), SaBE3 and SaKKH-BE3 at genomic loci with NNNRRT PAMs ( FIG. 92D ), VQR-BE3 and EQR-BE3 at genomic loci with NGAG PAMs ( FIG. 92E ), and with NGAH PAMs ( FIG. 92F ), and VRER-BE3 at genomic loci with NGCG PAMs ( FIG. 92G ). Values and error bars reflect the mean and standard deviation of at least two biological replicates. 
         FIGS. 93A to 93C  demonstrate that base editors with mutations in the cytidine deaminase domain exhibit narrowed editing windows.  FIGS. 93A to 93C  show HEK293T cells transfected with plasmids expressing mutant base editors and an appropriate sgRNA. Three days after transfection, genomic DNA was extracted and analyzed by high-throughput DNA sequencing at the indicated loci. The percentage of total DNA sequencing reads (without enrichment for transfected cells) with C changed to T at the target positions indicated are shown for the EMX1 site, HEK293 site 3, FANCF site, HEK293 site 2, site A, and site B loci.  FIG. 93A  illustrates certain cytidine deaminase mutations which narrow the base editing window. See  FIG. 98  for the characterization of additional mutations.  FIG. 93B  shows the effect of cytidine deaminase mutations which effect the editing window width on genomic loci. Combining beneficial mutations has an additive effect on narrowing the editing window.  FIG. 93C  shows that YE1-BE3, YE2-BE3, EE-BE3, and YEE-BE3 effect the product distribution of base editing, producing predominantly singly-modified products in contrast with BE3. Values and error bars reflect the mean and standard deviation of at least two biological replicates. 
         FIGS. 94A and 94B  show genetic variants from ClinVar that in principle can be corrected by the base editors developed in this work. The NCBI ClinVar database of human genetic variations and their corresponding phenotypes was searched for genetic diseases that in theory can be corrected by base editing.  FIG. 94A  demonstrates improvement in base editing targeting scope among all pathogenic T→C mutations in the ClinVar database through the use of base editors with altered PAM specificities. The white fractions denote the proportion of pathogenic T-C mutations accessible on the basis of the PAM requirements of either BE3, or BE3 together with the five modified-PAM base editors developed in this work.  FIG. 94B  shows improvement in base editing targeting scope among all pathogenic T-C mutations in the ClinVar database through the use of base editors with narrowed activity windows. BE3 was assumed to edit Cs in positions 4-8 with comparable efficiency as shown in  FIGS. 93A to 93C . YEE-BE3 was assumed to edit with C5&gt;C6&gt;C7&gt;others preference within its activity window. The white fractions denote the proportion of pathogenic T-C mutations that can be edited BE3 without comparable editing of other Cs (left), or that can be edited BE3 or YEE-BE3 without comparable editing of other Cs (right). 
         FIGS. 95A and 95B  show the effect of truncated guide RNAs on base editing window width. HEK293T cells were transfected with plasmids expressing BE3 and sgRNAs of different 5′ truncation lengths. The treated cells were analyzed as described in the Examples.  FIG. 95A  shows protospacer and PAM sequence (top, SEQ ID NO: 4270) and cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, at a site within the EMX1 genomic locus. At this site, the base editing window was altered through the use of a 17-nt truncated gRNA. 
         FIG. 95B  shows protospacer and PAM sequences (top, SEQ ID NO: 4270) and cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, at sites within the HEK site 3 and site 4 genomic loci. At these sites, no change in the base editing window was observed, but a linear decrease in editing efficiency for all substrate bases as the sgRNA is truncated was noted. 
         FIG. 96  shows the effect of APOBEC1-Cas9 linker lengths on base editing window width. HEK293T cells were transfected with plasmids expressing base editors with rAPOBEC1-Cas9 linkers of XTEN, GGS, (GGS) 3  (SEQ ID NO: 596), (GGS) 5  (SEQ ID NO: 4271), or (GGS) 7  (SEQ ID NO: 597) and an sgRNA. The treated cells were analyzed as described in the Examples. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, are shown for the various base editors with different linkers. 
         FIGS. 97A to 97C  show the effect of rAPOBEC mutations on base editing window width.  FIG. 97C  shows HEK293T cells transfected with plasmids expressing an sgRNA targeting either Site A or Site B and the BE3 point mutants indicated. The treated cells were analyzed as described in the Examples. All C&#39;s in the protospacer and within three basepairs of the protospacer are displayed and the cellular C to T conversion percentages are shown. The ‘editing window widths’, defined as the calculated number of nucleotides within which editing efficiency exceeds the half-maximal value, are displayed for all tested mutants. 
         FIG. 98  shows the effect of APOBEC1 mutation son product distributions of base editing in mammalian cells. HEK293T cells were transfected with plasmids expressing BE3 or its mutants and an appropriate sgRNAs. The treated cells were analyzed as described in the Examples. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, are shown (left). Percent of total sequencing reads containing the C to T conversion is shown on the right. The BE3 point mutants do not significantly affect base editing efficiencies at HEK site 4, a site with only one target cytidine. 
         FIG. 99  shows a comparison of on-target editing plasma delivery in BE3 and HF-BE3. 
         FIG. 100  shows a comparison of on-target editing in protein and plasma delivery of BE3. 
         FIG. 101  shows a comparison of on-target editing in protein and plasma delivery of HF-BE3. 
         FIG. 102  shows that both lipofection and installing HF mutations decrease off-target deamination events. The diamond indicates no off targets were detected and the specificity ratio was set to 100. 
         FIG. 103  shows in vitro C to T editing on a synthetic substrate with Cs placed at even positions in the protospacer (NNNNTC 2 TC 4 TC 6 TC 8 TC 10 TC 12 TC 14 TC 16 TC 18 TC 20 NGG, SEQ ID NO: 4272). 
         FIG. 104  shows in vitro C to T editing on a synthetic substrate with Cs placed at odd positions in the protospacer (NNNNTC 2 TC 4 TC 6 TC 8 TC 10 TC 12 TC 14 TC 16 TC 18 TC 20 NGG, SEQ ID NO: 4272). 
         FIG. 105  includes two graphs depicting the specificity ratio of base editing with plasmid vs. protein delivery. 
         FIGS. 106A to 106B  shows BE3 activity on non-NGG PAM sites. HEK293T cells were transfected with plasmids expressing BE3 and appropriate sgRNA. The treated cells were analyzed as described in the Examples.  FIG. 106A  shows BE3 activity on sites can be efficiently targeted by SaBE3 or SaKKH-BE3. BE3 shows low but significant activity on the NAG PAM.  FIG. 106B  shows BE3 has significantly reduced editing at sites with NGA or NGCG PAMs, in contrast to VQR-BE3 or VRER-BE3. 
         FIGS. 107A to 107B  show the effect of APOBEC1 mutations on VQR-BE3 and SaKKH-BE3. HEK293T cells were transfected with plasmids expressing VQR-BE3, SaKKH-BE3 or its mutants and an appropriate sgRNAs. The treated cells were analyzed as described in the Methods. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, are shown.  FIG. 107A  shows that the window-modulating mutations can be applied to VQR-BE3 to enable selective base editing at sites targetable by NGA PAM.  FIG. 107B  shows that, when applied to SaKKH-BE3, the mutations cause overall decrease in base editing efficiency without conferring base selectivity within the target window. 
         FIG. 108  shows a schematic representation of nucleotide editing. The following abbreviations are used: (MMR)—mismatch repair, (BE3 Nickase)—refers to base editor 3, which comprises a Cas9 nickase domain, (UGI)—uracil glycosylase inhibitor, UDG)—uracil DNA glycosylase, (APOBEC)—refers to an APOBEC cytidine deaminase. 
     
    
    
     DEFINITIONS 
     As used herein and in the claims, the singular forms “a,” “an,” and “the” include the singular and the plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to “an agent” includes a single agent and a plurality of such agents. 
     The term “Cas9” or “Cas9 nuclease” 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. 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, 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. 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 embodiments, a Cas9 nuclease has an inactive (e.g., an inactivated) DNA cleavage domain, that is, the Cas9 is a nickase. 
     A nuclease-inactivated Cas9 protein may interchangeably be referred to as a “dCas9” protein (for nuclease-“dead” 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, proteins comprising fragments of Cas9 are provided. For example, in some embodiments, a protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or fragments thereof are referred to as “Cas9 variants.” A Cas9 variant shares homology to Cas9, or a fragment thereof. For example a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild type Cas9. In some embodiments, the Cas9 variant may have 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA-cleavage domain), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid length of a corresponding wild type Cas9. 
     In some embodiments, the fragment is at least 100 amino acids in length. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or at least 1300 amino acids in length. In some embodiments, wild type Cas9 corresponds to Cas9 from  Streptococcus pyogenes  (NCBI Reference Sequence: NC_017053.1, SEQ ID NO:1 (nucleotide); SEQ ID NO:2 (amino acid)). 
     
       
         
           
               
            
               
                 (SEQ ID NO: 1) 
               
               
                 ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGA 
               
               
                   
               
               
                 TGGGCGGTGATCACTGATGATTATAAGGTTCCGTCTAAAAAGTTCAAGGTT 
               
               
                   
               
               
                 CTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTT 
               
               
                   
               
               
                 TTATTTGGCAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCT 
               
               
                   
               
               
                 CGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATT 
               
               
                   
               
               
                 TTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAA 
               
               
                   
               
               
                 GAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTT 
               
               
                   
               
               
                 GGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTAT 
               
               
                   
               
               
                 CATCTGCGAAAAAAATTGGCAGATTCTACTGATAAAGCGGATTTGCGCTTA 
               
               
                   
               
               
                 ATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATT 
               
               
                   
               
               
                 GAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAG 
               
               
                   
               
               
                 TTGGTACAAATCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGT 
               
               
                   
               
               
                 AGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGA 
               
               
                   
               
               
                 TTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAGAAATGGCTTGTTT 
               
               
                   
               
               
                 GGGAATCTCATTGCTTTGTCATTGGGATTGACCCCTAATTTTAAATCAAAT 
               
               
                   
               
               
                 TTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGAT 
               
               
                   
               
               
                 GATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTG 
               
               
                   
               
               
                 TTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTA 
               
               
                   
               
               
                 AGAGTAAATAGTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAG 
               
               
                   
               
               
                 CGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGA 
               
               
                   
               
               
                 CAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAAC 
               
               
                   
               
               
                 GGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAA 
               
               
                   
               
               
                 TTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTG 
               
               
                   
               
               
                 AAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGC 
               
               
                   
               
               
                 TCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGA 
               
               
                   
               
               
                 CAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAA 
               
               
                   
               
               
                 ATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAAT 
               
               
                   
               
               
                 AGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGG 
               
               
                   
               
               
                 AATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAA 
               
               
                   
               
               
                 CGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAA 
               
               
                   
               
               
                 CATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTC 
               
               
                   
               
               
                 AAATATGTTACTGAGGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAG 
               
               
                   
               
               
                 AAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTT 
               
               
                   
               
               
                 AAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTT 
               
               
                   
               
               
                 GAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGCGCCTACCAT 
               
               
                   
               
               
                 GATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAAT 
               
               
                   
               
               
                 GAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGG 
               
               
                   
               
               
                 GGGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAG 
               
               
                   
               
               
                 GTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCT 
               
               
                   
               
               
                 CGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTA 
               
               
                   
               
               
                 GATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATC 
               
               
                   
               
               
                 CATGATGATAGTTTGACATTTAAAGAAGATATTCAAAAAGCACAGGTGTCT 
               
               
                   
               
               
                 GGACAAGGCCATAGTTTACATGAACAGATTGCTAACTTAGCTGGCAGTCCT 
               
               
                   
               
               
                 GCTATTAAAAAAGGTATTTTACAGACTGTAAAAATTGTTGATGAACTGGTC 
               
               
                   
               
               
                 AAAGTAATGGGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAA 
               
               
                   
               
               
                 AATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGA 
               
               
                   
               
               
                 ATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCT 
               
               
                   
               
               
                 GTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTACAA 
               
               
                   
               
               
                 AATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGT 
               
               
                   
               
               
                 GATTATGATGTCGATCACATTGTTCCACAAAGTTTCATTAAAGACGATTCA 
               
               
                   
               
               
                 ATAGACAATAAGGTACTAACGCGTTCTGATAAAAATCGTGGTAAATCGGAT 
               
               
                   
               
               
                 AACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAA 
               
               
                   
               
               
                 CTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAA 
               
               
                   
               
               
                 GCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGC 
               
               
                   
               
               
                 CAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGAT 
               
               
                   
               
               
                 AGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTT 
               
               
                   
               
               
                 AAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTC 
               
               
                   
               
               
                 CAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCG 
               
               
                   
               
               
                 TATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTT 
               
               
                   
               
               
                 GAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATG 
               
               
                   
               
               
                 ATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTT 
               
               
                   
               
               
                 TACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGA 
               
               
                   
               
               
                 GAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATT 
               
               
                   
               
               
                 GTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATG 
               
               
                   
               
               
                 CCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCC 
               
               
                   
               
               
                 AAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAA 
               
               
                   
               
               
                 AAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCT 
               
               
                   
               
               
                 TATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTA 
               
               
                   
               
               
                 AAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTT 
               
               
                   
               
               
                 GAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAA 
               
               
                   
               
               
                 AAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAAC 
               
               
                   
               
               
                 GGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAG 
               
               
                   
               
               
                 CTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTAT 
               
               
                   
               
               
                 GAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTG 
               
               
                   
               
               
                 GAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTT 
               
               
                   
               
               
                 TCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCA 
               
               
                   
               
               
                 TATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATT 
               
               
                   
               
               
                 CATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTT 
               
               
                   
               
               
                 GATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGAT 
               
               
                   
               
               
                 GCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGAT 
               
               
                   
               
               
                 TTGAGTCAGCTAGGAGGTGACTGA 
               
               
                   
               
               
                 (SEQ ID NO: 2) 
               
               
                 MDKK YSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGAL   
               
               
                   
               
               
                   LFGSGET AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE 
               
               
                   
               
               
                 ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRL 
               
               
                   
               
               
                 IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINAS 
               
               
                   
               
               
                 RVDAKAILSARLSKSRRLENLIAQLPGEKRNGLFGNLIALSLGLTPNFKSN 
               
               
                   
               
               
                 FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL 
               
               
                   
               
               
                 RVNSEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN 
               
               
                   
               
               
                 GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG 
               
               
                   
               
               
                 SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN 
               
               
                   
               
               
                 SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK 
               
               
                   
               
               
                 HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV 
               
               
                   
               
               
                 KQLKEDYFKKIECFDSVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEEN 
               
               
                   
               
               
                 EDILEDIVLTLTLFEDRGMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS 
               
               
                   
               
               
                 RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS 
               
               
                   
               
               
                 GQG HSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMAR E 
               
               
                   
               
               
                 NQTTQK GQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ   
               
               
                   
               
               
                 
                   NGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSD 
                 
               
               
                   
               
               
                   NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK AERG GLSELDKAGFIKR   
               
               
                   
               
               
                 
                   QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF 
                 
               
               
                   
               
               
                 
                   QFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM 
                 
               
               
                   
               
               
                 
                   IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI 
                 
               
               
                   
               
               
                   VWDKGRDFATVRKVLSMPQVNIVKKTEVQT GGFSKESILPKRNSDKLIARK 
               
               
                   
               
               
                 KDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF 
               
               
                   
               
               
                 EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE 
               
               
                   
               
               
                 LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEF 
               
               
                   
               
               
                 SKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF 
               
               
                   
               
               
                 DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                 (single underline: HNH domain; double underline: 
               
               
                 RuvC domain) 
               
            
           
         
       
     
     In some embodiments, wild type Cas9 corresponds to, or comprises SEQ ID NO:3 (nucleotide) and/or SEQ ID NO: 4 (amino acid): 
     
       
         
           
               
            
               
                 (SEQ ID NO: 3) 
               
               
                 ATGGATAAAAAGTATTCTATTGGTTTAGACATCGGCACTAATTCCGTTGGA 
               
               
                   
               
               
                 TGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTG 
               
               
                   
               
               
                 TTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCCTC 
               
               
                   
               
               
                 CTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCT 
               
               
                   
               
               
                 CGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATT 
               
               
                   
               
               
                 TTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAA 
               
               
                   
               
               
                 GAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTT 
               
               
                   
               
               
                 GGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTAT 
               
               
                   
               
               
                 CACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTA 
               
               
                   
               
               
                 ATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATT 
               
               
                   
               
               
                 GAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAG 
               
               
                   
               
               
                 TTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGT 
               
               
                   
               
               
                 GGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGG 
               
               
                   
               
               
                 CTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTC 
               
               
                   
               
               
                 GGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAAC 
               
               
                   
               
               
                 TTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGAT 
               
               
                   
               
               
                 GACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTA 
               
               
                   
               
               
                 TTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTG 
               
               
                   
               
               
                 AGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAA 
               
               
                   
               
               
                 AGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGT 
               
               
                   
               
               
                 CAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAAC 
               
               
                   
               
               
                 GGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAG 
               
               
                   
               
               
                 TTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTA 
               
               
                   
               
               
                 AAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGT 
               
               
                   
               
               
                 AGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGG 
               
               
                   
               
               
                 CAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAA 
               
               
                   
               
               
                 ATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAAC 
               
               
                   
               
               
                 TCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGG 
               
               
                   
               
               
                 AATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAG 
               
               
                   
               
               
                 AGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAG 
               
               
                   
               
               
                 CACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTT 
               
               
                   
               
               
                 AAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAG 
               
               
                   
               
               
                 AAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGTT 
               
               
                   
               
               
                 AAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTC 
               
               
                   
               
               
                 GAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCAT 
               
               
                   
               
               
                 GACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAAT 
               
               
                   
               
               
                 GAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGG 
               
               
                   
               
               
                 GAAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGACGATAAG 
               
               
                   
               
               
                 GTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCG 
               
               
                   
               
               
                 CGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTC 
               
               
                   
               
               
                 GATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATC 
               
               
                   
               
               
                 CATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCC 
               
               
                   
               
               
                 GGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCA 
               
               
                   
               
               
                 GCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTT 
               
               
                   
               
               
                 AAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGC 
               
               
                   
               
               
                 GAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAG 
               
               
                   
               
               
                 AGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCAT 
               
               
                   
               
               
                 CCTGTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTA 
               
               
                   
               
               
                 CAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTA 
               
               
                   
               
               
                 TCTGATTACGACGTCGATCACATTGTACCCCAATCCTTTTTGAAGGACGAT 
               
               
                   
               
               
                 TCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGT 
               
               
                   
               
               
                 GACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGG 
               
               
                   
               
               
                 CAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACT 
               
               
                   
               
               
                 AAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAA 
               
               
                   
               
               
                 CGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATACTA 
               
               
                   
               
               
                 GATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAA 
               
               
                   
               
               
                 GTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGAT 
               
               
                   
               
               
                 TTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGAC 
               
               
                   
               
               
                 GCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAG 
               
               
                   
               
               
                 CTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAG 
               
               
                   
               
               
                 ATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTC 
               
               
                   
               
               
                 TTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAAC 
               
               
                   
               
               
                 GGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAA 
               
               
                   
               
               
                 ATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCC 
               
               
                   
               
               
                 ATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTT 
               
               
                   
               
               
                 TCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGT 
               
               
                   
               
               
                 AAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTT 
               
               
                   
               
               
                 GCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAA 
               
               
                   
               
               
                 CTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCT 
               
               
                   
               
               
                 TTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTA 
               
               
                   
               
               
                 AAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAA 
               
               
                   
               
               
                 AATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAAC 
               
               
                   
               
               
                 GAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCAT 
               
               
                   
               
               
                 TACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTT 
               
               
                   
               
               
                 GTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAA 
               
               
                   
               
               
                 TTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGC 
               
               
                   
               
               
                 GCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATT 
               
               
                   
               
               
                 ATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTAT 
               
               
                   
               
               
                 TTTGACACAACGATAGATCGCAAACGATACACTTCTACCAAGGAGGTGCTA 
               
               
                   
               
               
                 GACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATA 
               
               
                   
               
               
                 GATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTC 
               
               
                   
               
               
                 TCGAGCGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGAT 
               
               
                   
               
               
                 TACAAGGATGACGATGACAAGGCTGCAGGA 
               
               
                   
               
               
                 (SEQ ID NO: 4) 
               
               
                 MDKK YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL   
               
               
                   
               
               
                   LFDSGET AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE 
               
               
                   
               
               
                 ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL 
               
               
                   
               
               
                 IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS 
               
               
                   
               
               
                 GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN 
               
               
                   
               
               
                 FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL 
               
               
                   
               
               
                 RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN 
               
               
                   
               
               
                 GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG 
               
               
                   
               
               
                 SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN 
               
               
                   
               
               
                 SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK 
               
               
                   
               
               
                 HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV 
               
               
                   
               
               
                 KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN 
               
               
                   
               
               
                 EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS 
               
               
                   
               
               
                 RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS 
               
               
                   
               
               
                 GQG DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA R 
               
               
                   
               
               
                 ENQTTQK GQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL   
               
               
                   
               
               
                 
                   QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKS 
                 
               
               
                   
               
               
                   DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK AERG GLSELDKAGFIK   
               
               
                   
               
               
                 
                   RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD 
                 
               
               
                   
               
               
                 
                   FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK 
                 
               
               
                   
               
               
                 
                   MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGE 
                 
               
               
                   
               
               
                   IVWDKGRDFATVRKVLSMPQVNIVKKTEVQT GGFSKESILPKRNSDKLIAR 
               
               
                   
               
               
                 KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS 
               
               
                   
               
               
                 FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN 
               
               
                   
               
               
                 ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE 
               
               
                   
               
               
                 FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY 
               
               
                   
               
               
                 FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                 (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, SEQ ID NO: 8 (nucleotide); and Uniport Reference Sequence: Q99ZW2, SEQ ID NO: 10 (amino acid). 
     
       
         
           
               
            
               
                 (SEQ ID NO: 8) 
               
               
                 ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGA 
               
               
                   
               
               
                 TGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTT 
               
               
                   
               
               
                 CTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTT 
               
               
                   
               
               
                 TTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCT 
               
               
                   
               
               
                 CGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATT 
               
               
                   
               
               
                 TTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAA 
               
               
                   
               
               
                 GAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTT 
               
               
                   
               
               
                 GGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTAT 
               
               
                   
               
               
                 CATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTA 
               
               
                   
               
               
                 ATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATT 
               
               
                   
               
               
                 GAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAG 
               
               
                   
               
               
                 TTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGT 
               
               
                   
               
               
                 GGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGA 
               
               
                   
               
               
                 TTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTT 
               
               
                   
               
               
                 GGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAAT 
               
               
                   
               
               
                 TTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGAT 
               
               
                   
               
               
                 GATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTG 
               
               
                   
               
               
                 TTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTA 
               
               
                   
               
               
                 AGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAA 
               
               
                   
               
               
                 CGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGA 
               
               
                   
               
               
                 CAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAAC 
               
               
                   
               
               
                 GGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAA 
               
               
                   
               
               
                 TTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTG 
               
               
                   
               
               
                 AAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGC 
               
               
                   
               
               
                 TCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGA 
               
               
                   
               
               
                 CAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAA 
               
               
                   
               
               
                 ATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAAT 
               
               
                   
               
               
                 AGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGG 
               
               
                   
               
               
                 AATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAA 
               
               
                   
               
               
                 CGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAA 
               
               
                   
               
               
                 CATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTC 
               
               
                   
               
               
                 AAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAG 
               
               
                   
               
               
                 AAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTT 
               
               
                   
               
               
                 AAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTT 
               
               
                   
               
               
                 GAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCAT 
               
               
                   
               
               
                 GATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAAT 
               
               
                   
               
               
                 GAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGG 
               
               
                   
               
               
                 GAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAG 
               
               
                   
               
               
                 GTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCT 
               
               
                   
               
               
                 CGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTA 
               
               
                   
               
               
                 GATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATC 
               
               
                   
               
               
                 CATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCT 
               
               
                   
               
               
                 GGACAAGGCGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCT 
               
               
                   
               
               
                 GCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTC 
               
               
                   
               
               
                 AAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGT 
               
               
                   
               
               
                 GAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAA 
               
               
                   
               
               
                 CGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCAT 
               
               
                   
               
               
                 CCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTC 
               
               
                   
               
               
                 CAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTA 
               
               
                   
               
               
                 AGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGAT 
               
               
                   
               
               
                 TCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCG 
               
               
                   
               
               
                 GATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGA 
               
               
                   
               
               
                 CAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACG 
               
               
                   
               
               
                 AAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAA 
               
               
                   
               
               
                 CGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTG 
               
               
                   
               
               
                 GATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAG 
               
               
                   
               
               
                 GTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGAT 
               
               
                   
               
               
                 TTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGAT 
               
               
                   
               
               
                 GCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAA 
               
               
                   
               
               
                 CTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAA 
               
               
                   
               
               
                 ATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTC 
               
               
                   
               
               
                 TTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAAT 
               
               
                   
               
               
                 GGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAA 
               
               
                   
               
               
                 ATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCC 
               
               
                   
               
               
                 ATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTC 
               
               
                   
               
               
                 TCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGT 
               
               
                   
               
               
                 AAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTA 
               
               
                   
               
               
                 GCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAG 
               
               
                   
               
               
                 TTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCC 
               
               
                   
               
               
                 TTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTT 
               
               
                   
               
               
                 AAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAA 
               
               
                   
               
               
                 AACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAAT 
               
               
                   
               
               
                 GAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCAT 
               
               
                   
               
               
                 TATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTT 
               
               
                   
               
               
                 GTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAA 
               
               
                   
               
               
                 TTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGT 
               
               
                   
               
               
                 GCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATT 
               
               
                   
               
               
                 ATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATAT 
               
               
                   
               
               
                 TTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTA 
               
               
                   
               
               
                 GATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATT 
               
               
                   
               
               
                 GATTTGAGTCAGCTAGGAGGTGACTGA 
               
               
                   
               
               
                 (SEQ ID NO: 10) 
               
               
                 MDKK YSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL   
               
               
                   
               
               
                   LFDSGET AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE 
               
               
                   
               
               
                 ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL 
               
               
                   
               
               
                 IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS 
               
               
                   
               
               
                 GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN 
               
               
                   
               
               
                 FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL 
               
               
                   
               
               
                 RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN 
               
               
                   
               
               
                 GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG 
               
               
                   
               
               
                 SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN 
               
               
                   
               
               
                 SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK 
               
               
                   
               
               
                 HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV 
               
               
                   
               
               
                 KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN 
               
               
                   
               
               
                 EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS 
               
               
                   
               
               
                 RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS 
               
               
                   
               
               
                 GQG DSLHEHIANLAGSPAIKKGILQTVKVVDELV   KVMGRHKPENIVIEMA R 
               
               
                   
               
               
                 ENQTTQK GQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL   
               
               
                   
               
               
                 
                   QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKS 
                 
               
               
                   
               
               
                   DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK AERG GLSELDKAGFIK   
               
               
                   
               
               
                 
                   RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD 
                 
               
               
                   
               
               
                 
                   FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK 
                 
               
               
                   
               
               
                 
                   MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGE 
                 
               
               
                   
               
               
                   IVWDKGRDFATVRKVLSMPQVNIVKKTEVQT GGFSKESILPKRNSDKLIAR 
               
               
                   
               
               
                 KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS 
               
               
                   
               
               
                 FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN 
               
               
                   
               
               
                 ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE 
               
               
                   
               
               
                 FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY 
               
               
                   
               
               
                 FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                 (single underline: HNH domain; double underline: 
               
               
                 RuvC domain) 
               
            
           
         
       
     
     In some embodiments, Cas9 refers to Cas9 from:  Corynebacterium ulcerans  (NCBI Refs: NC_015683.1, NC_017317.1);  Corynebacterium diphtheria  (NCBI Refs: NC_016782.1, NC_016786.1);  Spiroplasma syrphidicola  (NCBI Ref: NC_021284.1);  Prevotella intermedia  (NCBI Ref: NC_017861.1);  Spiroplasma taiwanense  (NCBI Ref: NC_021846.1);  Streptococcus iniae  (NCBI Ref: NC_021314.1);  Belliella baltica  (NCBI Ref: NC_018010.1);  Psychroflexus torquis  I (NCBI Ref: NC_018721.1);  Streptococcus thermophilus  (NCBI Ref: YP_820832.1),  Listeria innocua  (NCBI Ref: NP_472073.1),  Campylobacter jejuni  (NCBI Ref: YP_002344900.1) or  Neisseria. meningitidis  (NCBI Ref: YP_002342100.1) or to a Cas9 from any of the organisms listed in Example 5. 
     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/or H840A mutation. 
     dCas9 (D10A and H840A): 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 9) 
                   
               
               
                 MDKK   YSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDS     
                   
               
               
                   
               
               
                     GET   AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK 
               
               
                   
               
               
                 HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLI 
               
               
                   
               
               
                 EGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQ 
               
               
                   
               
               
                 LPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQ 
               
               
                   
               
               
                 YADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL 
               
               
                   
               
               
                 PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK 
               
               
                   
               
               
                 QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSR 
               
               
                   
               
               
                 FAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF 
               
               
                   
               
               
                 TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIEC 
               
               
                   
               
               
                 FDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE 
               
               
                   
               
               
                 RLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     NTQLCINEKLYLYYLCINGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKV 
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 TVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI 
               
               
                   
               
               
                 IKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDN 
               
               
                   
               
               
                 EQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH 
               
               
                   
               
               
                 LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                 (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 in SEQ ID NO: 10, or at corresponding positions in any of the amino acid sequences provided in SEQ ID NOs: 11-260. Without wishing to be bound by any particular theory, the presence of the catalytic residue H840 restores the activity of the Cas9 to cleave the non-edited (e.g., non-deaminated) strand containing a G opposite the targeted C. Restoration of H840 (e.g., from A840) does not result in the cleavage of the target strand containing the C. 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 G to A change on the non-edited strand. A schematic representation of this process is shown in  FIG. 108 . Briefly, the C of a C-G basepair can be deaminated to a U by a deaminase, e.g., an APOBEC deamonase. Nicking the non-edited strand, having the G, facilitates removal of the G via mismatch repair mechanisms. UGI inhibits UDG, which prevents removal of the U. 
     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 H820, 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 (e.g., variants of SEQ ID NO: 10) are provided which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to SEQ ID NO: 10. In some embodiments, variants of dCas9 (e.g., variants of SEQ ID NO: 10) are provided having amino acid sequences which are shorter, or longer than SEQ ID NO: 10, by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more. 
     In some embodiments, Cas9 fusion proteins as provided herein comprise the full-length amino acid sequence of a Cas9 protein, e.g., one of the Cas9 sequences provided herein. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length Cas9 sequence, but only a fragment thereof. For example, in some embodiments, a Cas9 fusion protein provided herein comprises a Cas9 fragment, wherein the fragment binds crRNA and tracrRNA or sgRNA, but does not comprise a functional nuclease domain, e.g., in that it comprises only a truncated version of a nuclease domain or no nuclease domain at all. 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. 
     In some embodiments, Cas9 refers to Cas9 from:  Corynebacterium ulcerans  (NCBI Refs: NC_015683.1, NC_017317.1);  Corynebacterium diphtheria  (NCBI Refs: NC_016782.1, NC_016786.1);  Spiroplasma syrphidicola  (NCBI Ref: NC_021284.1);  Prevotella intermedia  (NCBI Ref: NC_017861.1);  Spiroplasma taiwanense  (NCBI Ref: NC_021846.1);  Streptococcus iniae  (NCBI Ref: NC_021314.1);  Belliella baltica  (NCBI Ref: NC_018010.1);  Psychroflexus torquis  I (NCBI Ref: NC_018721.1);  Streptococcus thermophilus  (NCBI Ref: YP_820832.1);  Listeria innocua  (NCBI Ref: NP_472073.1);  Campylobacter jejuni  (NCBI Ref: YP_002344900.1); or  Neisseria. meningitidis  (NCBI Ref: YP_002342100.1). 
     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 cytidine deaminase domain, catalyzing the hydrolytic deamination of cytosine to uracil. In some embodiments, the deaminase or deaminase domain is 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 is a variant of a naturally-occurring deaminase from an organism, that 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 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring deaminase from an organism. 
     The term “effective amount,” as used herein, refers to an amount of a biologically active agent that is sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of a nuclease may refer to the amount of the nuclease that is sufficient to induce cleavage of a target site specifically bound and cleaved by the nuclease. In some embodiments, an effective amount of a fusion protein provided herein, e.g., of a fusion protein comprising a nuclease-inactive Cas9 domain and a nucleic acid editing domain (e.g., a deaminase domain) may refer to the amount of the fusion protein that is sufficient to induce editing of a target site specifically bound and edited by the fusion protein. As will be appreciated by the skilled artisan, the effective amount of an agent, e.g., a fusion protein, a nuclease, a deaminase, a recombinase, a hybrid protein, a protein dimer, a complex of a protein (or protein dimer) and a polynucleotide, or a polynucleotide, may vary depending on various factors as, for example, on the desired biological response, e.g., on the specific allele, genome, or target site to be edited, on the cell or tissue being targeted, and on the agent being used. 
     The term “linker,” as used herein, refers to a chemical group or a molecule linking two molecules or moieties, e.g., two domains of a fusion protein, such as, for example, a nuclease-inactive Cas9 domain and a nucleic acid editing domain (e.g., a deaminase domain). 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 nucleic-acid editing protein. Typically, 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 5-100 amino acids in length, for example, 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. Longer or shorter linkers are also contemplated. 
     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  (4 th  ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). 
     The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g. nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or including non-naturally occurring nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5′ to 3′ direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(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 editing domain,” as used herein refers to a protein or enzyme capable of making one or more modifications (e.g., deamination of a cytidine residue) to a nucleic acid (e.g., DNA or RNA). Exemplary nucleic acid editing domains include, but are not limited to a deaminase, a nuclease, a nickase, a recombinase, a methyltransferase, a methylase, an acetylase, an acetyltransferase, a transcriptional activator, or a transcriptional repressor domain. In some embodiments the nucleic acid editing domain is a deaminase (e.g., a cytidine deaminase, such as an APOBEC or an AID deaminase). 
     The term “proliferative disease,” as used herein, refers to any disease in which cell or tissue homeostasis is disturbed in that a cell or cell population exhibits an abnormally elevated proliferation rate. Proliferative diseases include hyperproliferative diseases, such as pre-neoplastic hyperplastic conditions and neoplastic diseases. Neoplastic diseases are characterized by an abnormal proliferation of cells and include both benign and malignant neoplasias. Malignant neoplasia is also referred to as cancer. 
     The terms “protein,” “peptide,” and “polypeptide” 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 may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may 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 modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may 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 may 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 may 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. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may 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  (4 th  ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference. 
     The term “RNA-programmable nuclease,” and “RNA-guided nuclease” are used interchangeably herein and refer to a nuclease that forms a complex with (e.g., binds or associates with) one or more RNA 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); 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. 
     Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNA hybridization to target DNA cleavage sites, these proteins are able to be targeted, in principle, to any sequence specified by the guide RNA. Methods of using RNA-programmable nucleases, such as Cas9, for site-specific cleavage (e.g., to modify a genome) are known in the art (see e.g., Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems.  Science  339, 819-823 (2013);  Mali , P. et al. RNA-guided human genome engineering via Cas9 . Science  339, 823-826 (2013); Hwang, W. Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system.  Nature biotechnology  31, 227-229 (2013); Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, J. E. et al. Genome engineering in  Saccharomyces cerevisiae  using CRISPR-Cas systems.  Nucleic acids research  (2013); Jiang, W. et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems.  Nature biotechnology  31, 233-239 (2013); the entire contents of each of which are incorporated herein by reference). 
     The term “subject,” as used herein, refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development. 
     The term “target site” refers to a sequence within a nucleic acid molecule that is deaminated by a deaminase or a fusion protein comprising a deaminase, (e.g., a dCas9-deaminase fusion protein provided herein). 
     The terms “treatment,” “treat,” and “treating,” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. As used herein, the terms “treatment,” “treat,” and “treating” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence. 
     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. 
     The term “nucleobase editors (NBEs)” or “base editors (BEs),” as used herein, refers to the Cas9 fusion proteins described herein. In some embodiments, the fusion protein comprises a nuclease-inactive Cas9 (dCas9) fused to a deaminase. In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase. In some embodiments, the fusion protein comprises a nuclease-inactive Cas9 fused to a deaminase and further fused to a UGI domain. In some embodiments, the fusion protein comprises a Cas9 nickase fused to a deaminase and further fused to a UGI domain. In some embodiments, the dCas9 of the fusion protein comprises a D10A and a H840A mutation of SEQ ID NO: 10, or a corresponding mutation in any of SEQ ID NOs: 11-260, which inactivates nuclease activity of the Cas9 protein. In some embodiments, the fusion protein comprises a D10A mutation and comprises a histidine at residue 840 of SEQ ID NO: 10, or a corresponding mutation in any of SEQ ID NOs: 11-260, which renders Cas9 capable of cleaving only one strand of a nucleic acid duplex. An example of a Cas9 nickase is shown below in SEQ ID NO: 674. The terms “nucleobase editors (NBEs)” and “base editors (BEs)” may be used interchangeably. 
     The term “uracil glycosylase inhibitor” or “UGI,” as used herein, refers to a protein that is capable of inhibiting a uracil-DNA glycosylase base-excision repair enzyme. 
     The term “Cas9 nickase,” as used herein, refers to 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, a Cas9 nickase comprises a D10A mutation and has a histidine at position H840 of SEQ ID NO: 10, or a corresponding mutation in any of SEQ ID NOs: 11-260. For example, a Cas9 nickase may comprise the amino acid sequence as set forth in SEQ ID NO: 674. Such a Cas9 nickase has an active HNH nuclease domain and is able to cleave the non-targeted strand of DNA, i.e., the strand bound by the gRNA. Further, such a Cas9 nickase has an inactive RuvC nuclease domain and is not able to cleave the targeted strand of the DNA, i.e., the strand where base editing is desired. 
     Exemplary Cas9 nickase (Cloning vector pPlatTET-gRNA2; Accession No. BAV54124). 
     
       
         
           
               
            
               
                 (SEQ ID NO: 674) 
               
               
                 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL 
               
               
                   
               
               
                 LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE 
               
               
                   
               
               
                 ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL 
               
               
                   
               
               
                 IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS 
               
               
                   
               
               
                 GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN 
               
               
                   
               
               
                 FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL 
               
               
                   
               
               
                 RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN 
               
               
                   
               
               
                 GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG 
               
               
                   
               
               
                 SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN 
               
               
                   
               
               
                 SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK 
               
               
                   
               
               
                 HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV 
               
               
                   
               
               
                 KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN 
               
               
                   
               
               
                 EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS 
               
               
                   
               
               
                 RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS 
               
               
                   
               
               
                 GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR 
               
               
                   
               
               
                 ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL 
               
               
                   
               
               
                 QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKS 
               
               
                   
               
               
                 DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK 
               
               
                   
               
               
                 RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD 
               
               
                   
               
               
                 FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK 
               
               
                   
               
               
                 MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGE 
               
               
                   
               
               
                 IVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR 
               
               
                   
               
               
                 KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS 
               
               
                   
               
               
                 FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN 
               
               
                   
               
               
                 ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE 
               
               
                   
               
               
                 FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY 
               
               
                   
               
               
                 FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
            
           
         
       
     
     DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION 
     Some aspects of this disclosure provide fusion proteins that comprise a domain capable of binding to a nucleotide sequence (e.g., a Cas9, or a Cpf1 protein) and an enzyme domain, for example, a DNA-editing domain, such as, e.g., a deaminase domain. The deamination of a nucleobase by a deaminase can lead to a point mutation at the respective residue, which is referred to herein as nucleic acid editing. Fusion proteins comprising a Cas9 variant or domain and a DNA editing domain can thus be used for the targeted editing of nucleic acid sequences. Such fusion proteins are useful 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, e.g., the correction of genetic defects or the introduction of deactivating mutations in disease-associated genes in a subject. Typically, the Cas9 domain of the fusion proteins described herein does not have any nuclease activity but instead is a Cas9 fragment or a dCas9 protein or domain. Methods for the use of Cas9 fusion proteins as described herein are also provided. 
     Cas9 Domains of Nucleobase Editors 
     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 in SEQ ID NOs: 10-263. 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 in SEQ ID NOs: 10-263. In some embodiments, the Cas9 domain comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more or more mutations compared to any one of the amino acid sequences set forth in SEQ ID NOs: 10-263. 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 in SEQ ID NOs: 10-263. 
     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 in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, 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 in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. As one example, a nuclease-inactive Cas9 domain comprises the amino acid sequence set forth in SEQ ID NO: 263 (Cloning vector pPlatTET-gRNA2, Accession No. BAV54124). 
                    (SEQ ID NO: 263       MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL               LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLE               ESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL               IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINAS               GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN               FDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL               RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN               GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG               SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN               SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK               HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV               KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN               EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLS               RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS               GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAR               ENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL               QNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKS               DNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK               RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD               FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK               MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGE               IVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR               KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS               FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN               ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISE               FSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKY               FDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD;            
see, e.g., Qi et al., Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.  Cell.  2013; 152(5):1173-83, the entire contents of which are incorporated herein by reference).
 
     Additional suitable nuclease-inactive dCas9 domains will be apparent to those of skill in the art based on this disclosure and knowledge in the field, and are within the scope of this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A/H840A, D10A/D839A/H840A, and D10A/D839A/H840A/N863A mutant domains (See, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.  Nature Biotechnology.  2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference). In some embodiments 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 in SEQ ID NOs: 10-263. 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 leat 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 in SEQ ID NOs: 10-263. 
     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 of SEQ ID NO: 10, or a mutation in any of SEQ ID NOs: 11-260. For example, a Cas9 nickase may comprise the amino acid sequence as set forth in SEQ ID NO: 674. 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 of SEQ ID NO: 10, or a corresponding mutation in any of SEQ ID NOs: 11-260. 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. 
     Cas9 Domains with Reduced PAM Exclusivity 
     Some aspects of the disclosure provide Cas9 domains that have different PAM specificities. Typically, Cas9 proteins, such as Cas9 from  S. pyogenes  (spCas9), require a canonical NGG PAM sequence to bind a particular nucleic acid region. 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 where a target base is placed within a 4 base region (e.g., a “deamination window”), which is 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), the entire contents of which are hereby incorporated by reference. Accordingly, in some embodiments, any of the fusion proteins provided herein may contain a Cas9 domain that is capable of binding a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences have been described in the art and would be apparent to the skilled artisan. For example, Cas9 domains that bind non-canonical PAM sequences have been described in Kleinstiver, B. P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities”  Nature  523, 481-485 (2015); and Kleinstiver, B. P., et al., “Broadening the targeting range of  Staphylococcus aureus  CRISPR-Cas9 by modifying PAM recognition”  Nature Biotechnology  33, 1293-1298 (2015); the entire contents of each are hereby incorporated by reference. 
     In some embodiments, the Cas9 domain is a Cas9 domain from  Staphylococcus aureus  (SaCas9). In some embodiments, the SaCas9 domain is a nuclease active SaCas9, a nuclease inactive SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, the SaCas9 comprises the amino acid sequence SEQ ID NO: 4273. In some embodiments, the SaCas9 comprises a N579X mutation of SEQ ID NO: 4273, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid except for N. In some embodiments, the SaCas9 comprises a N579A mutation of SEQ ID NO: 4273, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. 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 PAM sequence. In some embodiments, the SaCas9 domain comprises one or more of a E781X, a N967X, and a R1014X mutation of SEQ ID NO: 4273, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, 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 of SEQ ID NO: 4273, or one or more corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the SaCas9 domain comprises a E781K, a N967K, or a R1014H mutation of SEQ ID NO: 4273, or corresponding mutations in any of the amino acid sequences provided in SEQ ID NOs: 11-260. 
     In some embodiments, the Cas9 domain 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 any one of SEQ ID NOs: 4273-4275. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises the amino acid sequence of any one of SEQ ID NOs: 4273-4275. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein consists of the amino acid sequence of any one of SEQ ID NOs: 4273-4275. 
                    Exemplary SaCas9 sequence       (SEQ ID NO: 4273)       KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRG               ARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEE               EFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQ               LERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLL               ETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLY               NALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNE               EDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIY               QSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWH               TNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKV               INAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRT               TGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRS               VSFDNSFNNKVLVKQEE   N   SKKGNRTPFQYLSSSDSKISYETFKKHILNLAK               GKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYF               RVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIF               KEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDF               KDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLK               KLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKY               SKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLD               NGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNND               LIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIA               SKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG            
Residue N579 of SEQ ID NO: 4273, which is underlined and in bold, may be mutated (e.g., to a A579) to yield a SaCas9 nickase.
 
                    Exemplary SaCas9n sequence       (SEQ ID NO: 4274)       KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR               GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLS               EEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVA               ELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTY               IDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY               NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK               EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQI               AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN               LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVK               RSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQT               NERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF               NYEVDHIIPRSVSFDNSFNNKVLVKQEE   A   SKKGNRTPFQYLSSSDSKISY               ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRY               ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH               AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYK               EIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLI               VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEK               NPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSR               NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK               KLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITY               REYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK               KG.            
Residue A579 of SEQ ID NO: xx, which can be mutated from N579 of SEQ ID NO: 4274 to yield a SaCas9 nickase, is underlined and in bold.
 
                    Exemplary SaKKH Cas9       (SEQ ID NO: 4275)       KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR               GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLS               EEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVA               ELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTY               IDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY               NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK               EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQI               AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN               LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVK               RSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQT               NERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF               NYEVDHIIPRSVSFDNSFNNKVLVKQEE   A   SKKGNRTPFQYLSSSDSKISY               ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRY               ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH               AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYK               EIFITPHQIKHIKDFKDYKYSHRVDKKPNR   K   LINDTLYSTRKDDKGNTLI               VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEK               NPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSR               NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK               KLKKISNQAEFIASFY   K   NDLIKINGELYRVIGVNNDLLNRIEVNMIDITY               REYLENMNDKRPP   H   IIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK               KG.            
Residue A579 of SEQ ID NO: 4275, which can be mutated from N579 of SEQ ID NO: 4275 to yield a SaCas9 nickase, is underlined and in bold. Residues K781, K967, and H1014 of SEQ ID NO: 4275, which can be mutated from E781, N967, and R1014 of SEQ ID NO: 4275 to yield a SaKKH Cas9 are underlined and in italics.
 
     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 the amino acid sequence SEQ ID NO: 4276. In some embodiments, the SpCas9 comprises a D9X mutation of SEQ ID NO: 4276, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid except for D. In some embodiments, the SpCas9 comprises a D9A mutation of SEQ ID NO: 4276, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. 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 a NGG, a NGA, or a NGCG PAM sequence. In some embodiments, the SpCas9 domain comprises one or more of a D1134X, a R1334X, and a T1336X mutation of SEQ ID NO: 4276, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1134E, R1334Q, and T1336R mutation of SEQ ID NO: 4276, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the SpCas9 domain comprises a D1134E, a R1334Q, and a T1336R mutation of SEQ ID NO: 4276, or corresponding mutations in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the SpCas9 domain comprises one or more of a D1134X, a R1334X, and a T1336X mutation of SEQ ID NO: 4276, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1134V, a R1334Q, and a T1336R mutation of SEQ ID NO: 4276, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the SpCas9 domain comprises a D1134V, a R1334Q, and a T1336R mutation of SEQ ID NO: 4276, or corresponding mutations in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the SpCas9 domain comprises one or more of a D1134X, a G1217X, a R1334X, and a T1336X mutation of SEQ ID NO: 4276, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of a D1134V, a G1217R, a R1334Q, and a T1336R mutation of SEQ ID NO: 4276, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the SpCas9 domain comprises a D1134V, a G1217R, a R1334Q, and a T1336R mutation of SEQ ID NO: 4276, or corresponding mutations in any of the amino acid sequences provided in SEQ ID NOs: 11-260. 
     In some embodiments, the Cas9 domain 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 any one of SEQ ID NOs: 4276-4280. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein comprises the amino acid sequence of any one of SEQ ID NOs: 4276-4280. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein consists of the amino acid sequence of any one of SEQ ID NOs: 4276-4280. 
                    Exemplary SpCas9       (SEQ ID NO: 4276)       DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL               FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEE               SFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI               YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASG               VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF               DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILR               VNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNG               YAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS               IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNS               RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH               SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK               QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENE               DILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSR               KLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSG               QGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE               NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ               NGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD               NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR               QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF               QFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM               IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI               VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARK               KDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF               EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE               LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEF               SKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF               DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD               Exemplary SpCas9n       (SEQ ID NO: 4277)       DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL               FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEE               SFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI               YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASG               VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF               DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILR               VNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNG               YAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS               IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNS               RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH               SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK               QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENE               DILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSR               KLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSG               QGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE               NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ               NGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD               NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR               QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF               QFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM               IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI               VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARK               KDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF               EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE               LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEF               SKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF               DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD               Exemplary SpEQR Cas9       (SEQ ID NO: 4278)       DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL               FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEE               SFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLI               YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASG               VDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF               DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILR               VNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNG               YAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS               IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNS               RFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKH               SLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK               QLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENE               DILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSR               KLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSG               QGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARE               NQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ               NGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD               NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKR               QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF               QFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKM               IAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI               VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARK               KDWDPKKYGGF   E   SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF               EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE               LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEF               SKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF               DTTIDRK   Q   Y   R   STKEVLDATLIHQSITGLYETRIDLSQLGGD            
Residues E1134, Q1334, and R1336 of SEQ ID NO: 4278, which can be mutated from D1134, R1334, and T1336 of SEQ ID NO: 4278 to yield a SpEQR Cas9, are underlined and in bold.
 
                    Exemplary SpVQR Cas9       (SEQ ID NO: 4279)       DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL               LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL               EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL               RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI               NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN               FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL               LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF               FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK               QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY               VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN               LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL               LFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKII               KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL               KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS               LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM               GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV               ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS               IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT               KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR               EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY               PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT               LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ               TGGFSKESILPKRNSDKLIARKKDWDPKKYGGF   V   SPTVAYSVLVVAKVEK               GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY               SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED               NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP               IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK   Q   Y   R   STKEVLDATLIHQS               ITGLYETRIDLSQLGGD            
Residues V1134, Q1334, and R1336 of SEQ ID NO: 4279, which can be mutated from D1134, R1334, and T1336 of SEQ ID NO: 4279 to yield a SpVQR Cas9, are underlined and in bold.
 
                    Exemplary SpVRER Cas9       (SEQ ID NO: 4280)       DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL               LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL               EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL               RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI               NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN               FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL               LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF               FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK               QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY               VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN               LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL               LFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKII               KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL               KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS               LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM               GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV               ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS               IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT               KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR               EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY               PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT               LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ               TGGFSKESILPKRNSDKLIARKKDWDPKKYGGF   V   SPTVAYSVLVVAKVEK               GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY               SLFELENGRKRMLASA   R   ELQKGNELALPSKYVNFLYLASHYEKLKGSPED               NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP               IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK   E   Y   R   STKEVLDATLIHQS               ITGLYETRIDLSQLGGD            
Residues V1134, R1217, Q1334, and R1336 of SEQ ID NO: 4280, which can be mutated from D1134, G1217, R1334, and T1336 of SEQ ID NO: 4280 to yield a SpVRER Cas9, are underlined and in bold.
 
     The following are exemplary fusion proteins (e.g., base editing proteins) capable of binding to a nucleic acid sequence having a non-canonical (e.g., a non-NGG) PAM sequence: 
     
       
         
           
               
               
            
               
                 Exemplary SaBE3 (rAPOBEC1-XTEN-SaCas9n-UGI-NLS) 
                   
               
               
                 (SEQ ID NO: 4281) 
                   
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVE 
                   
               
               
                   
               
               
                 VNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPR 
               
               
                   
               
               
                 NRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGL 
               
               
                   
               
               
                 PPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPES KRNYILG   
               
               
                   
               
               
                 
                   LDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLL 
                 
               
               
                   
               
               
                 
                   FDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELST 
                 
               
               
                   
               
               
                 
                   KEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSF 
                 
               
               
                   
               
               
                 
                   IDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALN 
                 
               
               
                   
               
               
                 
                   DLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEF 
                 
               
               
                   
               
               
                 
                   TNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNL 
                 
               
               
                   
               
               
                 
                   KGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPV 
                 
               
               
                   
               
               
                 
                   VKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTT 
                 
               
               
                   
               
               
                 
                   GKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLV 
                 
               
               
                   
               
               
                 
                   KQEEASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQ 
                 
               
               
                   
               
               
                 
                   KDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH 
                 
               
               
                   
               
               
                 
                   AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIK 
                 
               
               
                   
               
               
                 
                   DFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKL 
                 
               
               
                   
               
               
                 
                   LMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAH 
                 
               
               
                   
               
               
                 
                   LDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKL 
                 
               
               
                   
               
               
                 
                   KKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRI 
                 
               
               
                   
               
               
                   IKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG SGGSTNLSDIIEKETGKQLVIQESIL 
               
               
                   
               
               
                 MLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSG 
               
               
                   
               
               
                 GSPKKKRKV 
               
               
                   
               
               
                 Exemplary SaKKH-BE3 (rAPOBEC1-XTEN-SaCas9n-UGI-NLS) 
               
               
                 (SEQ ID NO: 4282) 
                   
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVE 
                   
               
               
                   
               
               
                 VNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPR 
               
               
                   
               
               
                 NRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGL 
               
               
                   
               
               
                 PPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPES KRNYILG   
               
               
                   
               
               
                 
                   LDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLL 
                 
               
               
                   
               
               
                 
                   FDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELST 
                 
               
               
                   
               
               
                 
                   KEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSF 
                 
               
               
                   
               
               
                 
                   IDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALN 
                 
               
               
                   
               
               
                 
                   DLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEF 
                 
               
               
                   
               
               
                 
                   TNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNL 
                 
               
               
                   
               
               
                 
                   KGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPV 
                 
               
               
                   
               
               
                 
                   VKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTT 
                 
               
               
                   
               
               
                 
                   GKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLV 
                 
               
               
                   
               
               
                 
                   KQEEASKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQ 
                 
               
               
                   
               
               
                 
                   KDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH 
                 
               
               
                   
               
               
                 
                   AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIK 
                 
               
               
                   
               
               
                 
                   DFKDYKYSHRVDKKPNR K LINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKL 
                 
               
               
                   
               
               
                 
                   LMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAH 
                 
               
               
                   
               
               
                 
                   LDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKL 
                 
               
               
                   
               
               
                 
                   KKISNQAEFIASFY K NDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPP H I 
                 
               
               
                   
               
               
                   IKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG SGGSTNLSDIIEKETGKQLVIQESIL 
               
               
                   
               
               
                 MLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSG 
               
               
                   
               
               
                 GSPKKKRKV 
               
               
                   
               
               
                 Exemplary EQR-BE3 (rAPOBEC1-XTEN-Cas9n-UGI-NLS) 
               
               
                 (SEQ ID NO: 4283) 
                   
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVE 
                   
               
               
                   
               
               
                 VNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPR 
               
               
                   
               
               
                 NRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGL 
               
               
                   
               
               
                 PPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPES DKKYSIG   
               
               
                   
               
               
                 
                   LAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRR 
                 
               
               
                   
               
               
                 
                   YTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT 
                 
               
               
                   
               
               
                 
                   IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE 
                 
               
               
                   
               
               
                 
                   ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAE 
                 
               
               
                   
               
               
                 
                   DAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIK 
                 
               
               
                   
               
               
                 
                   RYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT 
                 
               
               
                   
               
               
                 
                   EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIP 
                 
               
               
                   
               
               
                 
                   YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS 
                 
               
               
                   
               
               
                 
                   LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECF 
                 
               
               
                   
               
               
                 
                   DSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT 
                 
               
               
                   
               
               
                 
                   YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS 
                 
               
               
                   
               
               
                 
                   LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA 
                 
               
               
                   
               
               
                 
                   RENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE 
                 
               
               
                   
               
               
                 
                   LDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
                 
               
               
                   
               
               
                 
                   LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREV 
                 
               
               
                   
               
               
                 
                   KVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY 
                 
               
               
                   
               
               
                 DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRD 
               
               
                   
               
               
                 
                   FATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGF E SPTVAYS 
                 
               
               
                   
               
               
                 
                   VLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE 
                 
               
               
                   
               
               
                 
                   LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE 
                 
               
               
                   
               
               
                 
                   IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTI 
                 
               
               
                   
               
               
                   DRK Q Y R STKEVLDATLIHQSITGLYETRIDLSQLGGD SGGSTNLSDIIEKETGKQLVIQESIL 
               
               
                   
               
               
                 MLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSG 
               
               
                   
               
               
                 GSPKKKRKV 
               
               
                   
               
               
                 VQR-BE3 (rAPOBEC1-XTEN-Cas9n-UGI-NLS) 
               
               
                 (SEQ ID NO: 4284) 
                   
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVE 
                   
               
               
                   
               
               
                 VNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPR 
               
               
                   
               
               
                 NRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGL 
               
               
                   
               
               
                 PPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPES DKKYSIG   
               
               
                   
               
               
                 
                   LAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRR 
                 
               
               
                   
               
               
                 
                   YTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT 
                 
               
               
                   
               
               
                 
                   IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE 
                 
               
               
                   
               
               
                 
                   ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAE 
                 
               
               
                   
               
               
                 
                   DAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIK 
                 
               
               
                   
               
               
                 
                   RYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT 
                 
               
               
                   
               
               
                 
                   EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIP 
                 
               
               
                   
               
               
                 
                   YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS 
                 
               
               
                   
               
               
                 
                   LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECF 
                 
               
               
                   
               
               
                 
                   DSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT 
                 
               
               
                   
               
               
                 
                   YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS 
                 
               
               
                   
               
               
                 
                   LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA 
                 
               
               
                   
               
               
                 
                   RENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE 
                 
               
               
                   
               
               
                 
                   LDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
                 
               
               
                   
               
               
                 
                   LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREV 
                 
               
               
                   
               
               
                 
                   KVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY 
                 
               
               
                   
               
               
                 
                   DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRD 
                 
               
               
                   
               
               
                 
                   FATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGF V SPTVAYS 
                 
               
               
                   
               
               
                 
                   VLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE 
                 
               
               
                   
               
               
                 
                   LENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE 
                 
               
               
                   
               
               
                 
                   IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTI 
                 
               
               
                   
               
               
                   DRK Q Y R STKEVLDATLIHQSITGLYETRIDLSQLGGD SGGSTNLSDIIEKETGKQLVIQESIL 
               
               
                   
               
               
                 MLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSG 
               
               
                   
               
               
                 GSPKKKRKV 
               
               
                   
               
               
                 VRER-BE3 (rAPOBEC1-XTEN-Cas9n-UGI-NLS) 
               
               
                 (SEQ ID NO: 4285) 
                   
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVE 
                   
               
               
                   
               
               
                 VNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPR 
               
               
                   
               
               
                 NRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGL 
               
               
                   
               
               
                 PPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPES DKKYSIG   
               
               
                   
               
               
                 
                   LAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRR 
                 
               
               
                   
               
               
                 
                   YTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPT 
                 
               
               
                   
               
               
                 
                   IYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE 
                 
               
               
                   
               
               
                 
                   ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAE 
                 
               
               
                   
               
               
                 
                   DAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIK 
                 
               
               
                   
               
               
                 
                   RYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGT 
                 
               
               
                   
               
               
                 
                   EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIP 
                 
               
               
                   
               
               
                 
                   YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS 
                 
               
               
                   
               
               
                 
                   LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECF 
                 
               
               
                   
               
               
                 
                   DSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT 
                 
               
               
                   
               
               
                 
                   YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS 
                 
               
               
                   
               
               
                 
                   LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMA 
                 
               
               
                   
               
               
                 
                   RENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQE 
                 
               
               
                   
               
               
                 
                   LDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAK 
                 
               
               
                   
               
               
                 
                   LITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREV 
                 
               
               
                   
               
               
                 
                   KVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY 
                 
               
               
                   
               
               
                 
                   DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRD 
                 
               
               
                   
               
               
                 
                   FATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGF V SPTVAYS 
                 
               
               
                   
               
               
                 
                   VLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFE 
                 
               
               
                   
               
               
                 
                   LENGRKRMLASA R ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDE 
                 
               
               
                   
               
               
                 
                   IIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTI 
                 
               
               
                   
               
               
                   DRK E Y R STKEVLDATLIHQSITGLYETRIDLSQLGGD SGGSTNLSDIIEKETGKQLVIQESIL 
               
               
                   
               
               
                 MLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSG 
               
               
                   
               
               
                 GSPKKKRKV 
               
            
           
         
       
     
     High Fidelity Base Editors 
     Some aspects of the disclosure provide Cas9 fusion proteins (e.g., any of the fusion proteins provided herein) comprising a Cas9 domain that has high fidelity. Additional aspects of the disclosure provide Cas9 fusion proteins (e.g., any of the fusion proteins provided herein) comprising a Cas9 domain with decreased electrostatic interactions between the Cas9 domain and a sugar-phosphate backbone of a DNA, as compared to a wild-type Cas9 domain. In some embodiments, a Cas9 domain (e.g., a wild type Cas9 domain) comprises one or more mutations that decreases the association between the Cas9 domain and a sugar-phosphate backbone of a DNA. In some embodiments, any of the Cas9 fusion proteins provided herein comprise one or more of a N497X, a R661X, a Q695X, and/or a Q926X mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, 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 of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the Cas9 domain comprises a D10A mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the Cas9 domain (e.g., of any of the fusion proteins provided herein) comprises the amino acid sequence as set forth in SEQ ID NO: 325. In some embodiments, the fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 285. 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. 
     It should be appreciated that the base editors provided herein, for example base editor 2 (BE2) or base editor 3 (BE3), may be converted into high fidelity base editors by modifying the Cas9 domain as described herein to generate high fidelity base editors, for example high fidelity base editor 2 (HF-BE2) or high fidelity base editor 3 (HF-BE3). In some embodiments, base editor 2 (BE2) comprises a deaminase domain, a dCas9, and a UGI domain. In some embodiments, base editor 3 (BE3) comprises a deaminase domain an nCas9 domain and a UGI domain. 
     
       
         
           
               
            
               
                 Cas9 domain where mutations relative to Cas9 
               
               
                 of SEQ ID NO: 10 are shown in bold and 
               
               
                 underlines 
               
               
                 (SEQ ID NO: 325) 
               
               
                 DKKYSIGL   A   IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL 
               
               
                   
               
               
                 LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL 
               
               
                   
               
               
                 EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL 
               
               
                   
               
               
                 RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI 
               
               
                   
               
               
                 NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN 
               
               
                   
               
               
                 FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL 
               
               
                   
               
               
                 LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF 
               
               
                   
               
               
                 FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK 
               
               
                   
               
               
                 QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY 
               
               
                   
               
               
                 VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMT   A   FDKN 
               
               
                   
               
               
                 LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL 
               
               
                   
               
               
                 LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII 
               
               
                   
               
               
                 KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL 
               
               
                   
               
               
                 KRRRYTGWG   A   LSRKLINGIRDKQSGKTILDFLKSDGFANRNFM   A   LIHDDS 
               
               
                   
               
               
                 LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM 
               
               
                   
               
               
                 GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV 
               
               
                   
               
               
                 ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS 
               
               
                   
               
               
                 IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT 
               
               
                   
               
               
                 KAERGGLSELDKAGFIKRQLVETR   A   ITKHVAQILDSRMNTKYDENDKLIR 
               
               
                   
               
               
                 EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY 
               
               
                   
               
               
                 PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT 
               
               
                   
               
               
                 LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ 
               
               
                   
               
               
                 TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK 
               
               
                   
               
               
                 GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY 
               
               
                   
               
               
                 SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED 
               
               
                   
               
               
                 NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP 
               
               
                   
               
               
                 IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS 
               
               
                   
               
               
                 ITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 HF-BE3 
               
               
                 (SEQ ID NO: 285) 
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI 
               
               
                   
               
               
                 WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI 
               
               
                   
               
               
                 TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG 
               
               
                   
               
               
                 YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ 
               
               
                   
               
               
                 PQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYS 
               
               
                   
               
               
                 IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG 
               
               
                   
               
               
                 ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL 
               
               
                   
               
               
                 VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL 
               
               
                   
               
               
                 ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV 
               
               
                   
               
               
                 DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF 
               
               
                   
               
               
                 DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL 
               
               
                   
               
               
                 RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK 
               
               
                   
               
               
                 NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD 
               
               
                   
               
               
                 NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA 
               
               
                   
               
               
                 RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN 
               
               
                   
               
               
                 RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF 
               
               
                   
               
               
                 LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY 
               
               
                   
               
               
                 TGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMALIHDDSLTFKE 
               
               
                   
               
               
                 DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP 
               
               
                   
               
               
                 ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL 
               
               
                   
               
               
                 QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV 
               
               
                   
               
               
                 LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG 
               
               
                   
               
               
                 GLSELDKAGFIKRQLVETRAITKHVAQILDSRMNTKYDENDKLIREVKVI 
               
               
                   
               
               
                 TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLES 
               
               
                   
               
               
                 EFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE 
               
               
                   
               
               
                 IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS 
               
               
                   
               
               
                 KESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK 
               
               
                   
               
               
                 LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL 
               
               
                   
               
               
                 ENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ 
               
               
                   
               
               
                 LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA 
               
               
                   
               
               
                 ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY 
               
               
                   
               
               
                 ETRIDLSQLGGD 
               
            
           
         
       
     
     Cas9 Fusion Proteins 
     Any of the Cas9 domains (e.g., a nuclease active Cas9 protein, a nuclease-inactive dCas9 protein, or a Cas9 nickase protein) disclosed herein may be fused to a second protein, thus fusion proteins provided herein comprise a Cas9 domain as provided herein and a second protein, or a “fusion partner”. In some embodiments, the second protein is fused to the N-terminus of the Cas9 domain. However, in other embodiments, the second protein is fused to the C-terminus of the Cas9 domain. In some embodiments, the second protein that is fused to the Cas9 domain is a nucleic acid editing domain. In some embodiments, the Cas9 domain and the nucleic acid editing domain are fused via a linker, while in other embodiments the Cas9 domain and the nucleic acid editing domain are fused directly to one another. In some embodiments, the linker comprises (GGGS) n  (SEQ ID NO: 265), (GGGGS) n  (SEQ ID NO: 5), (G) n , (EAAAK) n  (SEQ ID NO: 6), (GGS) n , (SGGS) n (SEQ ID NO: 4288), SGSETPGTSESATPES (SEQ ID NO: 7), or (XP) n  motif, or a combination of any of these, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, the linker comprises a (GGS) n  motif, wherein n is 1, 3, or 7. In some embodiments, the linker comprises a (GGS) n  motif, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises an amino acid sequence of SGSETPGTSESATPES (SEQ ID NO: 7), also referred to as the XTEN linker in the Examples). The length of the linker can influence the base to be edited, as illustrated in the Examples. For example, a linker of 3-amino-acid long (e.g., (GGS) 1 ) may give a 2-5, 2-4, 2-3, 3-4 base editing window relative to the PAM sequence, while a 9-amino-acid linker (e.g., (GGS) 3  (SEQ ID NO: 596)) may give a 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, 5-6 base editing window relative to the PAM sequence. A 16-amino-acid linker (e.g., the XTEN linker) may give a 2-7, 2-6, 2-5, 2-4, 2-3, 3-7, 3-6, 3-5, 3-4, 4-7, 4-6, 4-5, 5-7, 5-6, 6-7 base window relative to the PAM sequence with exceptionally strong activity, and a 21-amino-acid linker (e.g., (GGS) 7  (SEQ ID NO: 597)) may give a 3-8, 3-7, 3-6, 3-5, 3-4, 4-8, 4-7, 4-6, 4-5, 5-8, 5-7, 5-6, 6-8, 6-7, 7-8 base editing window relative to the PAM sequence. The novel finding that varying linker length may allow the dCas9 fusion proteins of the disclosure to edit nucleobases different distances from the PAM sequence affords significant clinical importance, since a PAM sequence may be of varying distance to the disease-causing mutation to be corrected in a gene. It is to be understood that the linker lengths described as examples here are not meant to be limiting. 
     In some embodiments, the second protein comprises an enzymatic domain. In some embodiments, the enzymatic domain is a nucleic acid editing domain. Such a nucleic acid editing domain may be, without limitation, a nuclease, a nickase, a recombinase, a deaminase, a methyltransferase, a methylase, an acetylase, or an acetyltransferase. Non-limiting exemplary binding domains that may be used in accordance with this disclosure include transcriptional activator domains and transcriptional repressor domains. 
     Deaminase Domains 
     In some embodiments, second protein 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 a cytidine 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 APOBEC3A 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 (SEQ ID NO: 275). In some embodiments, the deaminase is a fragment of the human APOBEC3G (SEQ ID NO: 5740). In some embodiments, the deaminase is a human APOBEC3G variant comprising a D316R_D317R mutation (SEQ ID NO: 5739). In some embodiments, the deaminase is a frantment of the human APOBEC3G and comprising mutations corresponding to the D316R_D317R mutations in SEQ ID NO: 275 (SEQ ID NO: 5741). 
     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 one of SEQ ID NOs: 266-284, 607-610, 5724-5736, or 5738-5741. In some embodiments, the nucleic acid editing domain comprises the amino acid sequence of any one of SEQ ID NOs: 266-284, 607-610, 5724-5736, or 5738-5741. 
     Deaminase Domains that Modulate the Editing Window of Base Editors 
     Some aspects of the disclosure are based on the recognition that modulating the deaminase domain catalytic activity of any of the fusion proteins provided 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 may prevent unwanted deamination of residues adjacent of specific target residues, which may decrease or prevent off-target effects. 
     In some embodiments, any of the fusion proteins provided herein comprise a deaminase domain (e.g., a cytidine deaminase domain) that has reduced catalytic deaminase activity. In some embodiments, any of the fusion proteins provided herein comprise a deaminase domain (e.g., a cytidine deaminase domain) that has a reduced catalytic deaminase activity as compared to an appropriate control. For example, the appropriate control may be the deaminase activity of the deaminase prior to introducing one or more mutations into the deaminase. In other embodiments, the appropriate control may be a wild-type deaminase. In some embodiments, the appropriate control is a wild-type apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the appropriate control is an APOBEC1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, or an APOBEC3H deaminase. In some embodiments, the appropriate control is an activation induced deaminase (AID). In some embodiments, the appropriate control is a cytidine deaminase 1 from  Petromyzon marinus  (pmCDA1). In some embodiments, the deaminase domain may be a deaminase domain that has at least 1%, at least 5%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% less catalytic deaminase activity as compared to an appropriate control. 
     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 H121X, H122X, R126X, R126X, R118X, W90X, W90X, and R132X of rAPOBEC1 (SEQ ID NO: 284), 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 H121R, H122R, R126A, R126E, R118A, W90A, W90Y, and R132E of rAPOBEC1 (SEQ ID NO: 284), 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 one or more mutations selected from the group consisting of D316X, D317X, R320X, R320X, R313X, W285X, W285X, R326X of hAPOBEC3G (SEQ ID NO: 275), 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 (SEQ ID NO: 275), 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 H121R and a H122R mutation of rAPOBEC1 (SEQ ID NO: 284), 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 R126A mutation of rAPOBEC1 (SEQ ID NO: 284), 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 R126E mutation of rAPOBEC1 (SEQ ID NO: 284), 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 R118A mutation of rAPOBEC1 (SEQ ID NO: 284), 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 W90A mutation of rAPOBEC1 (SEQ ID NO: 284), 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 W90Y mutation of rAPOBEC1 (SEQ ID NO: 284), 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 R132E mutation of rAPOBEC1 (SEQ ID NO: 284), 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 W90Y and a R126E mutation of rAPOBEC1 (SEQ ID NO: 284), 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 R126E and a R132E mutation of rAPOBEC1 (SEQ ID NO: 284), 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 W90Y and a R132E mutation of rAPOBEC1 (SEQ ID NO: 284), 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 W90Y, R126E, and R132E mutation of rAPOBEC1 (SEQ ID NO: 284), 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 D316R and a D317R mutation of hAPOBEC3G (SEQ ID NO: 275), 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 (SEQ ID NO: 275), 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 R320E mutation of hAPOBEC3G (SEQ ID NO: 275), 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 R313A mutation of hAPOBEC3G (SEQ ID NO: 275), 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 W285A mutation of hAPOBEC3G (SEQ ID NO: 275), 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 W285Y mutation of hAPOBEC3G (SEQ ID NO: 275), 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 R326E mutation of hAPOBEC3G (SEQ ID NO: 275), 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 W285Y and a R320E mutation of hAPOBEC3G (SEQ ID NO: 275), 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 R320E and a R326E mutation of hAPOBEC3G (SEQ ID NO: 275), 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 W285Y and a R326E mutation of hAPOBEC3G (SEQ ID NO: 275), 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 W285Y, R320E, and R326E mutation of hAPOBEC3G (SEQ ID NO: 275), or one or more corresponding mutations in another APOBEC deaminase. 
     Some aspects of this disclosure provide fusion proteins comprising (i) a nuclease-inactive Cas9 domain; and (ii) a nucleic acid editing domain. In some embodiments, a nuclease-inactive Cas9 domain (dCas9), comprises an amino acid sequence that 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 amino acid sequence of a Cas9 as provided by any one of SEQ ID NOs: 10-263, and comprises mutations that inactivate the nuclease activity of Cas9. Mutations that render the nuclease domains of Cas9 inactive are well-known in the art. 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 of this disclosure comprises a D10A mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the dCas9 of this disclosure comprises a H840A mutation of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the dCas9 of this disclosure comprises both D10A and H840A mutations of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. In some embodiments, the Cas9 further comprises a histidine residue at position 840 of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. The presence of the catalytic residue H840 restores the activity of the Cas9 to cleave the non-edited strand containing a G opposite the targeted C. Restoration of H840 does not result in the cleavage of the target strand containing the C. In some embodiments, the dCas9 comprises an amino acid sequence of SEQ ID NO: 263. It is to be understood that other mutations that inactivate the nuclease domains of Cas9 may also be included in the dCas9 of this disclosure. 
     The Cas9 or dCas9 domains comprising the mutations disclosed herein, may be a full-length Cas9, or a fragment thereof. 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% 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, e.g., a Cas9 comprising the amino acid sequence of SEQ ID NO: 10. 
     Any of the Cas9 fusion proteins of this disclosure may further comprise a nucleic acid editing domain (e.g., an enzyme that is capable of modifying nucleic acid, such as a deaminase). In some embodiments, the nucleic acid editing domain is a DNA-editing domain. In some embodiments, the nucleic acid editing domain has deaminase activity. In some embodiments, the nucleic acid editing domain comprises or consists of a deaminase or deaminase domain. In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC1 family deaminase. In some embodiments, the deaminase is an activation-induced cytidine deaminase (AID). Some nucleic-acid editing domains as well as Cas9 fusion proteins including such domains are described in detail herein. Additional suitable nucleic acid editing domains will be apparent to the skilled artisan based on this disclosure and knowledge in the field. 
     Some aspects of the disclosure provide a fusion protein comprising a Cas9 domain fused to a nucleic acid editing domain, wherein the nucleic acid editing domain is fused to the N-terminus of the Cas9 domain. In some embodiments, the Cas9 domain and the nucleic acid editing-editing domain are fused via a linker. In some embodiments, the linker comprises a (GGGS) n (SEQ ID NO: 265), a (GGGGS) n  (SEQ ID NO: 5), a (G) n , an (EAAAK) n  (SEQ ID NO: 6), a (GGS) n , (SGGS) n (SEQ ID NO: 4288), an SGSETPGTSESATPES (SEQ ID NO: 7) motif (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 an (XP) n  motif, or a combination of any of these, wherein n is independently an integer between 1 and 30. In some embodiments, n is independently 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, or, if more than one linker or more than one linker motif is present, any combination thereof. In some embodiments, the linker comprises a (GGS) n  motif, wherein 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. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 7). Additional suitable linker motifs and linker configurations will be apparent to those of skill in the art. In some embodiments, suitable linker motifs and configurations include those described in Chen et al., Fusion protein linkers: property, design and functionality.  Adv Drug Deliv Rev.  2013; 65(10):1357-69, the entire contents of which are incorporated herein by reference. Additional suitable linker sequences will be apparent to those of skill in the art based on the instant disclosure. In some embodiments, the general architecture of exemplary Cas9 fusion proteins provided herein comprises the structure:
         [NH 2 ]-[nucleic acid editing domain]-[Cas9]-[COOH] or   [NH 2 ]-[nucleic acid editing domain]-[linker]-[Cas9]-[COOH],
 
wherein NH 2  is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein.
       

     The fusion proteins of the present disclosure may comprise one or more additional features. For example, in some embodiments, the fusion protein comprises a nuclear localization sequence (NLS). In some embodiments, the NLS of the fusion protein is localized between the nucleic acid editing domain and the Cas9 domain. In some embodiments, the NLS of the fusion protein is localized C-terminal to the Cas9 domain. 
     Other exemplary features that may be present 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. 
     In some embodiments, the nucleic acid editing domain is a deaminase. For example, in some embodiments, the general architecture of exemplary Cas9 fusion proteins with a deaminase domain comprises the structure:
         [NH 2 ]-[NLS]-[deaminase]-[Cas9]-[COOH],   [NH2]-[Cas9]-[deaminase]-[COOH],   [NH 2 ]-[deaminase]-[Cas9]-[COOH], or   [NH 2 ]-[deaminase]-[Cas9]-[NLS]-[COOH]
 
wherein NLS is a nuclear localization sequence, NH 2  is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein. 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, a NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 741) or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 742). In some embodiments, a linker is inserted between the Cas9 and the deaminase. In some embodiments, the NLS is located C-terminal of the Cas9 domain. In some embodiments, the NLS is located N-terminal of the Cas9 domain. In some embodiments, the NLS is located between the deaminase and the Cas9 domain. In some embodiments, the NLS is located N-terminal of the deaminase domain. In some embodiments, the NLS is located C-terminal of the deaminase domain.
       

     One exemplary suitable type of nucleic acid editing domain is a cytidine deaminase, for example, of the APOBEC family. The apolipoprotein B mRNA-editing complex (APOBEC) family of cytidine deaminase enzymes encompasses eleven proteins that serve to initiate mutagenesis in a controlled and beneficial manner. 29  One family member, activation-induced cytidine deaminase (AID), is responsible for the maturation of antibodies by converting cytosines in ssDNA to uracils in a transcription-dependent, strand-biased fashion. 30  The apolipoprotein B editing complex 3 (APOBEC3) enzyme provides protection to human cells against a certain HIV-1 strain via the deamination of cytosines in reverse-transcribed viral ssDNA. 31  These proteins all require a Zn 2+ -coordinating motif (His-X-Glu-X 23-26 -Pro-Cys-X 2-4 -Cys; SEQ ID NO: 598) and bound water molecule for catalytic activity. The Glu residue acts to activate the water molecule to a zinc hydroxide for nucleophilic attack in the deamination reaction. Each family member preferentially deaminates at its own particular “hotspot”, ranging from WRC (W is A or T, R is A or G) for hAID, to TTC for hAPOBEC3F. 32  A recent crystal structure of the catalytic domain of APOBEC3G revealed a secondary structure comprised of a five-stranded β-sheet core flanked by six α-helices, which is believed to be conserved across the entire family. 33  The active center loops have been shown to be responsible for both ssDNA binding and in determining “hotspot” identity. 34  Overexpression of these enzymes has been linked to genomic instability and cancer, thus highlighting the importance of sequence-specific targeting. 35    
     Some aspects of this disclosure relate to the recognition that the activity of cytidine deaminase enzymes such as APOBEC enzymes can be directed to a specific site in genomic DNA. Without wishing to be bound by any particular theory, advantages of using Cas9 as a recognition agent include (1) the sequence specificity of Cas9 can be easily altered by simply changing the sgRNA sequence; and (2) Cas9 binds to its target sequence by denaturing the dsDNA, resulting in a stretch of DNA that is single-stranded and therefore a viable substrate for the deaminase. It should be understood that other catalytic domains, or catalytic domains from other deaminases, can also be used to generate fusion proteins with Cas9, and that the disclosure is not limited in this regard. 
     Some aspects of this disclosure are based on the recognition that Cas9:deaminase fusion proteins can efficiently deaminate nucleotides at positions 3-11 according to the numbering scheme in  FIG. 3 . In view of the results provided herein regarding the nucleotides that can be targeted by Cas9:deaminase fusion proteins, a person of skill in the art will be able to design suitable guide RNAs to target the fusion proteins to a target sequence that comprises a nucleotide to be deaminated. 
     In some embodiments, the deaminase domain and the Cas9 domain are fused to each other via a linker. Various linker lengths and flexibilities between the deaminase domain (e.g., AID) and the Cas9 domain can be employed (e.g., ranging from very flexible linkers of the form (GGGGS) n  (SEQ ID NO: 5), (GGS) n , and (G) n  to more rigid linkers of the form (EAAAK) n  (SEQ ID NO: 6), (SGGS) n (SEQ ID NO: 4288), SGSETPGTSESATPES (SEQ ID NO: 7) (see, e.g., Guilinger J P, Thompson D B, Liu D R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification.  Nat. Biotechnol.  2014; 32(6): 577-82; the entire contents are incorporated herein by reference) and (XP) n ) 36  in order to achieve the optimal length for deaminase activity for the specific application. In some embodiments, the linker comprises a (GGS) n  motif, wherein n is 1, 3, or 7. In some embodiments, the linker comprises a (an SGSETPGTSESATPES (SEQ ID NO: 7) motif. 
     Some exemplary suitable nucleic-acid editing domains, e.g., deaminases and deaminase domains, that can be fused to Cas9 domains 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: 266) 
                   
               
               
                   MDSLLMNRRKFLYQFKNVRWAKGRRETYLC YVVKRRDSATSFSLDFGYLRNKNGCHVELLFLR 
                   
               
               
                   
               
               
                 YISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGL 
               
               
                   
               
               
                 RRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLP LYEVDDL   
               
               
                   
               
               
                 
                   RDAFRTLGL 
                 
               
               
                 (underline: nuclear localization sequence; 
               
               
                 double underline: nuclear export signal) 
               
               
                   
               
               
                 Mouse AID: 
               
               
                 (SEQ ID NO: 267) 
                   
               
               
                   MDSLLMKQKKFLYHFKNVRWAKGRHETYLC YVVKRRDSATSCSLDFGHLRNKSGCHVELLFLR 
                   
               
               
                   
               
               
                 YISDWDLDPGRCYRVTWFTSWSPCYDCARHVAEFLRWNPNLSLRIFTARLYFCEDRKAEPEGL 
               
               
                   
               
               
                 RRLHRAGVQIGIMTFKDYFYCWNTFVENRERTFKAWEGLHENSVRLTRQLRRILLP LYEVDDL   
               
               
                   
               
               
                 
                   RDAFRMLGF 
                 
               
               
                 (underline: nuclear localization sequence; 
               
               
                 double underline: nuclear export signal) 
               
               
                   
               
               
                 Dog AID: 
               
               
                 (SEQ ID NO: 268) 
                   
               
               
                   MDSLLMKQRKFLYHFKNVRWAKGRHETYLC YVVKRRDSATSFSLDFGHLRNKSGCHVELLFLR 
                   
               
               
                   
               
               
                 YISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFAARLYFCEDRKAEPEGL 
               
               
                   
               
               
                 RRLHRAGVQIAIMTFKDYFYCWNTFVENREKTFKAWEGLHENSVRLSRQLRRILLP LYEVDDL   
               
               
                   
               
               
                 
                   RDAFRTLGL 
                 
               
               
                 (underline: nuclear localization sequence; 
               
               
                 double underline: nuclear export signal) 
               
               
                   
               
               
                 Bovine AID: 
               
               
                 (SEQ ID NO: 269) 
                   
               
               
                   MDSLLKKQRQFLYQFKNVRWAKGRHETYLC YVVKRRDSPTSFSLDFGHLRNKAGCHVELLFLR 
                   
               
               
                   
               
               
                 YISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFTARLYFCDKERKAEPEG 
               
               
                   
               
               
                 LRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLP LYEVDD   
               
               
                   
               
               
                 
                   LRDAFRTLGL 
                 
               
               
                 (underline: nuclear localization sequence; 
               
               
                 double underline: nuclear export signal) 
               
               
                   
               
               
                 Rat AID 
               
               
                 (SEQ ID NO: 5725) 
                   
               
               
                   MAVGSKPKAALVGPHWERERIWCFLC STGLGTQQTGQTSRWLRPAATQDPVSPPRSLLMKQRK 
                   
               
               
                   
               
               
                 FLYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGYLRNKSGCHVELLFLRYISDWDLDPG 
               
               
                   
               
               
                 RCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLTGWGALPAGLMSPARPSDYFYCW 
               
               
                   
               
               
                 NTFV ENHERTFKAWEGLHENSVRLSRRLRRILLPLYEVDDLRDAFRTLGL   
               
               
                 (underline: nuclear localization sequence; 
               
               
                 double underline: nuclear export signal) 
               
               
                   
               
               
                 Mouse APOBEC-3: 
               
               
                 (SEQ ID NO: 270) 
                   
               
               
                 MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLGYAKGRKDTFLCYEVTRKDCDSPVSLHHGV 
                   
               
               
                   
               
               
                 FKNKDNI HAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFEC AEQIVRFLATHHNLSLDI 
               
               
                   
               
               
                 FSSRLYNVQDPETQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKRLLTNFRYQ 
               
               
                   
               
               
                 DSKLQEILRPCYIPVPSSSSSTLSNICLTKGLPETRFCVEGRRMDPLSEEEFYSQFYNQRVKH 
               
               
                   
               
               
                 LCYYHRMKPYLCYQLEQFNGQAPLKGCLLSEKGKQ HAEILFLDKIRSMELSQVTITCYLTWSP   
               
               
                   
               
               
                   CPNC AWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTN 
               
               
                   
               
               
                 FVNPKRPFWPWKGLEIISRRTQRRLRRIKESWGLQDLVNDFGNLQLGPPMS 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Rat APOBEC-3: 
               
               
                 (SEQ ID NO: 271) 
                   
               
               
                 MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLRYAIDRKDTFLCYEVTRKDCDSPVSLHHGV 
                   
               
               
                   
               
               
                 FKNKDNI HAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFEC AEQVLRFLATHHNLSLDI 
               
               
                   
               
               
                 FSSRLYNIRDPENQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKKLLTNFRYQ 
               
               
                   
               
               
                 DSKLQEILRPCYIPVPSSSSSTLSNICLTKGLPETRFCVERRRVHLLSEEEFYSQFYNQRVKH 
               
               
                   
               
               
                 LCYYHGVKPYLCYQLEQFNGQAPLKGCLLSEKGKQ HAEILFLDKIRSMELSQVIITCYLTWSP   
               
               
                   
               
               
                   CPNC AWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTN 
               
               
                   
               
               
                 FVNPKRPFWPWKGLEIISRRTQRRLHRIKESWGLQDLVNDFGNLQLGPPMS 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Rhesus macaque APOBEC-3G: 
               
               
                 (SEQ ID NO: 272) 
                   
               
               
                 
                   MVEPMDPRTFVSNFNNRPILSGLNTVWLCCEVKTKDPSGPPLDAKIFQGKVYSKAKY HPEM   
                   RF 
                 
                   
               
               
                   
               
               
                   LRWFHKWRQLHHDQEYKVTWYVSWSPCTRC ANSVATFLAKDPKVTLTIFVARLYYFWKPDYQQ 
               
               
                   
               
               
                 ALRILCQKRGGPHATMKIMNYNEFQDCWNKFVDGRGKPFKPRNNLPKHYTLLQATLGELLRHL 
               
               
                   
               
               
                 MDPGTFTSNFNNKPWVSGQHETYLCYKVERLHNDTWVPLNQHRGFLRNQAPNIHGFPKGR HAE   
               
               
                   
               
               
                   LCFLDLIPFWKLDGQQYRVTCFTSWSPCFSC AQEMAKFISNNEHVSLCIFAARIYDDQGRYQE 
               
               
                   
               
               
                 GLRALHRDGAKIAMMNYSEFEYCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAI 
               
               
                 (italic: nucleic acid editing domain; underline: 
               
               
                 cytoplasmic localization signal) 
               
               
                   
               
               
                 Chimpanzee APOBEC-3G: 
               
               
                 (SEQ ID NO: 273) 
                   
               
               
                   MKPHFRNPVERMYQDTFSDNFYNRPILSHRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYS KLK 
                   
               
               
                   
               
               
                 Y HPEMRFFHWFSKWRKLHRDQEYEVIWYISWSPCTK CTRDVATFLAEDPKVTLTIFVARLYYF 
               
               
                   
               
               
                 WDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIML 
               
               
                   
               
               
                 GEILRHSMDPPTFTSNFNNELWVRGRHETYLCYEVERLHNDTWVLLNQRRGFLCNQAPHKHGF 
               
               
                   
               
               
                 LEGR HAELCFLDVIPFWKLDLHQDYRVTCFTSWSPCFSC AQEMAKFISNNKHVSLCIFAARIY 
               
               
                   
               
               
                 DDQGRCQEGLRTLAKAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLEEHSQALSGRLRAI 
               
               
                   
               
               
                 LQNQGN 
               
               
                 (italic: nucleic acid editing domain; underline: 
               
               
                 cytoplasmic localization signal) 
               
               
                   
               
               
                 Green monkey APOBEC-3G: 
               
               
                 (SEQ ID NO: 274) 
                   
               
               
                   MNPQIRNMVEQMEPDIFVYYFNNRPILSGRNTVWLCYEVKTKDPSGPPLDANIFQGKLYP EAK 
                   
               
               
                   
               
               
                 D HPEMKFLHWFRKWRQLHRDQEYEVTWYVSWSPCTRC ANSVATFLAEDPKVTLTIFVARLYYF 
               
               
                   
               
               
                 WKPDYQQALRILCQERGGPHATMKIMNYNEFQHCWNEFVDGQGKPFKPRKNLPKHYTLLHATL 
               
               
                   
               
               
                 GELLRHVMDPGTFTSNFNNKPWVSGQRETYLCYKVERSHNDTWVLLNQHRGFLRNQAPDRHGF 
               
               
                   
               
               
                 PKGR HAELCFLDLIPFWKLDDQQYRVTCFTSWSPCFSC AQKMAKFISNNKHVSLCIFAARIYD 
               
               
                   
               
               
                 DQGRCQEGLRTLHRDGAKIAVMNYSEFEYCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAI 
               
               
                 (italic: nucleic acid editing domain; underline: 
               
               
                 cytoplasmic localization signal) 
               
               
                   
               
               
                 Human APOBEC-3G: 
               
               
                 (SEQ ID NO: 275) 
                   
               
               
                   MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYS ELK 
                   
               
               
                   
               
               
                 Y HPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKC TRDMATFLAEDPKVTLTIFVARLYYF 
               
               
                   
               
               
                 WDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIML 
               
               
                   
               
               
                 GEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGF 
               
               
                   
               
               
                 LEGR HAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSC AQEMAKFISKNKHVSLCIFTARIY 
               
               
                   
               
               
                 DDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAI 
               
               
                   
               
               
                 LQNQEN 
               
               
                 (italic: nucleic acid editing domain; underline: 
               
               
                 cytoplasmic localization signal) 
               
               
                   
               
               
                 Human APOBEC-3F: 
               
               
                 (SEQ ID NO: 276) 
                   
               
               
                 MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPRLDAKIFRGQ 
                   
               
               
                   
               
               
                 VYSQPEH HAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDC VAKLAEFLAEHPNVTL 
               
               
                   
               
               
                 TISAARLYYYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYSEGQPFMPW 
               
               
                   
               
               
                 YKFDDNYAFLHRTLKEILRNPMEAMYPHIFYFHFKNLRKAYGRNESWLCFTMEVVK 
               
               
                   
               
               
                 HHSPVSWKRGVFRNQVDPETHC HAERCFLSWFCDDILSPNTNYEVTWYTSWSPCPEC A 
               
               
                   
               
               
                 GEVAEFLARHSNVNLTIFTARLYYFWDTDYQEGLRSLSQEGASVEIMGYKDFKYCW 
               
               
                   
               
               
                 ENFVYNDDEPFKPWKGLKYNFLFLDSKLQEILE 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Human APOBEC-3B: 
               
               
                 (SEQ ID NO: 277) 
                   
               
               
                 MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFR 
                   
               
               
                   
               
               
                 GQVYFKPQY HAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDC VAKLAEFLSEHPN 
               
               
                   
               
               
                 VTLTISAARLYYYWERDYRRALCRLSQAGARVTIMDYEEFAYCWENFVYNEGQQF 
               
               
                   
               
               
                 MPWYKFDENYAFLHRTLKEILRYLMDPDTFTFNFNNDPLVLRRRQTYLCYEVERLD 
               
               
                   
               
               
                 NGTWVLMDQHMGFLCNEAKNLLCGFY GRHAELRFLDLVPSLQLDPAQIYRVTWFISWS   
               
               
                   
               
               
                   PCFSWGC AGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTY 
               
               
                   
               
               
                 DEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQNQGN 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Rat APOBEC-3B: 
               
               
                 (SEQ ID NO: 5729) 
                   
               
               
                 MQPQGLGPNAGMGPVCLGCSHRRPYSPIRNPLKKLYQQTFYFHFKNVRYAWGRKN 
                   
               
               
                   
               
               
                 NFLCYEVNGMDCALPVPLRQGVFRKQGHIHAELCFIYWFHDKVLRVLSPMEEFKVT 
               
               
                   
               
               
                 WYMSWSPCSKCAEQVARFLAAHRNLSLAIFSSRLYYYLRNPNYQQKLCRLIQEGVH 
               
               
                   
               
               
                 VAAMDLPEFKKCWNKFVDNDGQPFRPWMRLRINFSFYDCKLQEIFSRMNLLREDVF 
               
               
                   
               
               
                 YLQFNNSHRVKPVQNRYYRRKSYLCYQLERANGQEPLKGYLLYKKGEQHVEILFLE 
               
               
                   
               
               
                 KMRSMELSQVRITCYLTWSPCPNCARQLAAFKKDHPDLILRIYTSRLYFYWRKKFQK 
               
               
                   
               
               
                 GLCTLWRSGIHVDVMDLPQFADCWTNFVNPQRPFRPWNELEKNSWRIQRRLRRIKE 
               
               
                   
               
               
                 SWGL 
               
               
                   
               
               
                 Bovine APOBEC-3B: 
               
               
                 (SEQ ID NO: 5730) 
                   
               
               
                 DGWEVAFRSGTVLKAGVLGVSMTEGWAGSGHPGQGACVWTPGTRNTMNLLREVL 
                   
               
               
                   
               
               
                 FKQQFGNQPRVPAPYYRRKTYLCYQLKQRNDLTLDRGCFRNKKQRHAEIRFIDKINS 
               
               
                   
               
               
                 LDLNPSQSYKIICYITWSPCPNCANELVNFITRNNHLKLEIFASRLYFHWIKSFKMGLQ 
               
               
                   
               
               
                 DLQNAGISVAVMTHTEFEDCWEQFVDNQSRPFQPWDKLEQYSASIRRRLQRILTAPI 
               
               
                   
               
               
                 Chimpanzee APOBEC -3B: 
               
               
                 (SEQ ID NO: 5731) 
                   
               
               
                 MNPQIRNPMEWMYQRTFYYNFENEPILYGRSYTWLCYEVKIRRGHSNLLWDTGVFR 
                   
               
               
                   
               
               
                 GQMYSQPEHHAEMCFLSWFCGNQLSAYKCFQITWFVSWTPCPDCVAKLAKFLAEHP 
               
               
                   
               
               
                 NVTLTISAARLYYYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYNEGQPF 
               
               
                   
               
               
                 MPWYKFDDNYAFLHRTLKEIIRHLMDPDTFTFNFNNDPLVLRRHQTYLCYEVERLD 
               
               
                   
               
               
                 NGTWVLMDQHMGFLCNEAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFIS 
               
               
                   
               
               
                 WSPCFSWGCAGQVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIM 
               
               
                   
               
               
                 TYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQVRASSLCMVPHRPPP 
               
               
                   
               
               
                 PPQSPGPCLPLCSEPPLGSLLPTGRPAPSLPFLLTASFSFPPPASLPPLPSLSLSPGHLPVP 
               
               
                   
               
               
                 SFHSLTSCSIQPPCSSRIRETEGWASVSKEGRDLG 
               
               
                   
               
               
                 Human APOBEC-3C: 
               
               
                 (SEQ ID NO: 278) 
                   
               
               
                 MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVF 
                   
               
               
                   
               
               
                 RNQVDSETH CHAERCFLSWFCDDILSPNTKYQVIWYTSWSPCPDC AGEVAEFLARHSN 
               
               
                   
               
               
                 VNLTIFTARLYYFQYPCYQEGLRSLSQEGVAVEIMDYEDFKYCWENFVYNDNEPFKP 
               
               
                   
               
               
                 WKGLKTNFRLLKRRLRESLQ 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Gorilla APOB EC3C 
               
               
                 (SEQ ID NO: 5726) 
                   
               
               
                 MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVF 
                   
               
               
                   
               
               
                 RNQVDSETH CHAERCFLSWFCDDILSPNTNYQVIWYTSWSPCPEC AGEVAEFLARHSN 
               
               
                   
               
               
                 VNLTIFTARLYYFQDTDYQEGLRSLSQEGVAVKIMDYKDFKYCWENFVYNDDEPFK 
               
               
                   
               
               
                 PWKGLKYNFRFLKRRLQEILE 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Human APOBEC-3A: 
               
               
                 (SEQ ID NO: 279) 
                   
               
               
                 MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLH 
                   
               
               
                   
               
               
                 NQAKNLLCGFYGR HAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGC AGEVRAFLQ 
               
               
                   
               
               
                 ENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGC 
               
               
                   
               
               
                 PFQPWDGLDEHSQALSGRLRAILQNQGN 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Rhesus macaque APOBEC-3A: 
               
               
                 (SEQ ID NO: 5727) 
                   
               
               
                 MDGSPASRPRHLMDPNTFTFNFNNDLSVRGRHQTYLCYEVERLDNGTWVPMDERR 
                   
               
               
                   
               
               
                 GFLCNKAKNVPCGDYGC HVELRFLCEVPSWQLDPAQTYRVTWFISWSPC FRRGCAGQ 
               
               
                   
               
               
                 VRVFLQENKHVRLRIFAARIYDYDPLYQEALRTLRDAGAQVSIMTYEEFKHCWDTF 
               
               
                   
               
               
                 VDRQGRPFQPWDGLDEHSQALSGRLRAILQNQGN 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Bovine APOBEC-3A: 
               
               
                 (SEQ ID NO: 5728) 
                   
               
               
                 MDEYTFTENFNNQGWPSKTYLCYEMERLDGDATIPLDEYKGFVRNKGLDQPEKPC H   
                   
               
               
                   
               
               
                   AELYFLGKIHSWNLDRNQHYRLTCFISWSPC YDCAQKLTTFLKENHHISLHILASRIYTH 
               
               
                   
               
               
                 NRFGCHQSGLCELQAAGARITIMTFEDFKHCWETFVDHKGKPFQPWEGLNVKSQAL 
               
               
                   
               
               
                 CTELQAILKTQQN 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Human APOBEC-3H: 
               
               
                 (SEQ ID NO: 280) 
                   
               
               
                 MALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTPQNGSTPTRGYFENKKKC HAEI   
                   
               
               
                   
               
               
                   CFINEIKSMGLDETQCYQVTCYLTWSPCSSC AWELVDFIKAHDHLNLGIFASRLYYHWC 
               
               
                   
               
               
                 KPQQKGLRLLCGSQVPVEVMGFPKFADCWENFVDHEKPLSFNPYKMLEELDKNSRA 
               
               
                   
               
               
                 IKRRLERIKIPGVRAQGRYMDILCDAEV 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Rhesus macaque APOBEC-3H: 
               
               
                 (SEQ ID NO: 5732) 
                   
               
               
                 MALLTAKTFSLQFNNKRRVNKPYYPRKALLCYQLTPQNGSTPTRGHLKNKKKDHAE 
                   
               
               
                   
               
               
                 IRFINKIKSMGLDETQCYQVTCYLTWSPCPSCAGELVDFIKAHRHLNLRIFASRLYYH 
               
               
                   
               
               
                 WRPNYQEGLLLLCGSQVPVEVMGLPEFTDCWENFVDHKEPPSFNPSEKLEELDKNS 
               
               
                   
               
               
                 QAIKRRLERIKSRSVDVLENGLRSLQLGPVTPSSSIRNSR 
               
               
                   
               
               
                 Human APOBEC-3D: 
               
               
                 (SEQ ID NO: 281) 
                   
               
               
                 MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFR 
                   
               
               
                   
               
               
                 GPVLPKRQSNHRQEVYFRFEN HAEMCFLSWFCGNRLPANRRFQITWFVSWNPCLPC VV 
               
               
                   
               
               
                 KVTKFLAEHPNVTLTISAARLYYYRDRDWRWVLLRLHKAGARVKIMDYEDFAYCW 
               
               
                   
               
               
                 ENFVCNEGQPFMPWYKFDDNYASLHRTLKEILRNPMEAMYPHIFYFHFKNLLKACG 
               
               
                   
               
               
                 RNESWLCFTMEVTKHHSAVFRKRGVFRNQVDPETHC HAERCFLSWFCDDILSPNTNY   
               
               
                   
               
               
                   EVTWYTSWSPCPEC AGEVAEFLARHSNVNLTIFTARLCYFWDTDYQEGLCSLSQEGAS 
               
               
                   
               
               
                 VKIMGYKDFVSCWKNFVYSDDEPFKPWKGLQTNFRLLKRRLREILQ 
               
               
                 (italic: nucleic acid editing domain) 
               
               
                   
               
               
                 Human APOBEC-1: 
               
               
                 (SEQ ID NO: 282) 
               
               
                 MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKN 
               
               
                   
               
               
                 TTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYV 
               
               
                   
               
               
                 ARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYP 
               
               
                   
               
               
                 PLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIH 
               
               
                   
               
               
                 PSVAWR 
               
               
                   
               
               
                 Mouse APOBEC-1: 
               
               
                 (SEQ ID NO: 283) 
                   
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSVWRHTSQN 
                   
               
               
                   
               
               
                 TSNHVEVNFLEKFTTERYFRPNTRCSITWFLSWSPCGECSRAITEFLSRHPYVTLFIYIA 
               
               
                   
               
               
                 RLYHHTDQRNRQGLRDLISSGVTIQIMTEQEYCYCWRNFVNYPPSNEAYWPRYPHL 
               
               
                   
               
               
                 WVKLYVLELYCIILGLPPCLKILRRKQPQLTFFTITLQTCHYQRIPPHLLWATGLK 
               
               
                   
               
               
                 Rat APOBEC-1: 
               
               
                 (SEQ ID NO: 284) 
                   
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNT 
                   
               
               
                   
               
               
                 NKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIAR 
               
               
                   
               
               
                 LYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLW 
               
               
                   
               
               
                 VRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK 
               
               
                   
               
               
                 Human APOBEC-2: 
               
               
                 (SEQ ID NO: 5733) 
                   
               
               
                 MAQKEEAAVATEAASQNGEDLENLDDPEKLKELIELPPFEIVTGERLPANFFKFQFRN 
                   
               
               
                   
               
               
                 VEYSSGRNKTFLCYVVEAQGKGGQVQASRGYLEDEHAAAHAEEAFFNTILPAFDPALR 
               
               
                   
               
               
                 YNVTWYVSSSPCAACADRIIKTLSKTKNLRLLILVGRLFMWEEPEIQAALKKLKEAG 
               
               
                   
               
               
                 CKLRIMKPQDFEYVWQNFVEQEEGESKAFQPWEDIQENFLYYEEKLADILK 
               
               
                   
               
               
                 Mouse APOBEC-2: 
               
               
                 (SEQ ID NO: 5734) 
                   
               
               
                 MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFR 
                   
               
               
                   
               
               
                 NVEYSSGRNKTFLCYVVEVQSKGGQAQATQGYLEDEHAGAHAEEAFFNTILPAFDP 
               
               
                   
               
               
                 ALKYNVTWYVSSSPCAACADRILKTLSKTKNLRLLILVSRLFMWEEPEVQAALKKLK 
               
               
                   
               
               
                 EAGCKLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK 
               
               
                   
               
               
                 Rat APOBEC-2: 
               
               
                 (SEQ ID NO: 5735) 
                   
               
               
                 MAQKEEAAEAAAPASQNGDDLENLEDPEKLKELIDLPPFEIVTGVRLPVNFFKFQFR 
                   
               
               
                   
               
               
                 NVEYSSGRNKTFLCYVVEAQSKGGQVQATQGYLEDEHAGAHAEEAFFNTILPAFDP 
               
               
                   
               
               
                 ALKYNVTWYVSSSPCAACADRILKTLSKTKNLRLLILVSRLFMWEEPEVQAALKKLK 
               
               
                   
               
               
                 EAGCKLRIMKPQDFEYLWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK 
               
               
                   
               
               
                 Bovine APOBEC-2: 
               
               
                 (SEQ ID NO: 5736) 
                   
               
               
                 MAQKEEAAAAAEPASQNGEEVENLEDPEKLKELIELPPFEIVTGERLPAHYFKFQFRN 
                   
               
               
                   
               
               
                 VEYSSGRNKTFLCYVVEAQSKGGQVQASRGYLEDEHATNHAEEAFFNSIMPTFDPALR 
               
               
                   
               
               
                 YMVTWYVSSSPCAACADRIVKTLNKTKNLRLLILVGRLFMWEEPEIQAALRKLKEA 
               
               
                   
               
               
                 GCRLRIMKPQDFEYIWQNFVEQEEGESKAFEPWEDIQENFLYYEEKLADILK 
               
               
                   
               
               
                 Petromyzon marinus CDA1 (pmCDA1) 
               
               
                 (SEQ ID NO: 5738) 
                   
               
               
                 MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNK 
                   
               
               
                   
               
               
                 PQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRG 
               
               
                   
               
               
                 NGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQ 
               
               
                   
               
               
                 LNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV 
               
               
                   
               
               
                 Human APOBEC3G D316R_D317R 
               
               
                 (SEQ ID NO: 5739) 
                   
               
               
                 MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQ 
                   
               
               
                   
               
               
                 VYSELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDP 
               
               
                   
               
               
                 KVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQ 
               
               
                   
               
               
                 RELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVER 
               
               
                   
               
               
                 MHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTC 
               
               
                   
               
               
                 FTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYRRQGRCQEGLRTLAEAGAKISIMT 
               
               
                   
               
               
                 YSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN 
               
               
                   
               
               
                 Human APOBEC3G chain A 
               
               
                 (SEQ ID NO: 5740) 
                   
               
               
                 MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHG 
                   
               
               
                   
               
               
                 FLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCI 
               
               
                   
               
               
                 FTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLD 
               
               
                   
               
               
                 EHSQDLSGRLRAILQ 
               
               
                   
               
               
                 Human APOBEC3G chain A D120R_D121R 
               
               
                 (SEQ ID NO: 5741) 
                   
               
               
                 MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHG 
                   
               
               
                   
               
               
                 FLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCI 
               
               
                   
               
               
                 FTARIYRRQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDE 
               
               
                   
               
               
                 HSQDLSGRLRAILQ 
               
            
           
         
       
     
     In some embodiments, fusion proteins as provided herein comprise the full-length amino acid of a nucleic acid editing enzyme, e.g., one of the sequences provided above. In other embodiments, however, fusion proteins as provided herein do not comprise a full-length sequence of a nucleic acid editing enzyme, but only a fragment thereof. For example, in some embodiments, a fusion protein provided herein comprises a Cas9 domain and a fragment of a nucleic acid editing enzyme, e.g., wherein the fragment comprises a nucleic acid editing domain. Exemplary amino acid sequences of nucleic acid editing domains are shown in the sequences above as italicized letters, and additional suitable sequences of such domains will be apparent to those of skill in the art. 
     Additional suitable nucleic-acid editing enzyme sequences, e.g., deaminase enzyme and domain sequences, that can be used according to aspects of this invention, e.g., that can be fused to a nuclease-inactive Cas9 domain, will be apparent to those of skill in the art based on this disclosure. In some embodiments, such additional enzyme sequences include deaminase enzyme or deaminase domain sequences that are 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%, or at least 99% similar to the sequences provided herein. Additional suitable Cas9 domains, variants, and sequences will also be apparent to those of skill in the art. Examples of such additional suitable Cas9 domains include, but are not limited to, D10A, D10A/D839A/H840A, and D10A/D839A/H840A/N863A mutant domains (See, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838 the entire contents of which are incorporated herein by reference). In some embodiments, the Cas9 comprises a histidine residue at position 840 of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260. The presence of the catalytic residue H840 restores the acvitity of the Cas9 to cleave the non-edited strand containing a G opposite the targeted C. Restoration of H840 does not result in the cleavage of the target strand containing the C. 
     Additional suitable strategies for generating fusion proteins comprising a Cas9 domain and a deaminase domain will be apparent to those of skill in the art based on this disclosure in combination with the general knowledge in the art. Suitable strategies for generating fusion proteins according to aspects of this disclosure using linkers or without the use of linkers will also be apparent to those of skill in the art in view of the instant disclosure and the knowledge in the art. For example, Gilbert et al., CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.  Cell.  2013; 154(2):442-51, showed that C-terminal fusions of Cas9 with VP64 using 2 NLS&#39;s as a linker (SPKKKRKVEAS, SEQ ID NO: 599), can be employed for transcriptional activation. Mali et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.  Nat Biotechnol.  2013; 31(9):833-8, reported that C-terminal fusions with VP64 without linker can be employed for transcriptional activation. And Maeder et al., CRISPR RNA-guided activation of endogenous human genes.  Nat Methods.  2013; 10: 977-979, reported that C-terminal fusions with VP64 using a Gly4Ser (SEQ ID NO: 5) linker can be used as transcriptional activators. Recently, dCas9-FokI nuclease fusions have successfully been generated and exhibit improved enzymatic specificity as compared to the parental Cas9 enzyme (In 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, and in Tsai S Q, Wyvekens N, Khayter C, Foden J A, Thapar V, Reyon D, Goodwin M J, Aryee M J, Joung J K. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing.  Nat Biotechnol.  2014; 32(6):569-76. PMID: 24770325 a SGSETPGTSESATPES (SEQ ID NO: 7) or a GGGGS (SEQ ID NO: 5) linker was used in FokI-dCas9 fusion proteins, respectively). 
     Some aspects of this disclosure provide fusion proteins comprising (i) a Cas9 enzyme or domain (e.g., a first protein); and (ii) a nucleic acid-editing enzyme or domain (e.g., a second protein). In some aspects, the fusion proteins provided herein further include (iii) a programmable DNA-binding protein, for example, a zinc-finger domain, a TALE, or a second Cas9 protein (e.g., a third protein). Without wishing to be bound by any particular theory, fusing a programmable DNA-binding protein (e.g., a second Cas9 protein) to a fusion protein comprising (i) a Cas9 enzyme or domain (e.g., a first protein); and (ii) a nucleic acid-editing enzyme or domain (e.g., a second protein) may be useful for improving specificity of the fusion protein to a target nucleic acid sequence, or for improving specificity or binding affinity of the fusion protein to bind target nucleic acid sequence that does not contain a canonical PAM (NGG) sequence. In some embodiments, the third protein is a Cas9 protein (e.g, a second Cas9 protein). In some embodiments, the third protein is any of the Cas9 proteins provided herein. In some embodiments, the third protein is fused to the fusion protein N-terminal to the Cas9 protein (e.g., the first protein). In some embodiments, the third protein is fused to the fusion protein C-terminal to the Cas9 protein (e.g., the first protein). In some embodiments, the Cas9 domain (e.g., the first protein) and the third protein (e.g., a second Cas9 protein) are fused via a linker (e.g., a second linker). In some embodiments, the linker comprises a (GGGGS) n  (SEQ ID NO: 5), a (G) n , an (EAAAK) n  (SEQ ID NO: 6), a (GGS) n , (SGGS) n  (SEQ ID NO: 4288), an SGSETPGTSESATPES (SEQ ID NO: 7), or an (XP) n  motif, or a combination of any of these, wherein n is independently an integer between 1 and 30. In some embodiments, the general architecture of exemplary Cas9 fusion proteins provided herein comprises the structure:
         [NH2]-[nucleic acid-editing enzyme or domain]-[Cas9]-[third protein]-[COOH];   [NH2]-[third protein]-[Cas9]-[nucleic acid-editing enzyme or domain]-[COOH];   [NH2]-[Cas9]-[nucleic acid-editing enzyme or domain]-[third protein]-[COOH];   [NH2]-[third protein]-[nucleic acid-editing enzyme or domain]-[Cas9]-[COOH];   [NH2]-[UGI]-[nucleic acid-editing enzyme or domain]-[Cas9]-[third protein]-[COOH];   [NH2]-[UGI]-[third protein]-[Cas9]-[nucleic acid-editing enzyme or domain]-[COOH];   [NH2]-[UGI]-[Cas9]-[nucleic acid-editing enzyme or domain]-[third protein]-[COOH];   [NH2]-[UGI]-[third protein]-[nucleic acid-editing enzyme or domain]-[Cas9]-[COOH];   [NH2]-[nucleic acid-editing enzyme or domain]-[Cas9]-[third protein]-[UGI]-[COOH];   [NH2]-[third protein]-[Cas9]-[nucleic acid-editing enzyme or domain]-[UGI]-[COOH];   [NH2]-[Cas9]-[nucleic acid-editing enzyme or domain]-[third protein]-[UGI]-[COOH]; or   [NH2]-[third protein]-[nucleic acid-editing enzyme or domain]-[Cas9]-[UGI]-[COOH];
 
wherein NH2 is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein. In some embodiments, the “]-[” used in the general architecture above indicates the presence of an optional linker sequence. In other examples, the general architecture of exemplary Cas9 fusion proteins provided herein comprises the structure:
   [NH2]-[nucleic acid-editing enzyme or domain]-[Cas9]-[second Cas9 protein]-[COOH];   [NH2]-[second Cas9 protein]-[Cas9]-[nucleic acid-editing enzyme or domain]-[COOH];   [NH2]-[Cas9]-[nucleic acid-editing enzyme or domain]-[second Cas9 protein]-[COOH];   [NH2]-[second Cas9 protein]-[nucleic acid-editing enzyme or domain]-[Cas9]-[COOH];   [NH2]-[UGI]-[nucleic acid-editing enzyme or domain]-[Cas9]-[second Cas9 protein]-[COOH], [NH2]-[UGI]-[second Cas9 protein]-[Cas9]-[nucleic acid-editing enzyme or domain]-[COOH];   [NH2]-[UGI]-[Cas9]-[nucleic acid-editing enzyme or domain]-[second Cas9 protein]-[COOH];   [NH2]-[UGI]-[second Cas9 protein]-[nucleic acid-editing enzyme or domain]-[Cas9]-[COOH];   [NH2]-[nucleic acid-editing enzyme or domain]-[Cas9]-[second Cas9 protein]-[UGI]-[COOH];   [NH2]-[second Cas9 protein]-[Cas9]-[nucleic acid-editing enzyme or domain]-[UGI]-[COOH];   [NH2]-[Cas9]-[nucleic acid-editing enzyme or domain]-[second Cas9 protein]-[UGI]-[COOH]; or   [NH2]-[second Cas9 protein]-[nucleic acid-editing enzyme or domain]-[Cas9]-[UGI]-[COOH];
 
wherein NH 2  is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein. In some embodiments, the “]-[” used in the general architecture above indicates the presence of an optional linker sequence. In some embodiments, the second Cas9 is a dCas9 protein. In some examples, the general architecture of exemplary Cas9 fusion proteins provided herein comprises a structure as shown in  FIG. 3 . It should be appreciated that any of the proteins provided in any of the general architectures of exemplary Cas9 fusion proteins may be connected by one or more of the linkers provided herein. In some embodiments, the linkers are the same. In some embodiments, the linkers are different. In some embodiments, one or more of the proteins provided in any of the general architectures of exemplary Cas9 fusion proteins are not fused via a linker. In some embodiments, the fusion proteins further comprise a nuclear targeting sequence, for example a nuclear localization sequence. In some embodiments, 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 third protein. In some embodiments, the NLS is fused to the C-terminus of the third protein. In some embodiments, the NLS is fused to the N-terminus of the Cas9 protein. In some embodiments, the NLS is fused to the C-terminus of the Cas9 protein. In some embodiments, the NLS is fused to the N-terminus of the nucleic acid-editing enzyme or domain. In some embodiments, the NLS is fused to the C-terminus of the nucleic acid-editing enzyme or domain. In some embodiments, the NLS is fused to the N-terminus of the UGI protein. In some embodiments, the NLS is fused to the C-terminus of the UGI protein. 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
       

     Uracil Glycosylase Inhibitor Fusion Proteins 
     Some aspects of the disclosure relate to fusion proteins that comprise a uracil glycosylase inhibitor (UGI) domain. In some embodiments, any of the fusion proteins provided herein that comprise a Cas9 domain (e.g., a nuclease active Cas9 domain, a nuclease inactive dCas9 domain, or a Cas9 nickase) may be further fused to a UGI domain either directly or via a linker. Some aspects of this disclosure provide deaminase-dCas9 fusion proteins, deaminase-nuclease active Cas9 fusion proteins and deaminase-Cas9 nickase fusion proteins with increased nucleobase editing efficiency. Without wishing to be bound by any particular theory, cellular DNA-repair response to the presence of U:G heteroduplex DNA may be responsible for the decrease in nucleobase editing efficiency in cells. For example, uracil DNA glycosylase (UDG) catalyzes removal of U from DNA in cells, which may initiate base excision repair, with reversion of the U:G pair to a C:G pair as the most common outcome. As demonstrated in the Examples below, Uracil DNA Glycosylase Inhibitor (UGI) may inhibit human UDG activity. Thus, this disclosure contemplates a fusion protein comprising dCas9-nucleic acid editing domain further fused to a UGI domain. This disclosure also contemplates a fusion protein comprising a Cas9 nickase-nucleic acid editing domain further fused to a UGI domain. It should be understood that the use of a UGI domain may increase the editing efficiency of a nucleic acid editing domain that is capable of catalyzing a C to U change. For example, fusion proteins comprising a UGI domain may be more efficient in deaminating C residues. In some embodiments, the fusion protein comprises the structure:
         [deaminase]-[optional linker sequence]-[dCas9]-[optional linker sequence]-[UGI];   [deaminase]-[optional linker sequence]-[UGI]-[optional linker sequence]-[dCas9];   [UGI]-[optional linker sequence]-[deaminase]-[optional linker sequence]-[dCas9];   [UGI]-[optional linker sequence]-[dCas9]-[optional linker sequence]-[deaminase];   [dCas9]-[optional linker sequence]-[deaminase]-[optional linker sequence]-[UGI]; or   [dCas9]-[optional linker sequence]-[UGI]-[optional linker sequence]-[deaminase].
 
In other embodiments, the fusion protein comprises the structure:
   [deaminase]-[optional linker sequence]-[Cas9 nickase]-[optional linker sequence]-[UGI];   [deaminase]-[optional linker sequence]-[UGI]-[optional linker sequence]-[Cas9 nickase];   [UGI]-[optional linker sequence]-[deaminase]-[optional linker sequence]-[Cas9 nickase];   [UGI]-[optional linker sequence]-[Cas9 nickase]-[optional linker sequence]-[deaminase];   [Cas9 nickase]-[optional linker sequence]-[deaminase]-[optional linker sequence]-[UGI]; or   [Cas9 nickase]-[optional linker sequence]-[UGI]-[optional linker sequence]-[deaminase].       

     In some embodiments, the fusion proteins provided herein do not comprise a linker sequence. In some embodiments, one or both of the optional linker sequences are present. 
     In some embodiments, the “-” used in the general architecture above indicates the presence of an optional linker sequence. In some embodiments, the fusion proteins comprising a UGI further comprise a nuclear targeting sequence, for example a nuclear localization sequence. In some embodiments, 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 UGI protein. In some embodiments, the NLS is fused to the C-terminus of the UGI protein. In some embodiments, the NLS is fused to the N-terminus of the Cas9 protein. In some embodiments, the NLS is fused to the C-terminus of the Cas9 protein. 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 N-terminus of the second Cas9. In some embodiments, the NLS is fused to the C-terminus of the second Cas9. 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. In some embodiments, the NLS comprises an amino acid sequence as set forth in SEQ ID NO: 741 or SEQ ID NO: 742. 
     In some embodiments, a UGI domain comprises a wild-type UGI or a UGI as set forth in SEQ ID NO: 600. In some embodiments, the UGI proteins provided herein include fragments of UGI and proteins homologous to a UGI or a UGI fragment. For example, in some embodiments, a UGI domain comprises a fragment of the amino acid sequence set forth in SEQ ID NO: 600. In some embodiments, a UGI fragment comprises an amino acid sequence that comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the amino acid sequence as set forth in SEQ ID NO: 600. In some embodiments, a UGI comprises an amino acid sequence homologous to the amino acid sequence set forth in SEQ ID NO: 600 or an amino acid sequence homologous to a fragment of the amino acid sequence set forth in SEQ ID NO: 600. In some embodiments, proteins comprising UGI or fragments of UGI or homologs of UGI or UGI fragments are referred to as “UGI variants.” A UGI variant shares homology to UGI, or a fragment thereof. For example a UGI variant is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% identical to a wild type UGI or a UGI as set forth in SEQ ID NO: 600. In some embodiments, the UGI variant comprises a fragment of UGI, such that the fragment is at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% to the corresponding fragment of wild-type UGI or a UGI as set forth in SEQ ID NO: 600. In some embodiments, the UGI comprises the following amino acid sequence: &gt;sp|P14739|UNGI_BPPB2 Uracil-DNA glycosylase inhibitor MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLT SDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 600) 
     Suitable UGI protein and nucleotide sequences are provided herein and additional suitable UGI sequences are known to those in the art, and include, for example, those published in Wang et al., Uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 encodes a binding protein specific for uracil-DNA glycosylase. J. Biol. Chem. 264:1163-1171(1989); Lundquist et al., Site-directed mutagenesis and characterization of uracil-DNA glycosylase inhibitor protein. Role of specific carboxylic amino acids in complex formation with  Escherichia coli  uracil-DNA glycosylase. J. Biol. Chem. 272:21408-21419(1997); Ravishankar et al., X-ray analysis of a complex of  Escherichia coli  uracil DNA glycosylase (EcUDG) with a proteinaceous inhibitor. The structure elucidation of a prokaryotic UDG. Nucleic Acids Res. 26:4880-4887(1998); and Putnam et al., Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with  Escherichia coli  uracil-DNA glycosylase. J. Mol. Biol. 287:331-346(1999), the entire contents of each are incorporated herein by reference. 
     It should be appreciated that additional proteins may be uracil glycosylase inhibitors. For example, other proteins that are capable of inhibiting (e.g., sterically blocking) a uracil-DNA glycosylase base-excision repair enzyme are within the scope of this disclosure. Additionally, any proteins that block or inhibit base-excision repair as also within the scope of this disclosure. In some embodiments, a protein that binds DNA is used. In another embodiment, a substitute for UGI is used. In some embodiments, a uracil glycosylase inhibitor is a protein that binds single-stranded DNA. For example, a uracil glycosylase inhibitor may be a  Erwinia tasmaniensis  single-stranded binding protein. In some embodiments, the single-stranded binding protein comprises the amino acid sequence (SEQ ID NO: 322). In some embodiments, a uracil glycosylase inhibitor is a protein that binds uracil. In some embodiments, a uracil glycosylase inhibitor is a protein that binds uracil in DNA. In some embodiments, a uracil glycosylase inhibitor is a catalytically inactive uracil DNA-glycosylase protein. In some embodiments, a uracil glycosylase inhibitor is a catalytically inactive uracil DNA-glycosylase protein that does not excise uracil from the DNA. For example, a uracil glycosylase inhibitor is a UdgX. In some embodiments, the UdgX comprises the amino acid sequence (SEQ ID NO: 323). As another example, a uracil glycosylase inhibitor is a catalytically inactive UDG. In some embodiments, a catalytically inactive UDG comprises the amino acid sequence (SEQ ID NO: 324). It should be appreciated that other uracil glycosylase inhibitors would be apparent to the skilled artisan and are within the scope of this disclosure. In some embodiments, a uracil glycosylase inhibitor is a protein that is homologous to any one of SEQ ID NOs: 322-324. In some embodiments, a uracil glycosylase inhibitor is a protein that is at least 50% identical, at least 55% identical at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical to any one of SEQ ID NOs: 322-324. 
     
       
         
           
               
            
               
                   Erwinia tasmaniensis  SSB 
               
               
                 (themostable single-stranded DNA binding protein) 
               
               
                 (SEQ ID NO: 322) 
               
               
                 MASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKQTGETK 
               
               
                   
               
               
                 EKTEWHRVVLFGKLAEVAGEYLRKGSQVYIEGALQTRKWTDQAGVEKYTT 
               
               
                   
               
               
                 EVVVNVGGTMQMLGGRSQGGGASAGGQNGGSNNGWGQPQQPQGGNQFSGG 
               
               
                   
               
               
                 AQQQARPQQQPQQNNAPANNEPPIDFDDDIP 
               
               
                   
               
               
                 UdgX (binds to Uracil in DNA but does not excise) 
               
               
                 (SEQ ID NO: 323) 
               
               
                 MAGAQDFVPHTADLAELAAAAGECRGCGLYRDATQAVFGAGGRSARIMMI 
               
               
                   
               
               
                 GEQPGDKEDLAGLPFVGPAGRLLDRALEAADIDRDALYVTNAVKHFKFTR 
               
               
                   
               
               
                 AAGGKRRIHKTPSRTEVVACRPWLIAEMTSVEPDVVVLLGATAAKALLGN 
               
               
                   
               
               
                 DFRVTQHRGEVLHVDDVPGDPALVATVHPSSLLRGPKEERESAFAGLVDD 
               
               
                   
               
               
                 LRVAADVRP 
               
               
                   
               
               
                 UDG (catalytically inactive human UDG, binds to 
               
               
                 Uracil in DNA but does not excise) 
               
               
                 (SEQ ID NO: 324) 
               
               
                 MIGQKTLYSFFSPSPARKRHAPSPEPAVQGTGVAGVPEESGDAAAIPAKK 
               
               
                   
               
               
                 APAGQEEPGTPPSSPLSAEQLDRIQRNKAAALLRLAARNVPVGFGESWKK 
               
               
                   
               
               
                 HLSGEFGKPYFIKLMGFVAEERKHYTVYPPPHQVFTWTQMCDIKDVKVVI 
               
               
                   
               
               
                 LGQEPYHGPNQAHGLCFSVQRPVPPPPSLENIYKELSTDIEDFVHPGHGD 
               
               
                   
               
               
                 LSGWAKQGVLLLNAVLTVRAHQANSHKERGWEQFTDAVVSWLNQNSNGLV 
               
               
                   
               
               
                 FLLWGSYAQKKGSAIDRKRHHVLQTAHPSPLSVYRGFFGCRHFSKTNELL 
               
               
                   
               
               
                 QKSGKKPIDWKEL 
               
            
           
         
       
     
     In some embodiments, the nucleic acid editing domain is a deaminase domain. In some embodiments, the deaminase is a cytosine deaminase or a cytidine 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 APOBEC3A 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 rat APOBEC1 (SEQ ID NO: 282). In some embodiments, the deaminase is a human APOBEC1 (SEQ ID No: 284). In some embodiments, the deaminase is a  Petromyzon marinus  cytidine deaminase 1 (pmCDA1). In some embodiments, the deaminase is a human APOBEC3G (SEQ ID NO: 275). In some embodiments, the deaminase is a fragment of the human APOBEC3G (SEQ ID NO: 5740). In some embodiments, the deaminase is a human APOBEC3G variant comprising a D316R_D317R mutation (SEQ ID NO: 5739). In some embodiments, the deaminase is a frantment of the human APOBEC3G and comprising mutations corresponding to the D316R_D317R mutations in SEQ ID NO: 275 (SEQ ID NO: 5741). 
     In some embodiments, the linker comprises a (GGGS) n  (SEQ ID NO: 265), (GGGGS) n  (SEQ ID NO: 5), a (G) n , an (EAAAK) n  (SEQ ID NO: 6), a (GGS) n , an SGSETPGTSESATPES (SEQ ID NO: 7), or an (XP) n  motif, or a combination of any of these, wherein n is independently an integer between 1 and 30. 
     Suitable UGI protein and nucleotide sequences are provided herein and additional suitable UGI sequences are known to those in the art, and include, for example, those published in Wang et al., Uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 encodes a binding protein specific for uracil-DNA glycosylase.  J. Biol. Chem.  264:1163-1171(1989); Lundquist et al., Site-directed mutagenesis and characterization of uracil-DNA glycosylase inhibitor protein. Role of specific carboxylic amino acids in complex formation with  Escherichia coli  uracil-DNA glycosylase.  J. Biol. Chem.  272:21408-21419(1997); Ravishankar et al., X-ray analysis of a complex of  Escherichia coli  uracil DNA glycosylase (EcUDG) with a proteinaceous inhibitor. The structure elucidation of a prokaryotic UDG.  Nucleic Acids Res.  26:4880-4887(1998); and Putnam et al., Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with  Escherichia coli  uracil-DNA glycosylase.  J. Mol. Biol.  287:331-346(1999), the entire contents of which are incorporated herein by reference. In some embodiments, the optional linker comprises a (GGS) n  motif, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the optional linker comprises a (GGS) n  motif, wherein n is 1, 3, or 7. In some embodiments, the optional linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 7), which is also referred to as the XTEN linker in the Examples. 
     In some embodiments, a Cas9 nickase may further facilitate the removal of a base on the non-edited strand in an organism whose genome is edited in vivo. The Cas9 nickase, as described herein, may comprise a D10A mutation in SEQ ID NO: 10, or a corresponding mutation in any of SEQ ID NOs: 11-260. In some embodiments, the Cas9 nickase of this disclosure may comprise a histidine at mutation 840 of SEQ ID NO: 10, or a corresponding residue in any of SEQ ID NOs: 11-260. Such fusion proteins comprising the Cas9 nickase, can cleave a single strand of the target DNA sequence, e.g., the strand that is not being edited. Without wishing to be bound by any particular theory, this cleavage may inhibit mis-match repair mechanisms that reverse a C to U edit made by the deaminase. 
     Cas9 Complexes with Guide RNAs 
     Some aspects of this disclosure provide complexes comprising any of the fusion proteins provided herein, and a guide RNA bound to a Cas9 domain (e.g., a dCas9, a nuclease active Cas9, or a Cas9 nickase) of fusion protein. 
     In some embodiments, the 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, or40 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 mammal. 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 guide RNA is complementary to a sequence associated with a disease or disorder. In some embodiments, the guide RNA is complementary to a sequence associated with a disease or disorder having a mutation in a gene selected from the genes disclosed in any one of Tables 1-3. In some embodiments, the guide RNA comprises a nucleotide sequence of any one of the guide sequences provided in Table 2 or Table 3. Exemplary sequences in the human genome that may be targeted by the complexes of this disclosure are provided herein in Tables 1-3. 
     Methods of Using Cas9 Fusion Proteins 
     Some aspects of this disclosure provide methods of using the Cas9 proteins, fusion proteins, or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule (a) with any of the Cas9 proteins or 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; or (b) with a Cas9 protein, a Cas9 fusion protein, or a Cas9 protein or fusion protein complex with at least one gRNA as provided herein. In some embodiments, the 3′ end of the target sequence is not immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3′ end of the target sequence is immediately adjacent to an AGC, GAG, TTT, GTG, or CAA sequence. 
     In some embodiments, the target DNA sequence comprises a sequence associated with a disease or disorder. In some embodiments, the target DNA sequence comprises a point mutation associated with a disease or disorder. In some embodiments, the activity of the Cas9 protein, the Cas9 fusion protein, or the complex results in a correction of the point mutation. 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 wherein 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 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 contacting is in vivo in a subject. In some embodiments, the subject has or has been diagnosed with a disease or disorder. In some embodiments, the disease or disorder is cystic fibrosis, phenylketonuria, epidermolytic hyperkeratosis (EHK), Charcot-Marie-Toot disease type 4J, neuroblastoma (NB), von Willebrand disease (vWD), myotonia congenital, hereditary renal amyloidosis, dilated cardiomyopathy (DCM), hereditary lymphedema, familial Alzheimer&#39;s disease, HIV, Prion disease, chronic infantile neurologic cutaneous articular syndrome (CINCA), desmin-related myopathy (DRM), a neoplastic disease associated with a mutant PI3KCA protein, a mutant CTNNB1 protein, a mutant HRAS protein, or a mutant p53 protein. 
     Some embodiments provide methods for using the Cas9 DNA editing fusion proteins provided herein. In some embodiments, the fusion protein is used to introduce a point mutation into a nucleic acid by deaminating a target nucleobase, e.g., a C residue. In some embodiments, the deamination of the target nucleobase results in the correction of a genetic defect, e.g., in the correction of a point mutation that leads to a loss of function in a gene product. In some embodiments, the genetic defect is associated with a disease or disorder, e.g., a lysosomal storage disorder or a metabolic disease, such as, for example, type I diabetes. In some embodiments, the methods provided herein are used to introduce a deactivating point mutation into a gene or allele that encodes a gene product that is associated with a disease or disorder. For example, in some embodiments, methods are provided herein that employ a Cas9 DNA editing fusion protein to introduce a deactivating point mutation into an oncogene (e.g., in the treatment of a proliferative disease). A deactivating mutation may, in some embodiments, generate a premature stop codon in a coding sequence, which results in the expression of a truncated gene product, e.g., a truncated protein lacking the function of the full-length protein. 
     In some embodiments, the purpose of the methods provide herein is to restore the function of a dysfunctional gene via genome editing. The Cas9 deaminase fusion 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 fusion proteins provided herein, e.g., the fusion proteins comprising a Cas9 domain and a nucleic acid deaminase domain can be used to correct any single point T-&gt;C or A-&gt;G mutation. In the first case, deamination of the mutant C back to U corrects the mutation, and in the latter case, deamination of the C that is base-paired with the mutant G, followed by a round of replication, corrects the mutation. 
     An exemplary disease-relevant mutation that can be corrected by the provided fusion proteins in vitro or in vivo is the H1047R (A3140G) polymorphism in the PI3KCA protein. The phosphoinositide-3-kinase, catalytic alpha subunit (PI3KCA) protein acts to phosphorylate the 3-OH group of the inositol ring of phosphatidylinositol. The PI3KCA gene has been found to be mutated in many different carcinomas, and thus it is considered to be a potent oncogene. 37  In fact, the A3140G mutation is present in several NCI-60 cancer cell lines, such as, for example, the HCT116, SKOV3, and T47D cell lines, which are readily available from the American Type Culture Collection (ATCC). 38    
     In some embodiments, a cell carrying a mutation to be corrected, e.g., a cell carrying a point mutation, e.g., an A3140G point mutation in exon 20 of the PI3KCA gene, resulting in a H1047R substitution in the PI3KCA protein, is contacted with an expression construct encoding a Cas9 deaminase fusion protein and an appropriately designed sgRNA targeting the fusion protein to the respective mutation site in the encoding PI3KCA gene. Control experiments can be performed where the sgRNAs are designed to target the fusion enzymes to non-C residues that are within the PI3KCA gene. Genomic DNA of the treated cells can be extracted, and the relevant sequence of the PI3KCA genes PCR amplified and sequenced to assess the activities of the fusion proteins in human cell culture. 
     It will be understood that the example of correcting point mutations in PI3KCA is provided for illustration purposes and is not meant to limit the instant disclosure. The skilled artisan will understand that the instantly disclosed DNA-editing fusion proteins can be used to correct other point mutations and mutations associated with other cancers and with diseases other than cancer including other proliferative diseases. 
     The successful correction of point mutations in disease-associated genes and alleles opens up new strategies for gene correction with applications in therapeutics and basic research. Site-specific single-base modification systems like the disclosed fusions of Cas9 and deaminase enzymes or domains also have applications in “reverse” gene therapy, where certain gene functions are purposely suppressed or abolished. In these cases, site-specifically mutating Trp (TGG), Gln (CAA and CAG), or Arg (CGA) residues to premature stop codons (TAA, TAG, TGA) can be used to abolish protein function in vitro, ex vivo, or in vivo. 
     The instant 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 Cas9 DNA editing fusion protein provided herein. For example, in some embodiments, a method is provided that comprises administering to a subject having such a disease, e.g., a cancer associated with a PI3KCA point mutation as described above, an effective amount of a Cas9 deaminase fusion protein that corrects the point mutation or introduces a deactivating mutation into 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. Other diseases that can be treated by correcting a point mutation or introducing a deactivating mutation into a disease-associated gene will be known to those of skill in the art, and the disclosure is not limited in this respect. 
     The instant disclosure provides 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. Some 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. Exemplary suitable diseases and disorders are listed below. 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. Exemplary suitable diseases and disorders include, without limitation, cystic fibrosis (see, e.g., Schwank et al., Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients.  Cell stem cell.  2013; 13: 653-658; and Wu et. al., Correction of a genetic disease in mouse via use of CRISPR-Cas9 . Cell stem cell.  2013; 13: 659-662, neither of which uses a deaminase fusion protein to correct the genetic defect); phenylketonuria—e.g., phenylalanine to serine mutation at position 835 (mouse) or 240 (human) or a homologous residue in phenylalanine hydroxylase gene (T&gt;C mutation)—see, e.g., McDonald et al.,  Genomics.  1997; 39:402-405; Bernard-Soulier syndrome (BSS)—e.g., phenylalanine to serine mutation at position 55 or a homologous residue, or cysteine to arginine at residue 24 or a homologous residue in the platelet membrane glycoprotein IX (T&gt;C mutation)—see, e.g., Noris et al.,  British Journal of Haematology.  1997; 97: 312-320, and Ali et al.,  Hematol.  2014; 93: 381-384; epidermolytic hyperkeratosis (EHK)—e.g., leucine to proline mutation at position 160 or 161 (if counting the initiator methionine) or a homologous residue in keratin 1 (T&gt;C mutation)—see, e.g., Chipev et al.,  Cell.  1992; 70: 821-828, see also accession number P04264 in the UNIPROT database at www[dot]uniprot[dot]org; chronic obstructive pulmonary disease (COPD)—e.g., leucine to proline mutation at position 54 or 55 (if counting the initiator methionine) or a homologous residue in the processed form of α 1 -antitrypsin or residue 78 in the unprocessed form or a homologous residue (T&gt;C mutation)—see, e.g., Poller et al.,  Genomics.  1993; 17: 740-743, see also accession number P01011 in the UNIPROT database; Charcot-Marie-Toot disease type 4J—e.g., isoleucine to threonine mutation at position 41 or a homologous residue in  FIG. 4  (T&gt;C mutation)—see, e.g., Lenk et al., PLoS Genetics. 2011; 7: e1002104; neuroblastoma (NB)—e.g., leucine to proline mutation at position 197 or a homologous residue in Caspase-9 (T&gt;C mutation)—see, e.g., Kundu et al., 3  Biotech.  2013, 3:225-234; von Willebrand disease (vWD)—e.g., cysteine to arginine mutation at position 509 or a homologous residue in the processed form of von Willebrand factor, or at position 1272 or a homologous residue in the unprocessed form of von Willebrand factor (T&gt;C mutation)—see, e.g., Lavergne et al.,  Br. J. Haematol.  1992, see also accession number P04275 in the UNIPROT database; 82: 66-72; myotonia congenital—e.g., cysteine to arginine mutation at position 277 or a homologous residue in the muscle chloride channel gene CLCN1 (T&gt;C mutation)—see, e.g., Weinberger et al.,  The J. of Physiology.  2012; 590: 3449-3464; hereditary renal amyloidosis—e.g., stop codon to arginine mutation at position 78 or a homologous residue in the processed form of apolipoprotein All or at position 101 or a homologous residue in the unprocessed form (T&gt;C mutation)—see, e.g., Yazaki et al.,  Kidney Int.  2003; 64: 11-16; dilated cardiomyopathy (DCM)—e.g., tryptophan to Arginine mutation at position 148 or a homologous residue in the FOXD4 gene (T&gt;C mutation), see, e.g., Minoretti et. al.,  Int. J. of Mol. Med.  2007; 19: 369-372; hereditary lymphedema—e.g., histidine to arginine mutation at position 1035 or a homologous residue in VEGFR3 tyrosine kinase (A&gt;G mutation), see, e.g., Irrthum et al.,  Am. J. Hum. Genet.  2000; 67: 295-301; familial Alzheimer&#39;s disease—e.g., isoleucine to valine mutation at position 143 or a homologous residue in presenilin1 (A&gt;G mutation), see, e.g., Gallo et. al.,  J. Alzheimer&#39;s disease.  2011; 25: 425-431; Prion disease—e.g., methionine to valine mutation at position 129 or a homologous residue in prion protein (A&gt;G mutation)—see, e.g., Lewis et. al.,  J. of General Virology.  2006; 87: 2443-2449; chronic infantile neurologic cutaneous articular syndrome (CINCA)—e.g., Tyrosine to Cysteine mutation at position 570 or a homologous residue in cryopyrin (A&gt;G mutation)—see, e.g., Fujisawa et. al.  Blood.  2007; 109: 2903-2911; and desmin-related myopathy (DRM)—e.g., arginine to glycine mutation at position 120 or a homologous residue in αβ crystallin (A&gt;G mutation)—see, e.g., Kumar et al.,  J. Biol. Chem.  1999; 274: 24137-24141. The entire contents of all references and database entries is incorporated herein by reference. 
     The instant disclosure provides lists of genes comprising pathogenic T&gt;C or A&gt;G mutations. Provided herein, are the names of these genes, their respective SEQ ID NOs, their gene IDs, and sequences flanking the mutation site. (Tables 2 and 3). In some instances, the gRNA sequences that can be used to correct the mutations in these genes are disclosed (Tables 2 and 3). 
     In some embodiments, a Cas9-deaminase fusion protein recognizes canonical PAMs and therefore can correct the pathogenic T&gt;C or A&gt;G mutations with canonical PAMs, e.g., NGG (listed in Tables 2 and 3, SEQ ID NOs: 2540-2702 and 5084-5260), respectively, in the flanking sequences. For example, the Cas9 proteins that recognize canonical PAMs comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of  Streptococcus pyogenes  Cas9 as provided by SEQ ID NO: 10, or to a fragment thereof comprising the RuvC and HNH domains of SEQ ID NO: 10. 
     It will be apparent to those of skill in the art that in order to target a Cas9:nucleic acid editing enzyme/domain fusion protein as disclosed herein to a target site, e.g., a site comprising a point mutation to be edited, it is typically necessary to co-express the Cas9:nucleic acid editing enzyme/domain fusion protein together with a guide RNA, e.g., an sgRNA. As explained in more detail elsewhere herein, a guide RNA typically comprises a tracrRNA framework allowing for Cas9 binding, and a guide sequence, which confers sequence specificity to the Cas9:nucleic acid editing enzyme/domain fusion protein. In some embodiments, the guide RNA comprises a structure 5′-[guide sequence]-guuuuagagcuagaaauagcaaguuaaaauaaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuu uuu-3′ (SEQ ID NO: 601), 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 Cas9:nucleic acid editing enzyme/domain fusion proteins to specific target sequences are provided below. 
     Base Editor Efficiency 
     Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of modifying a specific nucleotide base without generating a significant proportion of indels. An “indel”, as used herein, refers to the insertion or deletion of a nucleotide base within a nucleic acid. Such insertions or deletions can lead to frame shift mutations within a coding region of a gene. In some embodiments, it is desirable to generate base editors that efficiently modify (e.g. mutate or deaminate) a specific nucleotide within a nucleic acid, without generating a large number of insertions or deletions (i.e., indels) in the nucleic acid. In certain embodiments, any of the base editors provided herein are capable of generating a greater proportion of intended modifications (e.g., point mutations or deaminations) versus indels. 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 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 200:1, at least 300:1, at least 400:1, at least 500:1, at least 600:1, at least 700:1, at least 800:1, at least 900:1, or at least 1000:1, or more. The number of intended mutations and indels may be determined using any suitable method, for example the methods used in the below Examples. 
     In some embodiments, the base editors provided herein are capable of limiting formation of indels in a region of a nucleic acid. In some embodiments, the region is at a nucleotide targeted by a base editor or a region within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nucleotide targeted by a base editor. In some embodiments, any of the base editors provided herein are capable of limiting the formation of indels at a region of a nucleic acid to less than 1%, less than 1.5%, less than 2%, less than 2.5%, less than 3%, less than 3.5%, less than 4%, less than 4.5%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 15%, or less than 20%. The number of indels formed at a nucleic acid region may depend on the amount of time a nucleic acid (e.g., a nucleic acid within the genome of a cell) is exposed to a base editor. In some embodiments, an number or proportion of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing a nucleic acid (e.g., a nucleic acid within the genome of a cell) to a base editor. 
     Some aspects of the disclosure are based on the recognition that any of the base editors provided herein are capable of efficiently generating an intended mutation, 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, a intended mutation is a mutation that is generated by a specific base editor bound to a 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 a cytosine (C) to thymine (T) point mutation associated with a disease or disorder. In some embodiments, the intended mutation is a guanine (G) to adenine (A) point mutation associated with a disease or disorder. In some embodiments, the intended mutation is a cytosine (C) to thymine (T) point mutation within the coding region of a gene. In some embodiments, the intended mutation is a guanine (G) to adenine (A) point mutation within the 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, the intended mutation is a mutation that alters the splicing of a gene. In some embodiments, the intended mutation is a mutation that alters the regulatory sequence of a gene (e.g., a gene promotor or gene repressor). 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. It should be appreciated that the characteristics of the base editors described in the “Base Editor Efficiency” section, herein, may be applied to any of the fusion proteins, or methods of using the fusion proteins provided herein. 
     Methods for Editing Nucleic Acids 
     Some aspects of the disclosure provide methods for editing a nucleic acid. In some embodiments, the method is a method for editing a nucleobase of a nucleic acid (e.g., a base pair of a double-stranded DNA sequence). In some embodiments, the method comprises the steps of: a) contacting a target region of a nucleic acid (e.g., a double-stranded DNA sequence) with a complex comprising a base editor (e.g., a Cas9 domain fused to a cytidine deaminase domain) and a guide nucleic acid (e.g., gRNA), wherein the target region comprises a targeted nucleobase pair, b) inducing strand separation of said target region, c) converting a first nucleobase of said target nucleobase pair in a single strand of the target region to a second nucleobase, and d) cutting no more than one strand of said target region, where a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase; and the method results in less than 20% indel formation in the nucleic acid. It should be appreciated that in some embodiments, step b is omitted. In some embodiments, the first nucleobase is a cytosine. In some embodiments, the second nucleobase is a deaminated cytosine, or a uracil. In some embodiments, the third nucleobase is a guanine. In some embodiments, the fourth nucleobase is an adenine. In some embodiments, the first nucleobase is a cytosine, the second nucleobase is a deaminated cytosine, or a uracil, the third nucleobase is a guanine, and the fourth nucleobase is an adenine. In some embodiments, the method results in less than 19%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, 0.2%, or less than 0.1% indel formation. In some embodiments, the method further comprises replacing the second nucleobase with a fifth nucleobase that is complementary to the fourth nucleobase, thereby generating an intended edited base pair (e.g., C:G-&gt;T:A). In some embodiments, the fifth nucleobase is a thymine. In some embodiments, at least 5% of the intended basepairs are edited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended basepairs are edited. 
     In some embodiments, the ratio of intended products to unintended products in the target nucleotide is at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 200:1, or more. In some embodiments, the ratio of intended point mutation to indel formation is greater than 1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1, or more. In some embodiments, the cut single strand (nicked strand) is hybridized to the guide nucleic acid. In some embodiments, the cut single strand is opposite to the strand comprising the first nucleobase. In some embodiments, the base editor comprises a Cas9 domain. In some embodiments, the first base is cytosine, and the second base is not a G, C, A, or T. In some embodiments, the second base is uracil. In some embodiments, the first base is cytosine. In some embodiments, the second base is not a G, C, A, or T. In some embodiments, the second base is uracil. 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 nickase activity. In some embodiments, the intended edited basepair is upstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended edited basepair is downstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream stream of the PAM site. In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the nucleobase editor comprises a linker. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length. In some embodiments, linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the target region comprises a target window, wherein the target window comprises the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotides in length. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended edited base pair is within the target window. In some embodiments, the target window comprises the intended edited base pair. In some embodiments, the method is performed using any of the base editors provided herein. In some embodiments, a target window is a deamination window 
     In some embodiments, the disclosure provides methods for editing a nucleotide. In some embodiments, the disclosure provides a method for editing a nucleobase pair of a double-stranded DNA sequence. In some embodiments, the method comprises a) contacting a target region of the double-stranded DNA sequence with a complex comprising a base editor and a guide nucleic acid (e.g., gRNA), where the target region comprises a target nucleobase pair, b) inducing strand separation of said target region, c) converting a first nucleobase of said target nucleobase pair in a single strand of the target region to a second nucleobase, d) cutting no more than one strand of said target region, wherein a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase, and the second nucleobase is replaced with a fifth nucleobase that is complementary to the fourth nucleobase, thereby generating an intended edited basepair, wherein the efficiency of generating the intended edited basepair is at least 5%. It should be appreciated that in some embodiments, step b is omitted. In some embodiments, at least 5% of the intended basepairs are edited. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended basepairs are edited. In some embodiments, the method causes less than 19%, 18%, 16%, 14%, 12%, 10%, 8%, 6%, 4%, 2%, 1%, 0.5%, 0.2%, or less than 0.1% indel formation. In some embodiments, the ratio of intended product to unintended products at the target nucleotide is at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or 200:1, or more. In some embodiments, the ratio of intended point mutation to indel formation is greater than 1:1, 10:1, 50:1, 100:1, 500:1, or 1000:1, or more. In some embodiments, the cut single strand is hybridized to the guide nucleic acid. In some embodiments, the cut single strand is opposite to the strand comprising the first nucleobase. In some embodiments, the first base is cytosine. In some embodiments, the second nucleobase is not G, C, A, or T. In some embodiments, the second base is uracil. 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 nucleobase editor comprises UGI activity. In some embodiments, the nucleobase edit comprises nickase activity. In some embodiments, the intended edited basepair is upstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended edited basepair is downstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream stream of the PAM site. In some embodiments, the method does not require a canonical (e.g., NGG) PAM site. In some embodiments, the nucleobase editor comprises a linker. In some embodiments, the linker is 1-25 amino acids in length. In some embodiments, the linker is 5-20 amino acids in length. In some embodiments, the linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the target region comprises a target window, wherein the target window comprises the target nucleobase pair. In some embodiments, the target window comprises 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotides in length. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the intended edited base pair occurs within the target window. In some embodiments, the target window comprises the intended edited base pair. In some embodiments, the nucleobase editor is any one of the base editors provided herein. 
     Kits, Vectors, Cells 
     Some aspects of this disclosure provide kits comprising a nucleic acid construct, comprising (a) a nucleotide sequence encoding a Cas9 protein or a Cas9 fusion protein as provided herein; and (b) a heterologous promoter that drives expression of the sequence of (a). In some embodiments, the kit further comprises an expression construct encoding a guide RNA backbone, wherein the construct comprises a cloning site positioned to allow the cloning of a nucleic acid sequence identical or complementary to a target sequence into the guide RNA backbone. 
     Some aspects of this disclosure provide polynucleotides encoding a Cas9 protein of a fusion protein as provided herein. Some aspects of this disclosure provide vectors comprising such polynucleotides. In some embodiments, the vector comprises a heterologous promoter driving expression of polynucleotide. 
     Some aspects of this disclosure provide cells comprising a Cas9 protein, a fusion protein, a nucleic acid molecule encoding the fusion protein, a complex comprise the Cas9 protein and the gRNA, and/or a vector as provided herein. 
     The description of exemplary embodiments of the reporter systems above is provided for illustration purposes only and not meant to be limiting. Additional reporter systems, e.g., variations of the exemplary systems described in detail above, are also embraced by this disclosure. 
     EXAMPLES 
     Example 1: Cas9 Deaminase Fusion Proteins 
     A number of Cas9:Deaminase fusion proteins were generated and deaminase activity of the generated fusions was characterized. The following deaminases were tested: 
     
       
         
           
               
            
               
                 Human AID (hAID): 
               
               
                 (SEQ ID NO: 607) 
               
               
                 MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLR 
               
               
                   
               
               
                 NKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRG 
               
               
                   
               
               
                 NPYLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNT 
               
               
                   
               
               
                 FVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGLLD 
               
               
                   
               
               
                 Human AID-DC (hAID-DC, truncated version of hAID 
               
               
                 with 7-fold increased activity): 
               
               
                 (SEQ ID NO: 608) 
               
               
                 MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLR 
               
               
                   
               
               
                 NKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRG 
               
               
                   
               
               
                 NPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNT 
               
               
                   
               
               
                 FVENHERTFKAWEGLHENSVRLSRQLRRILL 
               
               
                   
               
               
                 Rat APOBEC1 (rAPOBEC1): 
               
               
                 (SEQ ID NO: 284) 
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI 
               
               
                   
               
               
                 WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI 
               
               
                   
               
               
                 TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG 
               
               
                   
               
               
                 YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ 
               
               
                   
               
               
                 PQLTFFTIALQSCHYQRLPPHILWATGLK 
               
               
                   
               
               
                 Human APOBEC1 (hAPOBEC1) 
               
               
                 (SEQ ID NO: 5724) 
               
               
                 MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKI 
               
               
                   
               
               
                 WRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAI 
               
               
                   
               
               
                 REFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYY 
               
               
                   
               
               
                 HCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQ 
               
               
                   
               
               
                 NHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR 
               
               
                   
               
               
                 Petromyzon marinus (Lamprey) CDA1 (pmCDA1): 
               
               
                 (SEQ ID NO: 609) 
               
               
                 MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFW 
               
               
                   
               
               
                 GYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADC 
               
               
                   
               
               
                 AEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNV 
               
               
                   
               
               
                 MVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKIL 
               
               
                   
               
               
                 HTTKSPAV 
               
               
                   
               
               
                 Human APOBEC3G (hAPOBEC3G): 
               
               
                 (SEQ ID NO: 610) 
               
               
                 MELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLA 
               
               
                   
               
               
                 EDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQH 
               
               
                   
               
               
                 CWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNE 
               
               
                   
               
               
                 PWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAE 
               
               
                   
               
               
                 LCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCI 
               
               
                   
               
               
                 FTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQ 
               
               
                   
               
               
                 PWDGLDEHSQDLSGRLRAILQNQEN 
               
            
           
         
       
     
     Deaminase Activity on ssDNA. A USER (Uracil-Specific Excision Reagent) Enzyme-based assay for deamination was employed to test the activity of various deaminases on single-stranded DNA (ssDNA) substrates. USER Enzyme was obtained from New England Biolabs. An ssDNA substrate was provided with a target cytosine residue at different positions. Deamination of the ssDNA cytosine target residue results in conversion of the target cytosine to a uracil. The USER Enzyme excises the uracil base and cleaves the ssDNA backbone at that position, cutting the ssDNA substrate into two shorter fragments of DNA. In some assays, the ssDNA substrate is labeled on one end with a dye, e.g., with a 5′ Cy3 label (the * in the scheme below). Upon deamination, excision, and cleavage of the strand, the substrate can be subjected to electrophoresis, and the substrate and any fragment released from it can be visualized by detecting the label. Where Cy5 is images, only the fragment with the label will be visible via imaging. 
     In one USER Enzyme assay, ssDNA substrates were used that matched the target sequences of the various deaminases tested. Expression cassettes encoding the deaminases tested were inserted into a CMV backbone plasmid that has been used previously in the lab (Addgene plasmid 52970). The deaminase proteins were expressed using a TNT Quick Coupled Transcription/Translation System (Promega) according to the manufacturers recommendations. After 90 min of incubation, 5 mL of lysate was incubated with 5′ Cy3-labeled ssDNA substrate and 1 unit of USER Enzyme (NEB) for 3 hours. The DNA was resolved on a 10% TBE PAGE gel and the DNA was imaged using Cy-dye imaging. A schematic representation of the USER Enzyme assay is shown in  FIG. 41 . 
       FIG. 1  shows the deaminase activity of the tested deaminases on ssDNA substrates, such as Doench 1, Doench 2, G7′ and VEGF Target 2. The rAPOBEC1 enzyme exhibited a substantial amount of deamination on the single-stranded DNA substrate with a canonical NGG PAM, but not with a negative control non-canonical NNN PAM. Cas9 fusion proteins with APOBEC family deaminases were generated. The following fusion architectures were constructed and tested on ssDNA: 
     
       
         
           
               
               
            
               
                   rAPOBEC1 -GGS- dCas9  primary sequence 
                   
               
               
                 (SEQ ID NO: 611) 
                   
               
               
                 
                   MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNT 
                 
                   
               
               
                   
               
               
                 
                   NKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIAR 
                 
               
               
                   
               
               
                 
                   LYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLW 
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                   rAPOBEC1 -(GGS) 3 - dCas9  primary sequence 
               
               
                 (SEQ ID NO: 612) 
                   
               
               
                 
                   MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNT 
                 
                   
               
               
                   
               
               
                 
                   NKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIAR 
                 
               
               
                   
               
               
                 
                   LYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLW 
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 (SEQ ID NO: 613) 
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   VDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFI 
                 
               
               
                   
               
               
                 
                   EKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPR 
                 
               
               
                   
               
               
                 
                   NRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLEL 
                 
               
               
                   
               
               
                 
                   YCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK 
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 (SEQ ID NO: 614) 
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   ETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKH 
                 
               
               
                   
               
               
                 
                   VEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYH 
                 
               
               
                   
               
               
                 
                   HADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRL 
                 
               
               
                   
               
               
                 
                   YVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK 
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 (SEQ ID NO: 615) 
               
               
                 
                   MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNT 
                 
               
               
                   
               
               
                 
                   NKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIAR 
                 
               
               
                   
               
               
                 
                   LYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLW 
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
            
           
         
       
     
       FIG. 2  shows that the N-terminal deaminase fusions showed significant activity on the single stranded DNA substrates. For this reason, only the N-terminal architecture was chosen for further experiments. 
       FIG. 3  illustrates double stranded DNA substrate binding by deaminase-dCas9:sgRNA complexes. A number of double stranded deaminase substrate sequences were generated. The sequences are provided below. The structures according to  FIG. 3  are identified in these sequences (36 bp: underlined, sgRNA target sequence: bold; PAM: boxed; 21 bp: italicized). All substrates were labeled with a 5′-Cy3 label: 
                        (SEQ ID NO: 616)           2: GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGTC   CCGCGGATTTATTTATTT                                               (SEQ ID NO: 617)           3: GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCT   TCCGCGGATTTATTTATT                                               (SEQ ID NO: 618)           4: GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC   TTCCGCGGATTTATTTAT                                               (SEQ ID NO: 619)           5: GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC   ATTCCGCGGATTTATTTA                                               (SEQ ID NO: 620)           6: GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC   TATTCCGCGGATTTATTT                                               (SEQ ID NO: 621)           7: GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC   TTATTCCGCGGATTTATT                                               (SEQ ID NO: 622)           8: GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC   ATTATTCCGCGGATTTAT                                               (SEQ ID NO: 623)           9: GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC   TATTATTCCGCGGATTTA                                               (SEQ ID NO: 624)           10: GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC   ATTATATTCCGCGGATTT                                               (SEQ ID NO: 625)           11: GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC   TATTATATTCCGCGGATT                                               (SEQ ID NO: 626)           12: GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC   TTATTATATTCCGCGGAT                                               (SEQ ID NO: 627)           13: GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC   ATTATTATATTCCGCGGA                                               (SEQ ID NO: 628)           14: GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC   TATTATTATATTCCGCGG                                               (SEQ ID NO: 629)           15: GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC   ATTATTATTATTACCGCG                                               (SEQ ID NO: 630)           18: GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC   ATTATTATTATTATTACC                                               “-”:       (SEQ ID NO: 631)             GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGTA   ATATTAATTTATTTATTTAA                                               (SEQ ID NO: 632)           8U: GTAGGTAGTTAGGATGAATGGAAGGTTGGTGTAG   ATTATTATCUGCGGATTTA                                            
*In all substrates except for “8U”, the top strand in  FIG. 3  is the complement of the sequence specified here. In the case of “8U”, there is a “G” opposite the U.
 
       FIG. 4  shows the results of a double stranded DNA Deamination Assay. The fusions were expressed and purified with an N-terminal His6 tag via both Ni-NTA and sepharose chromatography. In order to assess deamination on dsDNA substrates, the various dsDNA substrates shown on the previous slide were incubated at a 1:8 dsDNA:fusion protein ratio and incubated at 37° C. for 2 hours. Once the dCas9 portion of the fusion binds to the DNA it blocks access of the USER enzyme to the DNA. Therefore, the fusion proteins were denatured following the incubation and the dsDNA was purified on a spin column, followed by incubation for 45 min with the USER Enzyme and resolution of the resulting DNA substrate and substrate fragments on a 10% TBE-urea gel. 
       FIG. 5  demonstrates that Cas9 fusions can target positions 3-11 of double-stranded DNA target sequences (numbered according to the schematic in  FIG. 3 ). Upper Gel: 1 μM rAPOBEC1-GGS-dCas9, 125 nM dsDNA, 1 eq sgRNA. Mid Gel: 1 μM rAPOBEC1-(GGS) 3 -dCas9, 125 nM dsDNA, 1 eq sgRNA. Lower Gel: 1.85 μM rAPOBEC1-XTEN-dCas9, 125 nM dsDNA, 1 eq sgRNA. Based on the data from these gels, positions 3-11 (according to the numbering in  FIG. 3 ) are sufficiently exposed to the activity of the deaminase to be targeted by the fusion proteins tested. Access of the deaminase to other positions is most likely blocked by the dCas9 protein. 
     The data further indicates that a linker of only 3 amino acids (GGS) is not optimal for allowing the deaminase to access the single stranded portion of the DNA. The 9 amino acid linker [(GGS) 3 ] (SEQ ID NO: 596) and the more structured 16 amino acid linker (XTEN) allow for more efficient deamination. 
       FIG. 6  demonstrates that the correct guide RNA, e.g., the correct sgRNA, is required for deaminase activity. The gel shows that fusing the deaminase to dCas9, the deaminase enzyme becomes sequence specific (e.g., using the fusion with an eGFP sgRNA results in no deamination), and also confers the capacity to the deaminase to deaminate dsDNA. The native substrate of the deaminase enzyme is ssDNA, and no deamination occurred when no sgRNA was added. This is consistent with reported knowledge that APOBEC deaminase by itself does not deaminate dsDNA. The data indicates that Cas9 opens the double-stranded DNA helix within a short window, exposing single-stranded DNA that is then accessible to the APOBEC deaminase for cytidine deamination. The sgRNA sequences used are provided below. sequences (36 bp: underlined, sgRNA target sequence: bold; PAM: boxed; 21 bp: italicized) 
     DNA Sequence 8: 
       
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 633) 
                   
               
               
                 5′-Cy3- 
                   
               
               
                 
                   GTAGGTAGTTAGGATGAATGGAAGGTTGGTATAGCC 
                   ATTATTCCGCGGATTTATT 
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 Correct sgRNA sequence (partial 3′ sequence): 
               
               
                 (SEQ ID NO: 634) 
                   
               
               
                 5′- AUUAUUCCGCGGAUUUAUUU GUUUUAGAGCUAG...-3′ 
                   
               
               
                   
               
               
                 eGFP sgRNA sequence (partial 3′-sequence): 
               
               
                 (SEQ ID NO: 635) 
                   
               
               
                 5′- CGUAGGCCAGGGUGGUCACG GUUUUAGAGCUAG...-3′ 
                   
               
            
           
         
       
     
     Example 2: Deamination of DNA Target Sequence 
     Exemplary deamination targets. The dCas9:deaminase fusion proteins described herein can be delivered to a cell in vitro or ex vivo or to a subject in vivo and can be used to effect C to T or G to A transitions when the target nucleotide is in positions 3-11 with respect to a PAM. Exemplary deamination targets include, without limitation, the following: CCR5 truncations: any of the codons encoding Q93, Q102, Q186, R225, W86, or Q261 of CCR5 can be deaminated to generate a STOP codon, which results in a nonfunctional truncation of CCR5 with applications in HIV treatment. APOE4 mutations: mutant codons encoding C11R and C57R mutant APOE4 proteins can be deaminated to revert to the wild-type amino acid with applications in Alzheimer&#39;s treatment. eGFP truncations: any of the codons encoding Q158, Q184, Q185 can be deaminated to generate a STOP codon, or the codon encoding M1 can be deaminated to encode I, all of which result in loss of eGFP fluorescence, with applications in reporter systems. eGFP restoration: a mutant codon encoding T65A or Y66C mutant GFP, which does not exhibit substantial fluorescence, can be deaminated to restore the wild-type amino acid and confer fluorescence. PIK3CA mutation: a mutant codon encoding K111E mutant PIK3CA can be deaminated to restore the wild-type amino acid residue with applications in cancer. CTNNB1 mutation: a mutant codon encoding T41A mutant CTNNB1 can be deaminated to restore the wild-type amino acid residue with applications in cancer. HRAS mutation: a mutant codon encoding Q61R mutant HRAS can be deaminated to restore the wild-type amino acid residue with applications in cancer. P53 mutations: any of the mutant codons encoding Y163C, Y236C, or N239D mutant p53 can be deaminated to encode the wild type amino acid sequence with applications in cancer. The feasibility of deaminating these target sequences in double-stranded DNA is demonstrated in  FIGS. 7 and 8 .  FIG. 7  illustrates the mechanism of target DNA binding of in vivo target sequences by deaminase-dCas9:sgRNA complexes. 
       FIG. 8  shows successful deamination of exemplary disease-associated target sequences. Upper Gel: CCR5 Q93: coding strand target in pos. 10 (potential off-targets at positions 2, 5, 6, 8, 9); CCR5 Q102: coding strand target in pos. 9 (potential off-targets at positions 1, 12, 14); CCR5 Q186: coding strand target in pos. 9 (potential off-targets at positions 1, 5, 15); CCR5 R225: coding strand target in pos. 6 (no potential off-targets); eGFP Q158: coding strand target in pos. 5 (potential off-targets at positions 1, 13, 16); eGFP Q184/185: coding strand target in pos. 4 and 7 (potential off-targets at positions 3, 12, 14, 15, 16, 17, 18); eGFP M1: template strand target in pos. 12 (potential off-targets at positions 2, 3, 7, 9, 11) (targets positions 7 and 9 to small degree); eGFP T65A: template strand target in pos. 7 (potential off-targets at positions 1, 8, 17); PIK3CA K111E: template strand target in pos. 2 (potential off-targets at positions 5, 8, 10, 16, 17); PIK3CA K111E: template strand target in pos. 13 (potential off-targets at positions 11, 16, 19) X. Lower Gel: CCR5 W86: template strand target in pos. 2 and 3 (potential off-targets at positions 1, 13) X; APOE4 C 11 R: coding strand target in pos. 11 (potential off-targets at positions 7, 13, 16, 17); APOE4 C 57 R: coding strand target in pos. 5) (potential off-targets at positions 7, 8, 12); eGFP Y66C: template strand target in pos. 11 (potential off-targets at positions 1, 4,6, 8, 9, 16); eGFP Y66C: template strand target in pos. 3 (potential off-targets at positions 1, 8, 17); CCR5 Q261: coding strand target in pos. 10 (potential off-targets at positions 3, 5, 6, 9, 18); CTNNB1 T41A: template strand target in pos. 7 (potential off-targets at positions 1, 13, 15, 16) X; HRAS Q61R: template strand target in pos. 6 (potential off-targets at positions 1, 2, 4, 5, 9, 10, 13); p53 Y163C: template strand target in pos. 6 (potential off-targets at positions 2, 13, 14); p53 Y236C: template strand target in pos. 8 (potential off-targets at positions 2, 4); p53 N239D: template strand target in pos. 4 (potential off-targets at positions 6, 8). Exemplary DNA sequences of disease targets are provided below (PAMs (5′-NGG-3′) and target positions are boxed): 
     
       
         
           
               
            
               
                 (SEQ ID NO: 636) 
               
               
                 CCR5 Q93: 5′-Cy3- 
               
               
                 
                   GTAGGTAGTTAGGATGAATGGAAGGTTGGTAACTAT 
                   GCTGCCGCC 
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 637) 
               
               
                 CCR5 Q102: 5′-Cy3- 
               
               
                 
                   GTAGGTAGTTAGGATGAATGGAAGGTTGGTAAAATA 
                   CAATGTGT 
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 638) 
               
               
                 CCR5 Q186: 5&#39;-Cy3- 
               
               
                 
                   GTAGGTAGTTAGGATGAATGGAAGGTTGGTATTTTC 
                   CATACAGT 
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 639) 
               
               
                 CCR5 R225: 5′-Cy3- 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 640) 
               
               
                 CCR5 W86: 5′-Cy3- 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 641) 
               
               
                 CCR5 Q261: 5′-Cy3- 
               
               
                 
                   GTAGGTAGTTAGGATGAATGGAAGGTTGGTATCCTG 
                   AACACCTT 
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 642) 
               
               
                 APOE4 C11R: 5′-Cy3- 
               
               
                 
                   GTAGGTAGTTAGGATGAATGGAAGGTTGGTAGACAT 
                   GGAGGAC 
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 643) 
               
               
                 APOE4 C57R: 5′-Cy3- 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 644) 
               
               
                 eGFP Q158: 5′-Cy3- 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 645) 
               
               
                 eGFP Q184/185: 5′-Cy3- 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 646) 
               
               
                 eGFP M1: 5′-Cy3- 
               
               
                 
                   GTAGGTAGTTAGGATGAATGGAAGGTTGGTACCTCG 
                   CCCTTGCTCA 
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 647) 
               
               
                 eGFP T65A: 5′-Cy3- 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 648) 
               
               
                 eGFP Y66C: 5&#39;-Cy3- 
               
               
                 
                   GTAGGTAGTTAGGATGAATGGAAGGTTGGTAAAGCA 
                   CTGCACTC 
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 649) 
               
               
                 eGFP Y66C: 5′-Cy3- 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 650) 
               
               
                 P1K3CA K111E: 5′-Cy3- 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 651) 
               
               
                 P1K3CA K111E: 5′-Cy3- 
               
               
                 
                   GTAGGTAGTTAGGATGAATGGAAGGTTGGTATTCTC 
                   GATTG 
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 652) 
               
               
                 CTNNB1 T41A: 5′-Cy3- 
               
               
                 
                   GTAGGTAGTTAGGATGAATGGAAGGTTGGTAAGGAG 
                   CTGTGG 
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 653) 
               
               
                 HRAS Q61R: 5′-Cy3- 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 654) 
               
               
                 p53 Y163C: 5′-Cy3- 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 655) 
               
               
                 p53 Y236C: 5′-Cy3- 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 (SEQ ID NO: 656) 
               
               
                 p53 N239D: 5′-Cy3- 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
            
           
         
       
     
     Example 3: Uracil Glycosylase Inhibitor Fusion Improves Deamination Efficiency 
     Direct programmable nucleobase editing efficiencies in mammalian cells by dCas9:deaminase fusion proteins can be improved significantly by fusing a uracil glycosylase inhibitor (UGI) to the dCas9:deaminase fusion protein. 
       FIG. 9  shows in vitro C→T editing efficiencies in human HEK293 cells using rAPOBEC1-XTEN-dCas9: 
                                                      (SEQ ID NO: 657)             MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNT                       NKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIAR                   LYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWV                   RLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK   SGSETP                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   KV              
Protospacer sequences were as follows:
 
                                EMX1:                         (SEQ ID NO: 293)                   FANCF:                         (SEQ ID NO: 294)               HEK293 site 2:                         (SEQ ID NO: 295)               HEK293 site 3:                         (SEQ ID NO: 296)               HEK293 site 4:                         (SEQ ID NO: 297)               RNF2:                         (SEQ ID NO: 298)            
*PAMs are boxed, residues within target window (positions 3-11) are numbered and bolded.
 
       FIG. 10  demonstrates that C→T editing efficiencies on the same protospacer sequences in HEK293T cells are greatly enhanced when a UGI domain is fused to the rAPOBEC1:dCas9 fusion protein. 
     
       
         
           
               
               
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                 (SEQ ID NO: 658) 
                   
               
               
                 
                   MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNT 
                 
                   
               
               
                   
               
               
                 
                   NKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIAR 
                 
               
               
                   
               
               
                 
                   LYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWV 
                 
               
               
                   
               
               
                 
                   RLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK 
                   SGSETP 
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     IIEKETGKOLVICIESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAP 
                   
                 
               
               
                   
               
               
                 
                   
                     EYKPWALVICIDSNGENKIKML 
                   
                   SGGSPKKKRKV 
                 
               
            
           
         
       
     
     The percentages in  FIGS. 9 and 10  are shown from sequencing both strands of the target sequence. Because only one of the strands is a substrate for deamination, the maximum possible deamination value in this assay is 50%. Accordingly, the deamination efficiency is double the percentages shown in the tables. E.g., a value of 50% relates to deamination of 100% of double-stranded target sequences. When a uracil glycosylase inhibitor (UGI) was fused to the dCas9:deaminase fusion protein (e.g., rAPOBEC1-XTEN-dCas9-[UGI]-NLS), a significant increase in editing efficiency in cells was observed. This result indicates that in mammalian cells, the DNA repair machinery that cuts out the uracil base in a U:G base pair is a rate-limiting process in DNA editing. Tethering UGI to the dVas9:deaminase fusion proteins greatly increases editing yields. 
     Without UGI, typical editing efficiencies in human cells were in the ˜2-14% yield range ( FIG. 9  and  FIG. 10 , “XTEN” entries). With UGI ( FIG. 10 , “UGI” entries) the editing was observed in the ˜6-40% range. Using a UGI fusion is thus more efficient than the current alternative method of correcting point mutations via HDR, which also creates an excess of indels in addition to correcting the point mutation. No indels resulting from treatment with the cas9:deaminase:UGI fusions were observed. 
     Example 4: Direct, Programmable Conversion of a Target Nucleotide in Genomic DNA without Double-Stranded DNA Cleavage 
     Current genome-editing technologies introduce double-stranded DNA breaks at a target locus of interest as the first step to gene correction. 39,40  Although most genetic diseases arise from mutation of a single nucleobase to a different nucleobase, current approaches to revert such changes are very inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus as a consequence of the cellular response to double-stranded DNA breaks. 39,40  Reported herein is the development of nucleobase editing, a new strategy for genome editing that enables the direct conversion of one target nucleobase into another in a programmable manner, without requiring double-stranded DNA backbone cleavage. Fusions of CRISPR/Cas9 were engineered and the cytidine deaminase enzyme APOBEC1 that retain the ability to be programmed with a guide RNA, do not induce double-stranded DNA breaks, and mediate the direct conversion of cytidine to uracil, thereby effecting a C→T (or G→A) substitution following DNA replication, DNA repair, or transcription if the template strand is targeted. The resulting “nucleobase editors” convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease in vitro. In four transformed human and murine cell lines, second- and third-generation nucleobase editors that fuse uracil glycosylase inhibitor (UGI), and that use a Cas9 nickase targeting the non-edited strand, respectively, can overcome the cellular DNA repair response to nucleobase editing, resulting in permanent correction of up to 37% or (˜15-75%) of total cellular DNA in human cells with minimal (typically ≤1%) indel formation. In contrast, canonical Cas9-mediated HDR on the same targets yielded an average of 0.7% correction with 4% indel formation. Nucleobase editors were used to revert two oncogenic p53 mutations into wild-type alleles in human breast cancer and lymphoma cells, and to convert an Alzheimer&#39;s Disease associated Arg codon in ApoE4 into a non-disease-associated Cys codon in mouse astrocytes. Base editing expands the scope and efficiency of genome editing of point mutations. 
     The clustered regularly interspaced short palindromic repeat (CRISPR) system is a prokaryotic adaptive immune system that has been adapted to mediate genome engineering in a variety of organisms and cell lines. 41  CRISPR/Cas9 protein-RNA complexes localize to a target DNA sequence through base pairing with a guide RNA, and natively create a DNA double-stranded break (DSB) at the locus specified by the guide RNA. In response to DSBs, endogenous DNA repair processes mostly result in random insertions or deletions (indels) at the site of DNA cleavage through non-homologous end joining (NHEJ). In the presence of a homologous DNA template, the DNA surrounding the cleavage site can be replaced through homology-directed repair (HDR). When simple disruption of a disease-associated gene is sufficient (for example, to treat some gain-of-function diseases), targeted DNA cleavage followed by indel formation can be effective. For most known genetic diseases, however, correction of a point mutation in the target locus, rather than stochastic disruption of the gene, is needed to address or study the underlying cause of the disease. 68    
     Motivated by this need, researchers have invested intense effort to increase the efficiency of HDR and suppress NHEJ. For example, a small-molecule inhibitor of ligase IV, an essential enzyme in the NHEJ pathway, has been shown to increase HDR efficiency. 42,43  However, this strategy is challenging in post-mitotic cells, which typically down-regulate HDR, and its therapeutic relevance is limited by the potential risks of inhibiting ligase IV in non-target cells. Enhanced HDR efficiency can also be achieved by the timed delivery of Cas9-guide RNA complexes into chemically synchronized cells, as HDR efficiency is highly cell-cycle dependent. 44  Such an approach, however, is limited to research applications in cell culture since synchronizing cells is highly disruptive. Despite these developments, current strategies to replace point mutations using HDR in most contexts are very inefficient (typically ˜0.1 to 5%), 42,43,45,46,75  especially in unmodified, non-dividing cells. In addition, HDR competes with NHEJ during the resolution of double-stranded breaks, and indels are generally more abundant outcomes than gene replacement. These observations highlight the need to develop alternative approaches to install specific modifications in genomic DNA that do not rely on creating double-stranded DNA breaks. A small-molecule inhibitor of ligase IV, an essential enzyme in the NHEJ pathway, has been shown to increase HDR efficiency. 42,43  However, this strategy is challenging in post-mitotic cells, which typically down-regulate HDR, and its therapeutic relevance is limited by the potential risks of inhibiting ligase IV in non-target cells. Enhanced HDR efficiency can also be achieved by the timed delivery of Cas9-guide RNA complexes into chemically synchronized cells, as HDR efficiency is highly cell-cycle dependent. 44  Such an approach, however, is limited to research applications in cell culture since synchronizing cells is highly disruptive. In some cases, it is possible to design HDR templates such that the product of successful HDR contains mutations in the PAM sequence and therefore is no longer a substrate for subsequent Cas9 modification, increasing the overall yield of HDR products, 75  although such an approach imposes constraints on the product sequences. Recently, this strategy has been coupled to the use of ssDNA donors that are complementary to the non-target strand and high-efficiency ribonucleoprotein (RNP) delivery to substantially increase the efficiency of HDR, but even in these cases the ratio of HDR to NHEJ outcomes is relatively low (&lt;2). 83    
     It was envisioned that direct catalysis of the conversion of one nucleobase to another at a programmable target locus without requiring DNA backbone cleavage could increase the efficiency of gene correction relative to HDR without introducing undesired random indels at the locus of interest. Catalytically dead Cas9 (dCas9), which contains Asp10Ala and His840Ala mutations that inactivate its nuclease activity, retains its ability to bind DNA in a guide RNA-programmed manner but does not cleave the DNA backbone. 16,47  In principle, conjugation of dCas9 with an enzymatic or chemical catalyst that mediates the direct conversion of one nucleobase to another could enable RNA-programmed nucleobase editing. The deamination of cytosine (C) is catalyzed by cytidine deaminases 29  and results in uracil (U), which has the base pairing properties of thymine (T). dCas9 was fused to cytidine deaminase enzymes in order to test their ability to convert C to U at a guide RNA-specified DNA locus. Most known cytidine deaminases operate on RNA, and the few examples that are known to accept DNA require single-stranded DNA. 48  Recent studies on the dCas9-target DNA complex reveal that at least nine nucleotides of the displaced DNA strand are unpaired upon formation of the Cas9:guide RNA:DNA “R-loop” complex. 12  Indeed, in the structure of the Cas9 R-loop complex the first 11 nucleotides of the protospacer on the displaced DNA strand are disordered, suggesting that their movement is not highly restricted. 76  It has also been speculated that Cas9 nickase-induced mutations at cytosines in the non-template strand might arise from their accessibility by cellular cytidine deaminase enzymes. 77  Recent studies on the dCas9-target DNA complex have revealed that at least 26 bases on the non-template strand are unpaired when Cas9 binds to its target DNA sequence. 49  It was reasoned that a subset of this stretch of single-stranded DNA in the R-loop might serve as a substrate for a dCas9-tethered cytidine deaminase to effect direct, programmable conversion of C to U in DNA ( FIG. 11A ). 
     Four different cytidine deaminase enzymes (hAID, hAPOBEC3G, rAPOBEC1, and pmCDA1) were expressed in a mammalian cell lysate-derived in vitro transcription-translation system and evaluated for ssDNA deamination. Of the four enzymes, rAPOBEC1 showed the highest deaminase activity under the tested conditions and was chosen for dCas9 fusion experiments ( FIG. 36A ). Although appending rAPOBEC1 to the C-terminus of dCas9 abolishes deaminase activity, fusion to the N-terminus of dCas9 preserves deaminase activity on ssDNA at a level comparable to that of the unfused enzyme. Four rAPOBEC1-dCas9 fusions were expressed and purified with linkers of different length and composition ( FIG. 36B ), and evaluated each fusion for single guide RNA (sgRNA)-programmed dsDNA deamination in vitro ( FIGS. 11A to 11C  and  FIGS. 15A to 15D ). Efficient, sequence-specific, sgRNA-dependent C to U conversion was observed in vitro ( FIGS. 11A to 11C ). Conversion efficiency was greatest using rAPOBEC1-dCas9 linkers over nine amino acids in length. The number of positions susceptible to deamination (the deamination “activity window”) increases with linker length was extended from three to 21 amino acids ( FIGS. 36C  to  36 F 15 A to  15 D). The 16-residue XTEN linker 50  was found to offer a promising balance between these two characteristics, with an efficient deamination window of approximately five nucleotides, from positions 4 to 8 within the protospacer, counting the end distal to the protospacer-adjacent motif (PAM) as position 1. The rAPOBEC1-XTEN-dCas9 protein served as the first-generation nucleobase editor (NBE1). 
     Elected were seven mutations relevant to human disease that in theory could be corrected by C to T nucleobase editing, synthesized double-stranded DNA 80-mers of the corresponding sequences, and assessed the ability of NBE1 to correct these mutations in vitro ( FIGS. 16A to 16B ). NBE1 yielded products consistent with efficient editing of the target C, or of at least one C within the activity window when multiple Cs were present, in six of these seven targets in vitro, with an average apparent editing efficiency of 44% ( FIGS. 16A to 16B ). In the three cases in which multiple Cs were present within the deamination window, evidence of deamination of some or all of these cytosines was observed. In only one of the seven cases tested were substantial yields of edited product observed ( FIGS. 16A to 16B ). Although the preferred sequence context for APOBEC1 substrates is reported to be CC or TC, 51  it was anticipated that the increased effective molarity of the deaminase and its single-stranded DNA substrate mediated by dCas9 binding to the target locus may relax this restriction. To illuminate the sequence context generality of NBE1, its ability to edit a 60-mer double-stranded DNA oligonucleotide containing a single fixed C at position 7 within the protospacer was assayed, as well as all 36 singly mutated variants in which protospacer bases 1-6 and 8-13 were individually varied to each of the other three bases. Each of these 37 sequences were treated with 1.9 μM NBE1, 1.9 μM of the corresponding sgRNA, and 125 nM DNA for 2 h, similar to standard conditions for in vitro Cas9 assays 52 . High-throughput DNA sequencing (HTS) revealed 50 to 80% C to U conversion of targeted strands (25 to 40% of total sequence reads arising from both DNA strands, one of which is not a substrate for NBE1) ( FIG. 12A ). The nucleotides surrounding the target C had little effect on editing efficiency was independent of sequence context unless the base immediately 5′ of the target C is a G, in which case editing efficiency was substantially lower ( FIGS. 12A to 12B ). NBE1 activity in vitro was assessed on all four NC motifs at positions 1 through 8 within the protospacer ( FIGS. 12A to 12B ). In general NBE1 activity on substrates was observed to follow the order TC≥CC≥AC&gt;GC, with maximum editing efficiency achieved when the target C is at or near position 7. In addition, it was observed that the nucleobase editor is highly processive, and will efficiently convert most of all Cs to Us on the same DNA strand within the 5-base activity window ( FIG. 17 ). 
     While BE1 efficiently processes substrates in a test tube, in cells a tree of possible DNA repair outcomes determines the fate of the initial U:G product of base editing ( FIG. 29A ). To test the effectiveness of nucleobase editing in human cells, NBE1 codon usage was optimized for mammalian expression, appended a C-terminal nuclear localization sequence (NLS), 53  and assayed its ability to convert C to T in human cells on 14Cs in six well-studied target sites throughout the human genome ( FIG. 37A ). 54  The editable Cs were confirmed within each protospacer in vitro by incubating NBE1 with synthetic 80-mers that correspond to the six different genomic sites, followed by HTS ( FIGS. 13A to 13C ,  FIG. 29B  and  FIG. 25 ). Next, HEK293T cells were transfected with plasmids encoding NBE1 and one of the six target sgRNAs, allowed three days for nucleobase editing to occur, extracted genomic DNA from the cells, and analyzed the loci by HTS. Although C to T editing in cells at the target locus was observed for all six cases, the efficiency of nucleobase editing was 1.1% to 6.3% or 0.8%-7.7% of total DNA sequences (corresponding to 2.2% to 12.6% of targeted strands), a 6.3-fold to 37-fold or 5-fold to 36-fold decrease in efficiency compared to that of in vitro nucleobase editing ( FIGS. 13A to 13C ,  FIG. 29B  and  FIG. 25 ). It was observed that some base editing outside of the typical window of positions 4 to 8 when the substrate C is preceded by a T, which we attribute to the unusually high activity of APOBEC1 for TC substrates. 48    
     It was asked whether the cellular DNA repair response to the presence of U:G heteroduplex DNA was responsible for the large decrease in nucleobase editing efficiency in cells ( FIG. 29A ). Uracil DNA glycosylase (UDG) catalyzes removal of U from DNA in cells and initiates base excision repair (BER), with reversion of the U:G pair to a C:G pair as the most common outcome ( FIG. 29A ). 5  Uracil DNA glycosylase inhibitor (UGI), an 83-residue protein from  B. subtilis  bacteriophage PBS1, potently blocks human UDG activity (IC 50 =12 μM). 56  UGI was fused to the C-terminus of NBE1 to create the second-generation nucleobase editor NBE2 and repeated editing assays on all six genomic loci. Editing efficiencies in human cells were on average 3-fold higher with NBE2 than with NBE1, resulting in gene conversion efficiencies of up to 22.8% of total DNA sequenced (up to 45.6% of targeted strands) ( FIGS. 13A to 13C  and  FIG. 29B ). To test base editing in human cells, BE1 codon usage was optimized for mammalian expression and appended a C-terminal nuclear localization sequence (NLS). 53    
     Similar editing efficiencies were observed when a separate plasmid overexpressing UGI was co-transfected with NBE1 ( FIGS. 18A to 18H ). However, while the direct fusion of UGI to NBE1 resulted in no significant increase in C to T mutations at monitored non-targeted genomic locations, overexpression of unfused UGI detectably increased the frequency of C to T mutations elsewhere in the genome ( FIGS. 18A to 18H ). The generality of NBE2-mediated nucleobase editing was confirmed by assessing editing efficiencies on the same six genomic targets in U2OS cells, and observed similar results with those in HEK293T cells ( FIG. 19 ). Importantly, NBE2 typically did not result in any detectable indels ( FIG. 13C  and  FIG. 29C ), consistent with the known mechanistic dependence of NHEJ on double-stranded DNA breaks. 57,78  Together, these results indicate that conjugating UGI to NBE1 can greatly increase the efficiency of nucleobase editing in human cells. 
     The permanence of nucleobase editing in human cells was confirmed by monitoring editing efficiencies over multiple cell divisions in HEK293T cells at two of the tested genomic loci. Genomic DNA was harvested at two time points: three days after transfection with plasmids expressing NBE2 and appropriate sgRNAs, and after passaging the cells and growing them for four additional days (approximately five subsequent cell divisions). No significant change in editing efficiency was observed between the non-passaged cells (editing observed in 4.6% to 6.6% of targeted strands for three different target Cs) and passaged cells (editing observed in 4.6% to 6.4% of targeted strands for the same three target Cs), confirming that the nucleobase edits became permanent following cell division ( FIG. 20 ). Indels will on rare occasion arise from the processing of U:G lesions by cellular repair processes, which involve single-strand break intermediates that are known to lead to indels. 84  Given that several hundred endogenous U:G lesions are generated every day per human cell from spontaneous cytidine deaminase, 85  it was anticipate that the total indel frequency from U:G lesion repair is unlikely to increase from BE1 or BE2 activity at a single target locus. 
     To further increase the efficiency of nucleobase editing in cells, it was anticipated that nicking the non-edited strand may result in a smaller fraction of edited Us being removed by the cell, since eukaryotic mismatch repair machinery uses strand discontinuity to direct DNA repair to any broken strand of a mismatched duplex ( FIG. 29A ). 58,79,80  The catalytic His residue was restored at position 840 in the Cas9 HNH domain, 47,59  resulting in the third-generation nucleobase editor NBE3 that nicks the non-edited strand containing a G opposite the targeted C, but does not cleave the target strand containing the C. Because NBE3 still contains the Asp10Ala mutation in Cas9, it does not induce double-stranded DNA cleavage. This strategy of nicking the non-edited strand augmented nucleobase editing efficiency in human cells by an additional 1.4- to 4.8-fold relative to NBE2, resulting in up to 36.3% of total DNA sequences containing the targeted C to T conversion on the same six human genomic targets in HEK293T cells ( FIGS. 13A to 13C  and  FIG. 29B ). Importantly, only a small frequency of indels, averaging 0.8% (ranging from 0.2% to 1.6% for the six different loci), was observed from NBE3 treatment ( FIG. 13C ,  FIG. 29C , and  FIG. 34 ). In contrast, when cells were treated with wild-type Cas9, sgRNA, and a single-stranded DNA donor template to mediate HDR at three of these loci C to T conversion efficiencies averaging only 0.7% were observed, with much higher relative indel formation averaging 3.9% ( FIGS. 13A to 13C  and  FIG. 29C ). The ratio of allele conversion to NHEJ outcomes averaged &gt;1,000 for BE2, 23 for BE3, and 0.17 for wild-type Cas9 ( FIG. 3 c   ). We confirmed the permanence of base editing in human cells by monitoring editing efficiencies over multiple cell divisions in HEK293T cells at the HEK293 site 3 and 4 genomic loci ( FIG. 38 ). These results collectively establish that nucleobase editing can effect much more efficient targeted single-base editing in human cells than Cas9-mediated HDR, and with much less (NBE3) or no (NBE2) indel formation. 
     Next, the off-target activity of NBE1, NBE2, and NBE3 in human cells was evaluated. The off-target activities of Cas9, dCas9, and Cas9 nickase have been extensively studied ( FIGS. 23 to 24 and 31 to 33 ). 54,60-62  Because the sequence preference of rAPOBEC1 has been shown to be independent of DNA bases more than one base from the target C, 63  consistent with the sequence context independence observed in  FIGS. 12A to 12B , it was assumed that potential off-target activity of nucleobase editors arises from off-target Cas9 binding. Since only a fraction of Cas9 off-target sites will have a C within the active window for nucleobase editing, off-target nucleobase editing sites should be a subset of the off-target sites of canonical Cas9 variants. For each of the six sites studied, the top ten known Cas9 off-target loci in human cells that were previously determined using the GUIDE-seq method were sequenced ( FIGS. 23 to 27 and 31 to 33 ). 54, 61  Detectable off-target nucleobase editing at only a subset (16/34, 47% for NBE1 and NBE2, and 17/34, 50% for NBE3) of known dCas9 off-target loci was observed. In all cases, the off-target base-editing substrates contained a C within the five-base target window. In general, off-target C to T conversion paralleled off-target Cas9 nuclease-mediated genome modification frequencies ( FIGS. 23 to 27 ). Also monitored were C to T conversions at 2,500 distinct cytosines surrounding the six on-target and 34 off-target loci tested, representing a total of 14,700,000 sequence reads derived from approximately 1.8×10 6  cells, and observed no detectable increase in C to T conversions at any of these other sites upon NBE1, NBE2, or NBE3 treatment compared to that of untreated cells ( FIG. 28 ). Taken together, these findings suggest that off-target substrates of nucleobase editors include a subset of Cas9 off-target substrates, and that nucleobase editors in human cells do not induce untargeted C to T conversion throughout the genome at levels that can be detected by the methods used here. No substantial change was observed in editing efficiency between non-passaged HEK293T cells (editing observed in 1.8% to 2.6% of sequenced strands for the three target Cs with BE2, and 6.2% to 14.3% with BE3) and cells that had undergone approximately five cell divisions after base editing (editing observed in 1.9% to 2.3% of sequenced strands for the same target Cs with BE2, and 6.4% to 14.5% with BE3), confirming that base edits in these cells are durable (Extended Data  FIG. 6 ). 
     Finally, the potential of nucleobase editing to correct three disease-relevant mutations in mammalian cells was tested. The apolipoprotein E gene variant APOE4 encodes two Arg residues at amino acid positions 112 and 158, and is the largest and most common genetic risk factor for late-onset Alzheimer&#39;s disease. 64  ApoE variants with Cys residues in positions 112 or 158, including APOE2 (Cys112/Cys158), APOE3 (Cys112/Arg158), and APOE3′ (Arg112/Cys158) have been shown 65  or are presumed 81  to confer substantially lower Alzheimer&#39;s disease risk than APOE4. Encouraged by the ability of NBE1 to convert APOE4 to APOE3′ in vitro ( FIGS. 16A to 16B ), this conversion was attempted in immortalized mouse astrocytes in which the endogenous murine APOE gene has been replaced by human APOE4 (Taconic). DNA encoding NBE3 and an appropriate sgRNA was delivered into these astrocytes by nucleofection (nucleofection efficiency of 25%), extracted genomic DNA from all treated cells two days later, and measured editing efficiency by HTS. Conversion of Arg158 to Cys158 was observed in 58-75% of total DNA sequencing reads (44% of nucleofected astrocytes) ( FIGS. 14A to 14C  and  FIG. 30A ). Also observed was 36-50% editing of total DNA at the third position of codon 158 and 38-55% editing of total DNA at the first position of Leu159, as expected since all three of these Cs are within the active nucleobase editing window. However, neither of the other two C→T conversions results in a change in the amino acid sequence of the ApoE3′ protein since both TGC and TGT encode Cys, and both CTG and TTG encode Leu. From &gt;1,500,000 sequencing reads derived from 1×10 6  cells evidence of 1.7% indels at the targeted locus following NBE3 treatment was observed ( FIG. 35 ). In contrast, identical treatment of astrocytes with wt Cas9 and donor ssDNA resulted in 0.1-0.3% APOE4 correction and 26-40% indels at the targeted locus, efficiencies consistent with previous reports of single-base correction using Cas9 and HDR 45,75  ( FIG. 30A  and  FIG. 40A ). Astrocytes treated identically but with an sgRNA targeting the VEGFA locus displayed no evidence of APOE4 base editing ( FIG. 34  and  FIG. 40A ). These results demonstrate how nucleobase editors can effect precise, single-amino acid changes in the coding sequence of a protein as the major product of editing, even when their processivity results in more than one nucleotide change in genomic DNA. The off-target activities of Cas9, dCas9, and Cas9 nickase have been extensively studied. 54, 60-62  In general, off-target C to T conversions by BE1, BE2, and BE3 paralleled off-target Cas9 nuclease-mediated genome modification frequencies. 
     The dominant-negative p53 mutations Tyr163Cys and Asn239Asp are strongly associated with several types of cancer. 66-67  Both of these mutations can be corrected by a C to T conversion on the template strand ( FIGS. 16A to 16B ). A human breast cancer cell line homozygous for the p53 Tyr163Cys mutation (HCC1954 cells) was nucleofected with DNA encoding NBE3 and an sgRNA programmed to correct Tyr163Cys. Because the nucleofection efficiency of HCC1954 cells was &lt;10%, a plasmid expressing IRFP was co-nucleofected into these cells to enable isolation of nucleofected cells by fluorescence-activated cell sorting two days after treatment. HTS of genomic DNA revealed correction of the Tyr163Cys mutation in 7.6% of nucleofected HCC1954 cells ( FIG. 30B  and  FIG. 40A to 40B ). Also nucleofected was a human lymphoma cell line that is heterozygous for p53 Asn239Asp (ST486 cells) with DNA encoding NBE2 and an sgRNA programmed to correct Asn239Asp with 92% nucleofection efficiency). Correction of the Asn239Asp mutation was observed in 11% of treated ST486 cells (12% of nucleofected ST486 cells). Consistent with the findings in HEK cells, no indels were observed from the treatment of ST486 cells with NBE2, and 0.6% indel formation from the treatment of HCC1954 cells with NBE3. No other DNA changes within at least 50 base pairs of both sides of the protospacer were detected at frequencies above that of untreated controls out of &gt;2,000,000 sequencing reads derived from 2×10 5  cells ( FIGS. 14A to 14C ,  FIG. 30B  and Table 1). These results collectively represent the conversion of three disease-associated alleles in genomic DNA into their wild-type forms with an efficiency and lack of other genome modification events that is, to our knowledge, not currently achievable using other methods. 
     To illuminate the potential relevance of nucleobase editors to address human genetic diseases, the NCBI ClinVar database 68  was searched for known genetic diseases that could in principle be corrected by this approach. ClinVar was filtered by first examining only single nucleotide polymorphisms (SNPs), then removing any nonpathogenic variants. Out of the 24,670 pathogenic SNPs, 3,956 are caused by either a T to C, or an A to G, substitution. This list was further filtered to only include variants with a nearby NGG PAM that would position the SNP within the deamination activity window, resulting in 1,089 clinically relevant pathogenic gene variants that could in principle be corrected by the nucleobase editors described here ( FIG. 21  and Table 1). To illuminate the potential relevance of base editors to address human genetic diseases, the NCBI ClinVar database 68  was searched for known genetic diseases that could in principle be corrected by this approach. ClinVar was filtered by first examining only single nucleotide polymorphisms (SNPs), then removing any non-pathogenic variants. Out of the 24,670 pathogenic SNPs, 3,956 are caused by either a T to C, or an A to G, substitution. This list was further filtered to only include variants with a nearby NGG PAM that would position the SNP within the deamination activity window, resulting in 911 clinically relevant pathogenic gene variants that could in principle be corrected by the base editors described here. Of these, 284 contain only one C within the base editing activity window. A detailed list of these pathogenic mutations can be found in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 List of 911 base-editable gene variants associated with human disease 
               
               
                 with an NGG PAM (SEQ ID NOs: 747 to 1868 appear from top to bottom below, 
               
               
                 respectively). The “Y” in the protospacer and PAM sequences indicates the base to be edited, 
               
               
                 e.g., C. (SEQ ID NOs: 747 to 1868 appear from top to bottom below, respectively) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Protospacer 
                   
               
               
                 dbSNP # 
                 Genotype 
                 and PAM sequence(s) 
                 Associated genetic disease 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 755445790 
                 NM_000391.3(TPP1):c.887-10A&gt;G 
                 TTTYTTTTTTTTTTTTTTTGAGG 
                 Ceroid lipofuscinosis, neuronal, 2 
               
               
                   
               
               
                 113994167 
                 NM_000018.3(ACADVL):c.848T&gt;C 
                 TTTGYGGTGGAGAGGGGCTTCGG, 
                 Very long chain acyl-CoA 
               
               
                   
                 (p.Val283Ala) 
                 TTGYGGTGGAGAGGGGCTTCGGG 
                 dehydrogenase deficiency 
               
               
                   
               
               
                 119470018 
                 NM_024996.5(GFM1):c.521A&gt;G 
                 TTGYTAATAAAAGTTAGAAACGG 
                 Combined oxidative phosphorylation 
               
               
                   
                 (p.Asn174Ser) 
                   
                 deficiency 1 
               
               
                   
               
               
                 115650537 
                 NM_000426.3(LAMA2):c.8282T&gt;C 
                 TTGAYAGGGAGCAAGCAGTTCGG, 
                 Merosin deficient congenital 
               
               
                   
                 (p.Ile2761Thr) 
                 TGAYAGGGAGCAAGCAGTTCGGG 
                 muscular dystrophy 
               
               
                   
               
               
                 587777752 
                 NM_014946.3(SPAST):c.1688- 
                 TTCYGTAAAACATAAAAGTCAGG 
                 Spastic paraplegia 4, autosomal dominant 
               
               
                   
               
               
                 794726821 
                 NM_001165963.1(SCN1A):c.4055T&gt;C 
                 TTCYGGTTTGTCTTATATTCTGG 
                 Severe myoclonic epilepsy in infancy 
               
               
                   
                 (p.Leu1352Pro) 
                   
                   
               
               
                   
               
               
                 397514745 
                 NM_001130089.1(KARS):c.517T&gt;C 
                 CTTCYATGATCTTCGAGGAGAGG, 
                 Deafness, autosomal recessive 89 
               
               
                   
                 (p.Tyr173His) 
                 TTCYATGATCTTCGAGGAGAGG 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 376960358 
                 NM_001202.3(BMP4):c.362A&gt;G 
                 TTCGTGGYGGAAGCTCCTCACGG 
                 Microphthalmia syndromic 6 
               
               
                   
                 (p.His121Arg) 
                   
                   
               
               
                   
               
               
                 606231280 
                 NM_001287223.1(SCN11A):c.1142T&gt;C 
                 CTTCAYTGTGGTCATTTTCCTGG, 
                 Episodic pain syndrome, familial, 3 
               
               
                   
                 (p.Ile381Thr) 
                 TTCAYTGTGGTCATTTTCCTGG 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 387906735 
                 m.608A&gt;G 
                 TTCAGYGTATTGCTTTGAGGAGG 
                   
               
               
                   
               
               
                 199474663 
                 m.3260A&gt;G 
                 TTAAGTTYTATGCGATTACCGGG 
                 Cardiomyopathy with or without 
               
               
                   
                   
                   
                 skeletal myopathy 
               
               
                   
               
               
                 104894962 
                 NM_003413.3(ZIC3):c.1213A&gt;G 
                 TGTGTTYGCGCAGGGAGCTCGGG, 
                 Heterotaxy, visceral, X-linked 
               
               
                   
                 (p.Lys405Glu) 
                 ATGTGTTYGCGCAGGGAGCTCG 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 796053181 
                 NM_021007.2(SCN2A):c.1271T&gt;C 
                 TGTGGYGGCCATGGCCTATGAGG 
                 not provided 
               
               
                   
                 (p.Val424Ala) 
                   
                   
               
               
                   
               
               
                 267606788 
                 NM_000129.3(F13A1):c.728T&gt;C 
                 TGTGAYGGACAGAGCACAAATGG 
                 Factor xiii, a subunit, deficiency of 
               
               
                   
                 (p.Met243Thr) 
                   
                   
               
               
                   
               
               
                 397514503 
                 NM_003863.3(DPM2):c.68A&gt;G 
                 TGTAGYAGGTGAAGATGATCAGG 
                 Congenital disorder of glycosylation type 
               
               
                   
                 (p.Tyr23Cys) 
                   
                 1u 
               
               
                   
               
               
                 104893973 
                 NM_000416.2(IFNGR1):c.260T&gt;C 
                 TGTAATAYTTCTGATCATGTTGG 
                 Disseminated atypical mycobacterial 
               
               
                   
                 (p.Ile87Thr) 
                   
                 infection, Mycobacterium tuberculosis, 
               
               
                   
                   
                   
                 susceptibility to 
               
               
                   
               
               
                 121908466 
                 NM_005682.6(ADGRG1):c.263A&gt;G 
                 TGGYAGAGGCCCCTGGGGTCAGG 
                 Polymicrogyria, bilateral frontoparietal 
               
               
                   
                 (p.Tyr88Cys) 
                   
                   
               
               
                   
               
               
                 147952488 
                 NM_002437.4(MPV17):c.186+2T&gt;C 
                 TGGYAAGTTCTCCCCTCAACAGG 
                 Navajo neurohepatopathy 
               
               
                   
               
               
                 21909537 
                 NM_001145.4(ANG):c.121A&gt;G 
                 TGGTTYGGCATCATAGTGCTGGG, 
                 Amyotrophic lateral sclerosis type 9 
               
               
                   
                 (p.Lys41Glu) 
                 GTGGTTYGGCATCATAGTGCTG 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 121918489 
                 NM_000141.4(FGFR2):c.1018T&gt;C 
                 TGGGGAAYATACGTGCTTGGCGG, 
                 Crouzon syndrome 
               
               
                   
                 (p.Tyr340His) 
                 GGGGAAYATACGTGCTTGGCGGG 
                   
               
               
                   
               
               
                 121434463 
                 m.12320A&gt;G 
                 GAGTYGCACCAAAATTTTTGGGG, 
                 Mitochondrial myopathy 
               
               
                   
                   
                 GGAGTYGCACCAAAATTTTTGGG, 
                   
               
               
                   
                   
                 TGGAGTYGCACCAAAATTTTTG 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 121908046 
                 NM_000403.3(GALE):c.101A&gt;G 
                 TGGAAGYTATCGATGACCACAGG 
                 UDPglucose-4-epimerase deficiency 
               
               
                   
                 (p.Asn34Ser) 
                   
                   
               
               
                   
               
               
                 431905512 
                 NM_003764.3(STX11):c.173T&gt;C 
                 TGCYGGTGGCCGACGTGAAGCGG 
                 Hemophagocytic lymphohistiocytosis, 
               
               
                   
                 (p.Leu58Pro) 
                   
                 familial, 4 
               
               
                   
               
               
                 121917905 
                 NM_000124.3(ERCC6):c.2960T&gt;C 
                 TGCYAAAAGACCCAAAACAAAGG 
                 Cerebro-oculo-facio-skeletal syndrome 
               
               
                   
                 (p.Leu987Pro) 
                   
                   
               
               
                   
               
               
                 121918500 
                 NM_000141.4(FGFR2):c.874A&gt;G 
                 TGCTYGATCCACTGGATGTGGGG, 
                 Crouzon syndrome 
               
               
                   
                 (p.Lys292Glu) 
                 GTGCTYGATCCACTGGATGTGGG, 
                   
               
               
                   
                   
                 CGTGCTYGATCCACTGGATGTG 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 60431989 
                 NM_000053.3(ATP7B):c.3443T&gt;C 
                 TGCTGAYTGGAAACCGTGAGTGG 
                 Wilson disease 
               
               
                   
                 (p.Ile1148Thr) 
                   
                   
               
               
                   
               
               
                 78950939 
                 NM_000250.1(MPO):c.518A&gt;G 
                 GTGCGGYATTTGTCCTGCTCCGG, 
                 Myeloperoxidase deficiency 
               
               
                   
                 (p.Tyr173Cys) 
                 TGCGGYATTTGTCCTGCTCCGG 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 115677373 
                 NM_201631.3(TGM5):c.763T&gt;C 
                 TGCGGAGYGGACGGGCAGCGTGG 
                 Peeling skin syndrome, acral type 
               
               
                   
                 (p.Trp255Arg) 
                   
                   
               
               
                   
               
               
                 5030804 
                 NM_000551.3(VHL):c.233A&gt;G 
                 GCGAYTGCAGAAGATGACCTGGG, 
                 Von Hippel-Lindau syndrome 
               
               
                   
                 (p.Asn78Ser) 
                 TGCGAYTGCAGAAGATGACCTG 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 397508328 
                 NM_000492.3(CFTR):c.1A&gt;G 
                 GCAYGGTCTCTCGGGCGCTGGGG, 
                 Cystic fibrosis 
               
               
                   
                 (p.Met1Val) 
                 TGCAYGGTCTCTCGGGCGCTGGG, 
                   
               
               
                   
                   
                 CTGCAYGGTCTCTCGGGCGCTGG 
                   
               
               
                   
               
               
                 137853299 
                 NM_000362.4(TIMP3):c.572A&gt;G 
                 TGCAGYAGCCGCCCTTCTGCCGG 
                 Sorsby fundus dystrophy 
               
               
                   
                 (p.Tyr191Cys) 
                   
                   
               
               
                   
               
               
                 121908549 
                 NM_000334.4(SCN4A):c.3478A&gt;G 
                 TGAYGGAGGGGATGGCGCCTAGG 
                   
               
               
                   
                 (p.Ile1160Val) 
                   
                   
               
               
                   
               
               
                 121909337 
                 NM_001451.2(FOXF1):c.1138T&gt;C 
                 TGATGYGAGGCTGCCGCCGCAGG 
                 Alveolar capillary dysplasia with 
               
               
                   
                 (p.Ter380Arg) 
                   
                 misalignment of pulmonary veins 
               
               
                   
               
               
                 281875320 
                 NM_005359.5(SMAD4):c.1500A&gt;G 
                 TGAGYATGCATAAGCGACGAAGG 
                 Myhre syndrome 
               
               
                   
                 (p.Ile500Met) 
                   
                   
               
               
                   
               
               
                 730880132 
                 NM_170707.3(LMNA):c.710T&gt;C 
                 TGAGTYTGAGAGCCGGCTGGCGG 
                 Primary dilated cardiomyopathy 
               
               
                   
                 (p.Phe237Ser) 
                   
                   
               
               
                   
               
               
                 281875322 
                 NM_005359.5(SMAD4):c.1498A&gt;G 
                 TGAGTAYGCATAAGCGACGAAGG 
                 Hereditary cancer-predisposing syndrome, 
               
               
                   
                 (p.Ile500Val) 
                   
                 Myhre syndrome 
               
               
                   
               
               
                 72556283 
                 NM_000531.5(OTC):c.527A&gt;G 
                 TGAGGYAATCAGCCAGGATCTGG 
                 not provided 
               
               
                   
                 (p.Tyr176Cys) 
                   
                   
               
               
                   
               
               
                 74315311 
                 NM_020435.3(WC2):c.857T&gt;C 
                 TGAGAYGGCCCACCTGGGCTTGG, 
                 Leukodystrophy, hypomyelinating, 2 
               
               
                   
                 (p.Met286Thr) 
                 GAGAYGGCCCACCTGGGCTTGGG 
                   
               
               
                   
               
               
                 121912495 
                 NM_170707.3(LMNA):c.1139T&gt;C 
                 TCTYGGAGGGCGAGGAGGAGAGG 
                 Congenital muscular dystrophy, LMNA-related 
               
               
                   
                 (p.Leu380Ser) 
                   
                   
               
               
                   
               
               
                 128620184 
                 NM_000061.2(BTK):c.1288A&gt;G 
                 TCTYGATGGCCACGTCGTACTGG 
                 X-linked agammaglobulinemia 
               
               
                   
                 (p.Lys430Glu) 
                   
                   
               
               
                   
               
               
                 118192252 
                 NM_004519.3(KCNQ3):c.1403A&gt;G 
                 TCTTTAYTGTTTAAGCCAACAGG 
                 Benign familial neonatal seizures 2, not 
               
               
                   
                 (p.Asn468Ser) 
                   
                 specified 
               
               
                   
               
               
                 121909142 
                 NM_001300.5(KLF6):c.190T&gt;C 
                 TCTGYGGACCAAAATCATTCTGG 
                   
               
               
                   
                 (p.Trp64Arg) 
                   
                   
               
               
                   
               
               
                 104895503 
                 NM_001127255.1(NLRP7):c.2738A&gt;G 
                 TCTGGYTGATACTCAAGTCCAGG 
                 Hydatidiform mole 
               
               
                   
                 (p.Asn913Ser) 
                   
                   
               
               
                   
               
               
                 587783035 
                 NM_000038.5(APC):c 1744-2A&gt;G 
                 TCCYAGTAAGAAACAGAATATGG 
                 Familial adenomatous polyposis 
               
               
                   
               
               
                 72556289 
                 NM_000531.5(OTC):c.541-2A&gt;G 
                 TCCYAAAAGGCACGGGATGAAGG 
                 not provided 
               
               
                   
               
               
                 28937313 
                 NM_005502.3(ABCA1):c.2804A&gt;G 
                 TCCAYTGTGGCCCAGGAAGGAGG, 
                 Tangier disease 
               
               
                   
                 (p.Asn935Ser) 
                 CGCTCCAYTGTGGCCCAGGAAGG 
                   
               
               
                   
               
               
                 143246552 
                 NM_001003811.1(TEX11):c.511A&gt;G 
                 TCCAYGGTCAAGTCAGCCTCAGG, 
                 Spermatogenic failure, X-linked, 2 
               
               
                   
                 (p.Met171Val) 
                 CCAYGGTCAAGTCAGCCTCAGGG 
                   
               
               
                   
               
               
                 587776451 
                 NM_002049.3(GATA1):c.2T&gt;C 
                 CTCCAYGGAGTTCCCTGGCCTGG, 
                 GATA-1-related thrombocytopenia 
               
               
                   
                 (p.Met1Thr) 
                 TCCAYGGAGTTCCCTGGCCTGGG, 
                 with dyserythropoiesis 
               
               
                   
                   
                 CCAYGGAGTTCCCTGGCCTGGGG 
                   
               
               
                   
               
               
                 121908403 
                 NM_021102.3(SPINT2):c.488A&gt;G 
                 TCCAYAGATGAAGTTATTGCAGG 
                 Diarrhea 3, secretory sodium, congenital, 
               
               
                   
                 (p.Tyr163Cys) 
                   
                 syndromic 
               
               
                   
               
               
                 281874738 
                 NM_000495.4(COL4A5):c.438+2T&gt;C 
                 CTCCAGYAAGTTATAAAATTTGG, 
                 Alport syndrome, X-linked recessive 
               
               
                   
                   
                 TCCAGYAAGTTATAAAATTTGG 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 730880279 
                 NM_030653.3(DDX11):c.2271+2T&gt;C 
                 TCCAGGYGCGGGCGTCATGCTGG, 
                 Warsaw breakage syndrome 
               
               
                   
                   
                 CCAGGYGCGGGCGTCATGCTGGG 
                   
               
               
                   
               
               
                 28940272 
                 NM_017890.4(VPS13B):c.8978A&gt;G 
                 TCAYTGATAAGCAGGGCCCAGGG, 
                 Cohen syndrome, not specified 
               
               
                   
                 (p.Asn2993Ser) 
                 TTCAYTGATAAGCAGGGCCCAGG 
                   
               
               
                   
               
               
                 137852375 
                 NM_000132.3(F8):c.5372T&gt;C 
                 TCAYGGTGAGTTAAGGACAGTGG 
                 Hereditary factor VIII deficiency disease 
               
               
                   
                 (p.Met1791Thr) 
                   
                   
               
               
                   
               
               
                 11567847 
                 NM_021961.5(TEAD1):c.1261T&gt;C 
                 TCATATTYACAGGCTTGTAAAGG 
                   
               
               
                   
                 (p.Tyr?His) 
                   
                   
               
               
                   
               
               
                 786203989 
                 NM_016069.9(PAM16):c.226A&gt;G 
                 CATAGTYCTGCAGAGGAGAGGGG, 
                 Chondrodysplasia, megarbane-dagher-melki 
               
               
                   
                 (p.Asn76Asp) 
                 TCATAGTYCTGCAGAGGAGAGGG 
                 type 
               
               
                   
               
               
                 587776437 
                 NC_012920.1:m.9478T&gt;C 
                 TCAGAAGYTTTTTTCTTCGCAGG 
                 Leigh disease 
               
               
                   
               
               
                 121912474 
                 NM_000424.3(KRT5):c.20T&gt;C 
                 TCAAGTGYGTCCTTCCGGAGCGG, 
                 Epidermolysis bullosa simplex, Koebner type 
               
               
                   
                 (p.Val7Ala) 
                 CAAGTGYGTCCTTCCGGAGCGGG, 
                   
               
               
                   
                   
                 AAGTGYGTCCTTCCGGAGCGGGG, 
                   
               
               
                   
                   
                 AGTGYGTCCTTCCGGAGCGGGGG 
                   
               
               
                   
               
               
                 104886461 
                 NM_020533.2(MCOLN1):c.406-2A&gt;G 
                 TACYGTGGGCAGAGAAGGGGAGG, 
                 Ganglioside sialidase deficiency 
               
               
                   
                   
                 AGGTACYGTGGGCAGAGAAGGGG, 
                   
               
               
                   
                   
                 CAGGTACYGTGGGCAGAGAAGGG 
                   
               
               
                   
               
               
                 104894275 
                 NM_000317.2(PTS):c.155A&gt;G 
                 TAAYTGTGCCCATGGCCATTTGG 
                 6-pyruvoyl-tetrahydropterin synthase deficiency 
               
               
                   
                 (p.Asn52Ser) 
                   
                   
               
               
                   
               
               
                 587777562 
                 NM_015599.2(PGM3):c.737A&gt;G 
                 TAAATGAYTGAGTTTGCCCTTGG 
                 Immunodeficiency 23 
               
               
                   
                 (p.Asn246Ser) 
                   
                   
               
               
                   
               
               
                 121964906 
                 NM_000027.3(AGA):c.916T&gt;C 
                 GTTATAYGTGCCAATGTGACTGG 
                 Aspartylglycosaminuria 
               
               
                   
                 (p.Cys306Arg) 
                   
                   
               
               
                   
               
               
                 28941769 
                 NM_000356.3(TCOF1):c.149A&gt;G 
                 GTGTGTAYAGATGTCCAGAAGGG 
                 Treacher collins syndrome 1 
               
               
                   
                 (p.Tyr50Cys) 
                   
                   
               
               
                   
               
               
                 121434464 
                 m.12297T&gt;C 
                 GTCYTAGGCCCCAAAAATTTTGG 
                 Cardiomyopathy, mitochondrial 
               
               
                   
               
               
                 121908407 
                 NM_054027.4(ANKH):c.143T&gt;C 
                 GTCGAGAYGCTGGCCAGCTACGG, 
                 Chondrocalcinosis 2 
               
               
                   
                 (p.Met48Thr) 
                 TCGAGAYGCTGGCCAGCTACGGG 
                   
               
               
                   
               
               
                 59151893 
                 NM_000422.2(KRT17):c.275A&gt;G 
                 GTCAYTGAGGTTCTGCATGGTGG, 
                 Pachyonychia congenita type 2 
               
               
                   
                 (p.Asn92Ser) 
                 GCGGTCAYTGAGGTTCTGCATGG 
                   
               
               
                   
               
               
                 121909499 
                 NM_002427.3(MMP13):c.272T&gt;C 
                 GTCAYGAAAAAGCCAAGATGCGG, 
                   
               
               
                   
                 (p.Met91Thr) 
                 TCAYGAAAAAGCCAAGATGCGG 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 61748478 
                 NM_000552.3(VWF):c.2384A&gt;G 
                 GTCAYAGTTCTGGCACGTTTTGG 
                 von Willebrand disease type 2N 
               
               
                   
                 (p.Tyr795Cys) 
                   
                   
               
               
                   
               
               
                 387906889 
                 NM_006796.2(AFG3L2):c.1847A&gt;G 
                 GTAYAGAGGTATTGTTCTTTTGG 
                 Spastic ataxia 5, autosomal recessive 
               
               
                   
                 (p.Tyr616Cys) 
                   
                   
               
               
                   
               
               
                 118203907 
                 NM_000130.4(F5):c.5189A&gt;G 
                 GTAGYAGGCCCAAGCCCGACAGG 
                 Factor V deficiency 
               
               
                   
                 (p.Tyr1730Cys) 
                   
                   
               
               
                   
               
               
                 118203945 
                 NM_013319.2(UBIAD1):c.305A&gt;G 
                 GTAAGTGYTGACCAAATTACCGG 
                 Schnyder crystalline corneal dystrophy 
               
               
                   
                 (p.Asn102Ser) 
                   
                   
               
               
                   
               
               
                 267607080 
                 NM_005633.3(SOSI):c.1294T&gt;C 
                 GGTYGGGAGGGAAAAGACATTGG 
                 Noonan syndrome 4, Rasopathy 
               
               
                   
                 (p.Trp432Arg) 
                   
                   
               
               
                   
               
               
                 137852953 
                 NM_012464.4(TLL1):c.1885A&gt;G 
                 GGTTAYGGTGCCGTTAAGTTTGG 
                 Atrial septal defect 6 
               
               
                   
                 (p.Ile629Val) 
                   
                   
               
               
                   
               
               
                 118203949 
                 NM_013319.2(UBIAD1):c.695A&gt;G 
                 GGTGTTGYTGGAATGGAGAATGG 
                 Schnyder crystalline corneal dystrophy 
               
               
                   
                 (p.Asn232Ser) 
                   
                   
               
               
                   
               
               
                 137852952 
                 NM_012464.4(TLL1):c.713T&gt;C 
                 GGGATTGYTGTTCATGAATTGGG 
                 Atrial septal defect 6 
               
               
                   
                 (p.Val238Ala) 
                   
                   
               
               
                   
               
               
                 41460449 
                 m.3394T&gt;C 
                 GGCYATATACAACTACGCAAAGG 
                 Leber optic atrophy 
               
               
                   
               
               
                 80357281 
                 NM_007294.3(BRCA1):c.5291T&gt;C 
                 GGGCYAGAAATCTGTTGCTATGG, 
                 Familial cancer of breast, Breast-ovarian 
               
               
                   
                 (p.Leu1764Pro) 
                 GGCYAGAAATCTGTTGCTATGGG 
                 cancer, familial 1 
               
               
                   
               
               
                 5030764 
                 NM_000174.4(GP9):c.182A&gt;G 
                 GGCTGYTGTTGGCCAGCAGAAGG 
                 Bernard-Soulier syndrome type C 
               
               
                   
                 (p.Asn61Ser) 
                   
                   
               
               
                   
               
               
                 72556282 
                 NM_000531.5(OTC):c.526T&gt;C 
                 GGCTGATYACCTCACGCTCCAGG, 
                 not provided 
               
               
                   
                 (p.Tyr176His) 
                 GATYACCTCACGCTCCAGGTTGG 
                   
               
               
                   
               
               
                 121913594 
                 NM_000530.6(MPZ):c.242A&gt;G 
                 GGCATAGYGGAAGATCTATGAGG 
                 Charcot-Marie-Tooth disease type 1B 
               
               
                   
                 (p.His8lArg) 
                   
                   
               
               
                   
               
               
                 587777736 
                 NM_017617.3(NOTCH1):c.1285T&gt;C 
                 GGCAAGYGCATCAACACGCTGGG, 
                 Adams-Oliver syndrome 1, Adams- 
               
               
                   
                 (p.Cys429Arg) 
                 GGGCAAGYGCATCAACACGCTGG 
                 Oliver syndrome 5 
               
               
                   
               
               
                 63750912 
                 NM_016835.4(MAPT):c.1839T&gt;C 
                 GGATAAYATCAAACACGTCCCGG, 
                 Frontotemporal dementia 
               
               
                   
                 (p.Asn613=) 
                 GATAAYATCAAACACGTCCCGG 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 121918075 
                 NM_000371.3(TTR):c.401A&gt;G 
                 GGAGYAGGGGCTCAGCAGGGCGG, 
                 Amyloidogenic transthyretin amyloidosis 
               
               
                   
                 (p.Tyr134Cys) 
                 ATAGGAGYAGGGGCTCAGCAGGG 
                   
               
               
                   
               
               
                 730882063 
                 NM_004523.3(KIF11):c.2547+2T&gt;C 
                 GGAGGYAATAACTTTGTAAGTGG 
                 Microcephaly with or without 
               
               
                   
                   
                   
                 chorioretinopathy, lymphedema, or 
               
               
                   
                   
                   
                 mental retardation 
               
               
                   
               
               
                 397516156 
                 NM_000257.3(MYH7):c.2546T&gt;C 
                 GGAGAYGGCCTCCATGAAGGAGG 
                 Primary familial hypertrophic 
               
               
                   
                 (p.Met849Thr) 
                   
                 cardiomyopathy, 
               
               
                   
               
               
                 118204430 
                 NM_000035.3(ALDOB):c.442T&gt;C 
                 GGAAGYGGCGTGCTGTGCTGAGG 
                 Hereditary fructosuria 
               
               
                   
                 (p.Trp148Arg) 
                   
                   
               
               
                   
               
               
                 200198778 
                 NM_013382.5(POMT2):c.1997A&gt;G 
                 GGAAGYAGTGGTGGAAGTAGAGG 
                 Congenital muscular dystrophy, Congenital 
               
               
                   
                 (p.Tyr666Cys) 
                   
                 muscular dystrophy-dystroglycanopathy with 
               
               
                   
                   
                   
                 brain and eye anomalies, type A2, Muscular 
               
               
                   
                   
                   
                 dystrophy, Congenital muscular 
               
               
                   
                   
                   
                 dystrophy-dystroglycanopathy with mental retardation, 
               
               
                   
                   
                   
                 type B2 
               
               
                   
               
               
                 754896795 
                 NM_004006.2(DMD):c.6982A&gt;T 
                 GCTTTTYTTCAAGCTGCCCAAGG 
                 Duchenne muscular dystrophy, Becker 
               
               
                   
                 (p.Lys2328Ter) 
                   
                 muscular dystrophy, Dilated cardiomyopathy 3B 
               
               
                   
               
               
                 148924904 
                 NM_000546.5(TP53):c.488A&gt;G 
                 GCTTGYAGATGGCCATGGCGCGG 
                 Hereditary cancer-predisposing syndrome 
               
               
                   
                 (p.Tyr163Cys) 
                   
                   
               
               
                   
               
               
                 786204770 
                 NM_016035.4(C0Q4):c.155T&gt;C 
                 GCTGTYGGCCGCCGGCTCCGCGG 
                 COENZYME Q10 DEFICIENCY, PRIMARY, 7 
               
               
                   
                 (p.Leu52Ser) 
                   
                   
               
               
                   
               
               
                 121909520 
                 NM_001100.3(ACTA1):c.350A&gt;G 
                 CGGYTGGCCTTGGGATTGAGGGG, 
                 Nemaline myopathy 3 
               
               
                   
                 (p.Asn117Ser) 
                 GCGGYTGGCCTTGGGATTGAGGG, 
                   
               
               
                   
                   
                 CGCGGYTGGCCTTGGGATTGAGG 
                   
               
               
                   
               
               
                 587776879 
                 NM_004656.3(BAP1):c.438-2A&gt;G 
                 GCCYGGGGAAAAACAGAGTCAGG 
                 Tumor predisposition syndrome 
               
               
                   
               
               
                 727504434 
                 NM_000501.3(ELN):c.890-2A&gt;G 
                 GCCYGAAAACACAGCCACAGAGG 
                 Supravalvar aortic stenosis 
               
               
                   
               
               
                 119455953 
                 NM_000391.3(TPP1):c.1093T&gt;C 
                 GCCGGGYGTTGGTCTGTCTCTGG 
                 Ceroid lipofuscinosis, neuronal, 2 
               
               
                   
                 (p.Cys365Arg) 
                   
                   
               
               
                   
               
               
                 121964983 
                 NM_000481.3(AMT):c.125A&gt;G 
                 GCCAGGYGGAAGTCATAGAGCGG 
                 Non-ketotichyperglycinemia 
               
               
                   
                 (p.His42Arg) 
                   
                   
               
               
                   
               
               
                 121908300 
                 NM_001005741.2(GBA):c.751T&gt;C 
                 GCCAGAYACTTTGTGAAGTAAGG, 
                 Gaucher disease, type 1 
               
               
                   
                 (p.Tyr251His) 
                 CCAGAYACTTTGTGAAGTAAGG 
                   
               
               
                   
               
               
                 786205083 
                 NM_003494.3(DYSF):c.3443-33A&gt;G 
                 GCCAGAGYGAGTGGCTGGAGTGG 
                 Limb-girdle muscular dystrophy, type 2B 
               
               
                   
               
               
                 121908133 
                 NM_175073.2(APTX):c.602A&gt;G 
                 GCCAAYGGTAACGGGCCTTTGGG, 
                 Adult onset ataxia with oculomotor apraxia 
               
               
                   
                 (p.His201Arg) 
                 AGCCAAYGGTAACGGGCCTTTGG 
                   
               
               
                   
               
               
                 587777195 
                 NM_005017.3(PCYT1A):c.571T&gt;C 
                 GCATGYTTGCTCCAACACAGAGG 
                 Spondylometaphyseal dysplasia with cone-rod 
               
               
                   
                 (p.Phe191Leu) 
                   
                 dystrophy 
               
               
                   
               
               
                 431905520 
                 NM_014714.3(IFT140):c.4078T&gt;C 
                 CAAGCAGYGTGAGCTGCTCCTGG, 
                 Renal dysplasia, retinal pigmentary dystrophy, 
               
               
                   
                 (p.Cys1360Arg) 
                 GCAGYGTGAGCTGCTCCTGGAGG 
                 cerebellar ataxia and skeletal dysplasia 
               
               
                   
               
               
                 121912889 
                 NM_001844.4(COL2A1):c.4172A&gt;G 
                 GCAGTGGYAGGTGATGTTCTGGG 
                 Spondyloperipheral dysplasia, Platyspondylic 
               
               
                   
                 (p.Tyr1391Cys) 
                   
                 lethal skeletal dysplasia Torrance type 
               
               
                   
               
               
                 137854492 
                 NM_001363.4(DKC1):c.1069A&gt;G 
                 GCAGGYAGAGATGACCGCTGTGG 
                 Dyskeratosis congenita X-linked 
               
               
                   
                 (p.Thr357Ala) 
                   
                   
               
               
                   
               
               
                 121434362 
                 NM_152783.4(D2HGDH):c.1315A&gt;G 
                 GCAGGTYACCATCTCCTGGAGGG, 
                 D-2-hydroxyglutaric aciduria 1 
               
               
                   
                 (p.Asn439Asp) 
                 TGCAGGTYACCATCTCCTGGAGG 
                   
               
               
                   
               
               
                 80338732 
                 NM_002764.3(PRPS1):c.344T&gt;C 
                 GCAAATAYGCTATCTGTAGCAGG 
                 Charcot-Marie-Tooth disease, X- 
               
               
                   
                 (p.Met115Thr) 
                   
                 linked recessive, type 5 
               
               
                   
               
               
                 387906675 
                 NM_000313.3(PROS1):c.701A&gt;G 
                 GATTAYATCTGTAGCCTTCGGGG, 
                 Thrombophilia due to protein S deficiency, 
               
               
                   
                 (p.Tyr234Cys) 
                 AGATTAYATCTGTAGCCTTCGGG, 
                 autosomal recessive 
               
               
                   
                   
                 GAGATTAYATCTGTAGCCTTCGG 
                   
               
               
                   
               
               
                 28935478 
                 NM_000061.2(BTK):c.1082A&gt;G 
                 GATGGYAGTTAATGAGCTCAGGG, 
                   
               
               
                   
                 (p.Tyr36ICys) 
                 TGATGGYAGTTAATGAGCTCAGG 
                   
               
               
                   
               
               
                 201777056 
                 NM_005050.3(ABCD4):c.956A&gt;G 
                 GATGAGGYAGATGCACACAAAGG 
                 METHYLMALONIC ACIDURIA 
               
               
                   
                 (p.Tyr319Cys) 
                   
                 AND HOMOCYSTINURIA, cblJ 
               
               
                   
               
               
                 121918528 
                 NM_000098.2(CPT2):c.359A&gt;G 
                 GATAGGYACATATCAAACCAGGG, 
                 Carnitine palmitoyltransferase II 
               
               
                   
                 (p.TyrI20Cys) 
                 AGATAGGYACATATCAAACCAG 
                 deficiency, infantile 
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 267607014 
                 NM_002942.4(ROBO2):c.2834T&gt;C 
                 GAGAYTGGAAATTTTGGCCGTGG 
                 Vesicoureteral reflux 2 
               
               
                   
                 (p.Ile945Thr) 
                   
                   
               
               
                   
               
               
                 281865192 
                 NM_025114.3(CEP290):c.2991+1655 
                 GATAYTCACAATTACAACTGGGG, 
                 Leber congenital amaurosis 10 
               
               
                   
                 A&gt;G 
                 AGATAYTCACAATTACAACTGGG, 
                   
               
               
                   
                   
                 GAGATAYTCACAATTACAACTG 
                   
               
               
                   
               
               
                 386833492 
                 NM_000112.3(SLC26A2):c.- 
                 GAGAGGYGAGAAGAGGGAAGCGG 
                 Diastrophic dysplasia 
               
               
                   
                 26+2T&gt;C 
                   
                   
               
               
                   
               
               
                 587779773 
                 NM_001101.3(ACTB):c.356T&gt;C 
                 GAGAAGAYGACCCAGGTGAGTGG 
                 Baraitser-Winter syndrome 1 
               
               
                   
                 (p.Met119Thr) 
                   
                   
               
               
                   
               
               
                 121913512 
                 NM_000222.2(KIT):c.1924A&gt;G 
                 GACTTYGAGTTCAGACATGAGGG, 
                   
               
               
                   
                 (p.Lys642Glu) 
                 GGACTTYGAGTTCAGACATGAGG 
                   
               
               
                   
               
               
                 28939072 
                 NM_006329.3(FBLN5):c.506T&gt;C 
                 GACAYTGATGAATGTCGCTATGG 
                 Age-related macular degeneration 3 
               
               
                   
                 (p.Ile169Thr) 
                   
                   
               
               
                   
               
               
                 104894248 
                 NM_000525.3(KCNJ11):c.776A&gt;G 
                 GACAYGGTAGATGATCAGCGGGG, 
                 Islet cell hyperplasia 
               
               
                   
                 (p.His259Arg) 
                 TGACAYGGTAGATGATCAGCGGG, 
                   
               
               
                   
                   
                 ATGACAYGGTAGATGATCAGCGG 
                   
               
               
                   
               
               
                 387907132 
                 NM_016464.4(TMEM138):c.287A&gt;G 
                 GACAYGAAGGGAGATGCTGAGGG, 
                 Joubert syndrome 16 
               
               
                   
                 (p.His96Arg) 
                 AGACAYGAAGGGAGATGCTGAGG 
                   
               
               
                   
               
               
                 121918170 
                 NM_000275.2(OCA2):c.1465A&gt;G 
                 GACATYTGGAGGGTCCCCGATGG 
                 Tyrosinase-positive oculocutaneous albinism 
               
               
                   
                 (p.Asn489Asp) 
                   
                   
               
               
                   
               
               
                 122467173 
                 NM_014009.3(FOXP3):c.970T&gt;C 
                 GACAGAGYTCCTCCACAACATGG 
                 Insulin-dependent diabetes mellitus secretory 
               
               
                   
                 (p.Phe324Leu) 
                   
                 diarrhea syndrome 
               
               
                   
               
               
                 137852268 
                 NM_000133.3(F9):c.1328T&gt;C 
                 GAAYATATACCAAGGTATCCCGG 
                 Hereditary factor LX deficiency disease 
               
               
                   
                 (p.Ile443Thr) 
                   
                   
               
               
                   
               
               
                 149054177 
                 NM_001999.3(FBN2):c.3740T&gt;C 
                 GAATGTAYGATAATGAACGGAGG 
                 not specified, Macular degeneration, early- 
               
               
                   
                 (p.Met1247Thr) 
                   
                 onset 
               
               
                   
               
               
                 137854488 
                 NM_212482.1(FN1):c.2918A&gt;G 
                 GAAGTAAYAGGTGACCCCAGGGG 
                 Glomerulopathy with fibronectin deposits 2 
               
               
                   
                 (p.Tyr973Cys) 
                   
                   
               
               
                 786204027 
                 NM_005957.4(MTHFR):c.1530+2T&gt;C 
                 GAAGGYGTGGTAGGGAGGCACGG, 
                 Homocysteinemia due to MTHFR deficiency 
               
               
                   
                   
                 AAGGYGTGGTAGGGAGGCACGGG, 
                   
               
               
                   
                   
                 AGGYGTGGTAGGGAGGCACGGGG 
                   
               
               
                   
               
               
                 104894223 
                 NM_012193.3(FZD4):c.766A&gt;G 
                 GAAATAYGATGGGGCGCTCAGGG, 
                 Retinopathy of prematurity 
               
               
                   
                 (p.Ile256Val) 
                 AGAAATAYGATGGGGCGCTCAGG 
                   
               
               
                   
               
               
                 137854474 
                 NM_000138.4(FBN1):c.3793T&gt;C 
                 CTTGYGTTATGATGGATTCATGG 
                 Marfan syndrome 
               
               
                   
                 (p.Cys1265Arg) 
                   
                   
               
               
                   
               
               
                 587784418 
                 NM_006306.3(SMC1A):c.3254A&gt;G 
                 CTTAYAGATCTCATCAATGTTGG 
                 Congenital muscular hypertrophy-cerebral 
               
               
                   
                 (p.Tyr1085Cys) 
                   
                 syndrome 
               
               
                   
               
               
                 81002805 
                 NM_000059.3(BRCA2):c.316+2T&gt;C 
                 CTTAGGYAAGTAATGCAATATGG 
                 Familial cancer of breast, Breast-ovarian 
               
               
                   
                   
                   
                 cancer, familial 2, Hereditary cancer-predisposing syndrome 
               
               
                   
               
               
                 121909653 
                 NM_182925.4(FLT4):c.3104A&gt;G 
                 CTGYGGATGCACTGGGGTGCGGG, 
                   
               
               
                   
                 (p.His1035Arg) 
                 TCTGYGGATGCACTGGGGTGCGG 
                   
               
               
                   
               
               
                 786205107 
                 NM_031226.2(CYP19A1):c.743+2T&gt;C 
                 CTGTGYAAGTAATACAACTTTGG 
                 Aromatase deficiency 
               
               
                   
               
               
                 587777037 
                 NM_001283009.1(RTEL1):c.3730T&gt;C 
                 CTGTGTGYGCCAGGGCTGTGGGG 
                 Dyskeratosis congenita, autosomal recessive, 5 
               
               
                   
                 (p.Cys1244Arg) 
                   
                   
               
               
                   
               
               
                 794728380 
                 NM_000238.3(KCNH2):c.1945+6T&gt;C 
                 CTGTGAGYGTGCCCAGGGGCGGG, 
                 Cardiac arrhythmia 
               
               
                   
                   
                 TGAGYGTGCCCAGGGGCGGGCGG 
                   
               
               
                   
               
               
                 267607987 
                 NM_000251.2(MSH2):c.2005+2T&gt;C 
                 CTGGYAAAAAACCTGGTTTTTGG, 
                 Hereditary Nonpolyposis Colorectal Neoplasms 
               
               
                   
                   
                 TGGYAAAAAACCTGGTTTTTGG 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 397509397 
                 NM_006876.2(B4GAT1):c.1168A&gt;G 
                 TGATYTTCAGCCTCCTTTTGGGG, 
                 Congenital muscular dystrophy-dystroglycanopathy 
               
               
                   
                 (p.Asn390Asp) 
                 CTGATYTTCAGCCTCCTTTTGGG, 
                 with brain and eye anomalies, type A13 
               
               
                   
                   
                 GCTGATYTTCAGCCTCCTTTTGG 
                   
               
               
                   
               
               
                 121918381 
                 NM_000040.1(APOC3):c.280A&gt;G 
                 CTGAAGYTGGTCTGACCTCAGGG, 
                   
               
               
                   
                 (p.Thr94Ala) 
                 GCTGAAGYTGGTCTGACCTCAGG 
                   
               
               
                   
               
               
                 104894919 
                 NM_001015877.1(PHF6):c.769A&gt;G 
                 CTCYTGATGTTGTTGTGAGCTGG 
                 Borjeson-Forssman-Lehmann syndrome 
               
               
                   
                 (p.Arg257Gly) 
                   
                   
               
               
                   
               
               
                 267606869 
                 NM_005144.4(HR):c.-218A&gt;G 
                 CTCYAGGGCCGCAGGTTGGAGGG, 
                 Marie Unna hereditary hypotrichosis 1 
               
               
                   
                   
                 GCTCYAGGGCCGCAGGTTGGAGG, 
                   
               
               
                   
                   
                 GGCGCTCYAGGGCCGCAGGTTGG 
                   
               
               
                   
               
               
                 139732572 
                 NM_000146.3(FTL):c.1A&gt;G 
                 CTCAYGGTTGGTTGGCAAGAAGG 
                 L-ferritin deficiency 
               
               
                   
                 (p.Met1Val) 
                   
                   
               
               
                   
               
               
                 397515418 
                 NM_018486.2(HDAC8):c.1001A&gt;G 
                 CTCAYGATCTGGGATCTCAGAGG 
                 Cornelia de Lange syndrome 5 
               
               
                   
                 (p.His334Arg) 
                   
                   
               
               
                   
               
               
                 372395294 
                 NM_198056.2(SCN5A):c.1247A&gt;G 
                 CTCAYAGGCCATTGCGACCACGG 
                 not provided 
               
               
                   
                 (p.Tyr416Cys) 
                   
                   
               
               
                   
               
               
                 104895304 
                 NM_000431.3(MVK):c.803T&gt;C 
                 CTCAAYAGATGCCATCTCCCTGG 
                 Hyperimmunoglobulin D with periodic 
               
               
                   
                 (p.Ile26811r) 
                   
                 fever, Mevalonic aciduria 
               
               
                   
               
               
                 587777188 
                 NM_001165899.1(PDE4D):c.1850T&gt;C 
                 CTATAYTGTTCATCCCCTCTGGG, 
                 Acrodysostosis 2, with or without hormone 
               
               
                   
                 (p.Ile617Thr) 
                 ACTATAYTGTTCATCCCCTCTGG 
                 resistance 
               
               
                   
               
               
                 398123026 
                 NM_003867.3(FGF17):c.560A&gt;G 
                 CGTGGYTGGGGAAGGGCAGCTGG 
                 Hypogonadotropic hypogonadism 20 with or 
               
               
                   
                 (p.Asn187Ser) 
                   
                 without anosmia 
               
               
                   
               
               
                 121964924 
                 NM_001385.2(DPYS):c.1078T&gt;C 
                 CGTAATAYGGGAAAAAGGCGTGG, 
                 Dihydropyrimidinase deficiency 
               
               
                   
                 (p.Trp360Arg) 
                 AATAYGGGAAAAAGGCGTGGTGG, 
                   
               
               
                   
                   
                 ATAYGGGAAAAAGGCGTGGTGGG 
                   
               
               
                   
               
               
                 587777301 
                 NM_199189.2(MATR3):c.1864A&gt;G 
                 CGGYTGAACTCTCAGTCTTCTGG 
                 Myopathy, distal, 2 
               
               
                   
                 (p.Thr622Ala) 
                   
                   
               
               
                   
               
               
                 200238879 
                 NM_000527.4(LDLR):c.694+2T&gt;C 
                 ACTGCGGYATGGGCGGGGCCAGG, 
                 Familial hypercholesterolemia 
               
               
                   
                   
                 CTGCGGYATGGGCGGGGCCAGGG, 
                   
               
               
                   
                   
                 CGGYATGGGCGGGGCCAGGGTGG 
                   
               
               
                   
               
               
                 142951029 
                 NM_145046.4(CALR3):c.245A&gt;G 
                 CGGTYTGAAGCGTGCAGAGATGG 
                 Arrhythmogenic right ventricular 
               
               
                   
                 (p.Lys82Arg) 
                   
                 cardiomyopathy, Familial 
               
               
                   
                   
                   
                 hypertrophic cardiomyopathy 19, 
               
               
                   
                   
                   
                 Hypertrophic cardiomyopathy 
               
               
                   
               
               
                 786200953 
                 NM_006785.3(MALTI):c.1019- 
                 CGCYTTGAAAAAAAAAGAAAGGG, 
                 Combined immunodeficiency 
               
               
                   
                 
                   
                 
                 TCGCYTTGAAAAAAAAAGAAAG 
                   
               
               
                   
               
               
                 120074192 
                 NM_000218.2(KCNQ1):c.418A&gt;G 
                 CGCYGAAGATGAGGCAGACCAGG 
                 Atrial fibrillation, familial, 3, Atrial fibrillation 
               
               
                   
                 (p.Ser140Gly) 
                   
                   
               
               
                   
               
               
                 267606887 
                 NM_005957.4(MTHFR):c.971A&gt;G 
                 CGCGGYTGAGGGTGTAGAAGTGG 
                 Homocystinuria due to MTHFR deficiency 
               
               
                   
                 (p.Asn324Ser) 
                   
                   
               
               
                   
               
               
                 118192117 
                 NM_000540.2(RYR1):c.1205T&gt;C 
                 CGCAYGATCCACAGCACCAATGG 
                 Congenital myopathy with fiber 
               
               
                   
                 (p.Met402Thr) 
                   
                 type disproportion, Central core 
               
               
                   
                   
                   
                 disease 
               
               
                   
               
               
                 199473625 
                 NM_198056.2(SCN5A):c.4978A&gt;G 
                 CGAYGTTGAAGAGGGCAGGCAGG, 
                 Brugada syndrome 
               
               
                   
                 (p.Ile1660Val) 
                 AGCCCGAYGTTGAAGAGGGCAGG 
                   
               
               
                   
               
               
                 794726865 
                 NM_000921.4(PDE3A):c.1333A&gt;G 
                 CGAGGYGGTGGTGGTCCAAGTGG 
                 Brachydactyly with hypertension 
               
               
                   
                 (p.Thr445Ala) 
                   
                   
               
               
                   
               
               
                 606231254 
                 NM_005740.2(DNAL4):c.153+2T&gt;C 
                 CGAGGYATTGCCAGCAGTGCAGG 
                 Mirror movements 3 
               
               
                   
               
               
                 786204826 
                 NM_004771.3(MMP20):c.611A&gt;G 
                 CGAAAYGTGTATCTCCTCCCAGG 
                 Amelogenesis imperfecta, hypomaturation type, 
               
               
                   
                 (p.His204Arg) 
                   
                 IIA2 
               
               
                   
               
               
                 796053139 
                 NM_021007.2(SCN2A):c.4308+2T&gt;C 
                 CGAAATGYAAGTCTAGTTAGAGG, 
                 not provided 
               
               
                   
                   
                 GAAATGYAAGTCTAGTTAGAGG 
                   
               
               
                   
               
               
                 137854494 
                 NM_005502.3(ABCA1):c.4429T&gt;C 
                 CCTGTGYGTCCCCCAGGGGCAGG, 
                 Tangier disease 
               
               
                   
                 (p.Cys1477Arg) 
                 CTGTGYGTCCCCCAGGGGCAGGG, 
                   
               
               
                   
                   
                 TGTGYGTCCCCCAGGGGCAGGGG, 
                   
               
               
                   
                   
                 GTGYGTCCCCCAGGGGCAGGGGG 
                   
               
               
                   
               
               
                 786205144 
                 NM_001103.3(ACTN2):c.683T&gt;C 
                 CCTAAAAYGTTGGATGCTGAAGG 
                 Dilated cardiomyopathy IAA 
               
               
                   
                 (p.Met228Thr) 
                   
                   
               
               
                   
               
               
                 199919568 
                 NM_007254.3(PNKP):c.1029+2T&gt;C 
                 CCGGYGAGGCCCTGGGGCGGGGG, 
                 not provided 
               
               
                   
                   
                 TCCGGYGAGGCCCTGGGGCGGGG, 
                   
               
               
                   
                   
                 ATCCGGYGAGGCCCTGGGGCGGG, 
                   
               
               
                   
                   
                 GATCCGGYGAGGCCCTGGGGCGG 
                   
               
               
                   
               
               
                 28939079 
                 NM_018965.3(TREM2):c.401A&gt;G 
                 TGAYCCAGGGGGTCTATGGGAGG, 
                 Polycystic lipomembranous osteodysplasia with 
               
               
                   
                 (p.Asp134Gly) 
                 CGGTGAYCCAGGGGGTCTATGGG, 
                 sclerosing leukoencephalopathy 
               
               
                   
                   
                 CCGGTGAYCCAGGGGGTCTATGG 
                   
               
               
                   
               
               
                 193302855 
                 NM_032520.4(GNPTG):c.610-2A&gt;G 
                 CCCYGAAGGTGGAGGATGCAGGG, 
                 Mucolipidosis III Gamma 
               
               
                   
                   
                 GCCCYGAAGGTGGAGGATGCAGG 
                   
               
               
                   
               
               
                 111033708 
                 NM_000155.3(GALT):c.499T&gt;C 
                 CCCTYGGGTGCAGGTTTGTGAGG 
                 Deficiency of UDPglucose-hexose-1-phosphate 
               
               
                   
                 (p.Trp167Arg) 
                   
                 uridylyltransferase 
               
               
                   
               
               
                 28933378 
                 NM_000174.4(GP9):c.70T&gt;C 
                 CCCAYGTACCTGCCGCGCCCTGG 
                 Bernard Soulier syndrome, Bernard-Soulier 
               
               
                   
                 (p.Cys24Arg) 
                   
                 syndrome type C 
               
               
                   
               
               
                 364897 
                 NM_000157.3(GBA):c.680A&gt;G 
                 CCAYTGGTCTTGAGCCAAGTGGG, 
                 Gaucher disease, Subacute neuronopathic 
               
               
                   
                 (p.Asn227Ser) 
                 TCCAYTGGTCTTGAGCCAAGTGG 
                 Gaucher disease, Gaucher disease, type 1 
               
               
                   
               
               
                 796052551 
                 NM_000833.4(GRIN2A):c.2449A&gt;G 
                 CCAYGTTGTCAATGTCCAGCTGG 
                 not provided 
               
               
                   
                 (p.Met817Val) 
                   
                   
               
               
                   
               
               
                 63751006 
                 NM_002087.3(GRN):c.2T&gt;C 
                 CCAYGTGGACCCTGGTGAGCTGG 
                 Frontotemporal dementia, ubiquitin-positive 
               
               
                   
                 (p.Met1Thr) 
                   
                   
               
               
                   
               
               
                 786203997 
                 NM_001031.4(RPS28):c.1A&gt;G 
                 TGTCCAYGATGGCGGCGCGGCGG, 
                 Diamond-Blackfan anemia with microtia and 
               
               
                   
                 (p.Met1Val) 
                 CCAYGATGGCGGCGCGGCGGCGG 
                 cleft palate 
               
               
                   
               
               
                 121908595 
                 NM_002755.3(MAP2K1):c.389A&gt;G 
                 CCAYAGAAGCCCACGATGTACGG 
                 Cardiofaciocutaneous syndrome 3, Rasopathy 
               
               
                   
                 (p.Tyr130Cys) 
                   
                   
               
               
                   
               
               
                 398122910 
                 NM_000431.3(MVK):c.1039+2T&gt;C 
                 CCAGGYATCCCGGGGGTAGGTGG, 
                 Porokeratosis, disseminated superficial actinic 
               
               
                   
                   
                 CAGGYATCCCGGGGGTAGGTGGG 
                 1 
               
               
                   
               
               
                 119474039 
                 NM_020365.4(EIF2B3):c.1037T&gt;C 
                 CCAGAYTGTCAGCAAACACCTGG 
                 Leukoencephalopathy with vanishing white 
               
               
                   
                 (p.Ile346Thr) 
                   
                 matter 
               
               
                   
               
               
                 587777866 
                 NM_000076.2(CDKN1C):c.*5+2T&gt;C 
                 CCAAGYGAGTACAGCGCACCTGG, 
                 Beckwith-Wiedemann syndrome 
               
               
                   
                   
                 CAAGYGAGTACAGCGCACCTGGG, 
                   
               
               
                   
                   
                 AAGYGAGTACAGCGCACCTGGGG 
                   
               
               
                   
               
               
                 121918530 
                 NM_005587.2(MEF2A):c.788A&gt;G 
                 AGAYTACCACCACCTGGTGGAGG, 
                   
               
               
                   
                 (p.Asn263Ser) 
                 CCAAGAYTACCACCACCTGGTGG 
                   
               
               
                   
               
               
                 483352818 
                 NM_000211.4(ITGB2):c.1877+2T&gt;C 
                 CATGYGAGTGCAGGCGGAGCAGG 
                 Leukocyte adhesion deficiency type 1 
               
               
                   
               
               
                 460184 
                 NM_000186.3(CFH):c.3590T&gt;C 
                 CAGYTGAATTTGTGTGTAAACGG 
                 Atypical hemolytic-uremic syndrome 1 
               
               
                   
                 (p.Val1197Ala) 
                   
                   
               
               
                   
               
               
                 121908423 
                 NM_004795.3(KL):c.578A&gt;G 
                 CAGYGGTACAGGGTGACCACGGG, 
                   
               
               
                   
                 (p.His193Arg) 
                 CCAGYGGTACAGGGTGACCACGG 
                   
               
               
                   
               
               
                 281860300 
                 NM_005247.2(FGF3):c.146A&gt;G 
                 CAGYAGAGCTTGCGGCGCCGGGG, 
                 Deafness with labyrinthine aplasia microtia and 
               
               
                   
                 (p.Tyr49Cys) 
                 GCAGYAGAGCTTGCGGCGCCGGG, 
                 microdontia (LAIVIM) 
               
               
                   
                   
                 CGCAGYAGAGCTTGCGGCGCCGG 
                   
               
               
                   
               
               
                 28935488 
                 NM_000169.2(GLA):c.806T&gt;C 
                 CAGTTAGYGATTGGCAACTTTGG 
                 Fabry disease 
               
               
                   
                 (p.Val269Ala) 
                   
                   
               
               
                   
               
               
                 587776514 
                 NM_173560.3(RFX6):c.380+2T&gt;C 
                 CAGTGGYGAGACTCGCCCGCAGG, 
                 Mitchell-Riley syndrome 
               
               
                   
                   
                 AGTGGYGAGACTCGCCCGCAGGG 
                   
               
               
                   
               
               
                 104894117 
                 NM_178138.4(LHX3):c.332A&gt;G 
                 CAGGTGGYACACGAAGTCCTGGG 
                 Pituitary hormone deficiency, combined 3 
               
               
                   
                 (p.Tyr111Cys) 
                   
                   
               
               
                   
               
               
                 34878913 
                 NM_000184.2(HBG2):c.125T&gt;C 
                 CAGAGGTYCTTTGACAGCTTTGG 
                 Cyanosis, transient neonatal 
               
               
                   
                 (p.Phe42Ser) 
                   
                   
               
               
                   
               
               
                 120074124 
                 NM_000543.4(SMPD1):c.911T&gt;C 
                 AGCACYTGTGAGGAAGTTCCTGG, 
                 Sphingomyelin/cholesterol lipidosis,Niemann- 
               
               
                   
                 (p.Leu304Pro) 
                 GCACYTGTGAGGAAGTTCCTGGG, 
                 Pick disease, type A, Niemann-Pick disease, 
               
               
                   
                   
                 CACYTGTGAGGAAGTTCCTGGGG 
                 type B 
               
               
                   
               
               
                 281860272 
                 NM_005211.3(CSF1R):c.2320-2A&gt;G 
                 CACYGAGGGAAAGCACTGCAGGG, 
                 Hereditary diffuse leukoencephalopathy with 
               
               
                   
                   
                 GCACYGAGGGAAAGCACTGCAGG 
                 spheroids 
               
               
                   
               
               
                 128624216 
                 NM_000033.3(ABCD1):c.443A&gt;G 
                 CACTGYTGACGAAGGTAGCAGGG, 
                 Adrenoleukodystrophy 
               
               
                   
                 (p.Asn148Ser) 
                 GCACTGYTGACGAAGGTAGCAGG 
                   
               
               
                   
               
               
                 398124257 
                 NM_012463.3(ATP6V0A2):c.825+2 
                 CACTGYGAGTAAGCTGGAAGTGG 
                 Cutis laxa with osteodystrophy 
               
               
                   
                 T&gt;C 
                   
                   
               
               
                   
               
               
                 267606679 
                 NM_004183.3(BEST I):c.704T&gt;C 
                 CACTGGYGTATACACAGGTGAGG 
                 Vitreoretinochoroidopathy dominant 
               
               
                   
                 (p.Val235Ala) 
                   
                   
               
               
                   
               
               
                 397514518 
                 NM_000344.3(SMNI):c.388T&gt;C 
                 CACTGGAYATGGAAATAGAGAGG 
                 Kugelberg-Welander disease 
               
               
                   
                 (p.Tyr130His) 
                   
                   
               
               
                   
               
               
                 143946794 
                 NM_001946.3(DUSP6):c.566A&gt;G 
                 CACTAYTGGGGTCTCGGTCAAGG 
                 Hypogonadotropic hypogonadism 19 with or 
               
               
                   
                 (p.Asn189Ser) 
                   
                 without anosmia 
               
               
                   
               
               
                 397516076 
                 NM_000256.3(MYBPC3):c.821+2T 
                 GCACGYGAGTGGCCATCCTCAGG, 
                 Familial hypertrophic cardiomyopathy 4, not 
               
               
                   
                 
                   
                 
                 CACGYGAGTGGCCATCCTCAGGG 
                 specified 
               
               
                 149977726 
                 NM_001257988.1(TYMP):c.665A&gt;G 
                 CACGAGTYTCTTACTGAGAATGG, 
                   
               
               
                   
                 (p.Lys222Arg) 
                 GAGTYTCTTACTGAGAATGGAGG 
                   
               
               
                   
               
               
                 121917770 
                 NM_003361.3(UMOD):c.383A&gt;G 
                 CACAYTGACACATGTGGCCAGGG, 
                 Familial juvenile gout 
               
               
                   
                 (p.Asn128Ser) 
                 CCACAYTGACACATGTGGCCAGG 
                   
               
               
                   
               
               
                 121909008 
                 NM_000492.3(CFTR):c.2738A&gt;G 
                 CACATAAYACGAACTGGTGCTGG 
                 Cystic fibrosis 
               
               
                   
                 (p.Tyr913Cys) 
                   
                   
               
               
                   
               
               
                 137852819 
                 NM_003688.3(CASK):c.2740T&gt;C 
                 CACAGYGGGTCCCTGTCTCCTGG, 
                 FG syndrome 4 
               
               
                   
                 (p.Trp914Arg) 
                 ACAGYGGGTCCCTGTCTCCTGGG 
                   
               
               
                   
               
               
                 74315320 
                 NM_024009.2(GM3):c.421A&gt;G 
                 CAAYGATGAGCTTGAAGATGAGG 
                 Deafness, autosomal recessive 
               
               
                   
                 (p.Ile141Val) 
                   
                   
               
               
                   
               
               
                 80356747 
                 NM_001701.3(BAAT):c.967A&gt;G 
                 CAAYGAAGAGGAATTGCCCCTGG 
                 Atypical hemolytic-uremic syndrome 1 
               
               
                   
                 (p.Ile323Val) 
                   
                   
               
               
                   
               
               
                 180177324 
                 NM_012203.1(GRHPR):c.934A&gt;G 
                 CAAGTYGTTAGCTGCCAACAAGG 
                 Primary hyperoxaluria, type II 
               
               
                   
                 (p.Asn312Asp) 
                   
                   
               
               
                   
               
               
                 281860274 
                 NM 005211.3(CSFIR):c.238IT&gt;C 
                 CAAGAYTGGGGACTTCGGGCTGG 
                 Hereditary diffuse leukoencephalopathy with 
               
               
                   
                 (p.Ile794Thr) 
                   
                 spheroids 
               
               
                   
               
               
                 398122908 
                 NM_005334.2(HCFC1):c.- 
                 CAAGAYGGCGGCTCCCAGGGAGG 
                 Mental retardation 3, X-linked 
               
               
                   
                 970T&gt;C 
                   
                   
               
               
                   
               
               
                 548076633 
                 NM_002693.2(POLG):c.3470A&gt;G 
                 CAAGAGGYTGGTGATCTGCAAGG 
                 not provided 
               
               
                   
                 (p.Asn1157Ser) 
                   
                   
               
               
                   
               
               
                 120074146 
                 NM_000019.3(ACAT1):c.935T&gt;C 
                 CAAGAAYAGTAGGTAAGGCCAGG 
                 Deficiency of acetyl-CoA acetyltransferase 
               
               
                   
                 (p.Ile312Thr) 
                   
                   
               
               
                   
               
               
                 397514489 
                 NM_005340.6(HINT1):c.250T&gt;C 
                 CAAGAAAYGTGCTGCTGATCTGG, 
                 Gamstorp-Wohlfart syndrome 
               
               
                   
                 (p.Cys84Arg) 
                 AAGAAAYGTGCTGCTGATCTGGG 
                   
               
               
                   
               
               
                 587783539 
                 NM_178151.2(DCX):c.2T&gt;C 
                 CAAAATAYGGAACTTGATTTTGG 
                 Heterotopia 
               
               
                   
                 (p.Met1Thr) 
                   
                   
               
               
                   
               
               
                 104894765 
                 NM_005448.2(BMP15):c.704A&gt;G 
                 ATTGAAAYAGAGTAACAAGAAGG 
                 Ovarian dysgenesis 2 
               
               
                   
                 (p.Tyr235Cys) 
                   
                   
               
               
                   
               
               
                 137852429 
                 NM_000132.3(F8):c.1892A&gt;G 
                 ATGYTGGAGGCTTGGAACTCTGG 
                 Hereditary factor VIII deficiency disease 
               
               
                   
                 (p.Asn631Ser) 
                   
                   
               
               
                   
               
               
                 72558441 
                 NM_000531.5(OTC):c.779T&gt;C 
                 ATGTATYAATTACAGACACTTGG 
                 not provided 
               
               
                   
                 (p.Leu260Ser) 
                   
                   
               
               
                   
               
               
                 398123765 
                 NM_003494.3(DYSF):c.1284+2T&gt;C 
                 ATGGYAAGGAGCAAGGGAGCAGG 
                 Limb-girdle muscular dystrophy, type 2B 
               
               
                   
               
               
                 387906924 
                 NM_020191.2(MRPS22):c.644T&gt;C 
                 ATCYTAGGGTAAGGTGACTTAGG 
                 Combined oxidative phosphorylation deficiency 
               
               
                   
                 (p.Leu215Pro) 
                   
                 5 
               
               
                   
               
               
                 397518039 
                 NM_206933.2(USH2A):c.8559- 
                 ATCYAAAGCAAAAGACAAGCAGG 
                 Retinitis pigmentosa, Usher syndrome, type 2A 
               
               
                   
                 2A&gt;G 
                   
                   
               
               
                   
               
               
                 5742905 
                 NM_000071.2(CBS):c.833T&gt;C 
                 ATCAYTGGGGTGGATCCCGAAGG, 
                 Homocystinuria due to CBS 
               
               
                   
                 (p.Ile278Thr) 
                 TCAYTGGGGTGGATCCCGAAGGG 
                 deficiency, Homocystinuria, 
               
               
                   
                   
                   
                 
                   
                 
               
               
                   
               
               
                 397507473 
                 NM_004333.4(BRAF):c.403T&gt;C 
                 ATCATYTGGAACAGTCTACAAGG, 
                 Cardiofaciocutaneous syndrome, Rasopathy 
               
               
                   
                 (p.Phe468Ser) 
                 TCATYTGGAACAGTCTACAAGG 
                   
               
               
                   
               
               
                 786204056 
                 NM_000264.3(PTCHH:c.3168+2T&gt;C 
                 ATCATTGYGAGTGTATTATAAGG, 
                 Gorlin syndrome 
               
               
                   
                   
                 TCATTGYGAGTGTATTATAAGGG, 
                   
               
               
                   
                   
                 CATTGYGAGTGTATTATAAGGG 
                   
               
               
                   
               
               
                 72558484 
                 NM_000531.5(OTC):c.1005+2T&gt;C 
                 ATCATGGYAAGCAAGAAACAAGG 
                 not provided 
               
               
                   
               
               
                 199473074 
                 NM_000335.4(SCN5A):c.688A&gt;G 
                 ATAYAGTTTTCAGGGCCCGGAGG, 
                 Brugada syndrome 
               
               
                   
                 (p.Ile230Val) 
                 CTGATAYAGTTTTCAGGGCCCGG 
                   
               
               
                   
               
               
                 111033273 
                 NM_206933.2(USH2A):c.1606T&gt;C 
                 ATATAGAYGCCTCTGCTCCCAGG 
                 Usher syndrome, type 2A 
               
               
                   
                 (p.Cys536Arg) 
                   
                   
               
               
                   
               
               
                 72556290 
                 NM_000531.5(OTC):c.542A&gt;G 
                 ATAGTGTYCCTAAAAGGCACGGG 
                 not provided 
               
               
                   
                 (p.Glu181Gly) 
                   
                   
               
               
                   
               
               
                 121918711 
                 NM_004612.3(TGFBR1):c.1199A&gt;G 
                 ATAGATGYCAGCACGTTTGAAGG 
                 Loeys-Dietz syndrome 1 
               
               
                   
                 (p.Asp400Gly) 
                   
                   
               
               
                   
               
               
                 104886288 
                 NM_000495.4(COL4A5):c.4699T&gt;C 
                 AGTAYGTGAAGCTCCAGCTGTGG 
                 Alport syndrome, X-linked recessive 
               
               
                   
                 (p.Cys1567Arg) 
                   
                   
               
               
                   
               
               
                 144637717 
                 NM_016725.2(FOLRH:c.493+2T&gt;C 
                 CTTCAGGYGAGGGCTGGGGTGGG, 
                 not provided 
               
               
                   
                   
                 AGGYGAGGGCTGGGGTGGGCAGG 
                   
               
               
                   
               
               
                 72558492 
                 NM_000531.5(OTC):c.1034A&gt;G 
                 AGGTGAGYAATCTGTCAGCAGGG 
                 not provided 
               
               
                   
                 (p.Tyr345Cys) 
                   
                   
               
               
                   
               
               
                 62638745 
                 NM_000121.3(EPOR):c.1460A&gt;G 
                 AGGGYTGGAGTAGGGGCCATCGG 
                 Acute myeloid leukemia, M6 type, 
               
               
                   
                 (p.Asn487Ser) 
                   
                 Familial erythrocytosis, 1 
               
               
                   
               
               
                 387907021 
                 NM_031427.3(DNAL1):c.449A&gt;G 
                 AGGGAYTGCCTACAAACACCAGG 
                 Kartagener syndrome, Ciliary dyskinesia, 
               
               
                   
                 (p.Asn150Ser) 
                   
                 primary, 16 
               
               
                   
               
               
                 397514488 
                 NM_001161581.1(POC1A):c.398T&gt;C 
                 AGCYGTGGGACAAGAGCAGCCGG 
                 Short stature, onychodysplasia, facial 
               
               
                   
                 (p.Leu133Pro) 
                   
                 dysmorphism, and hypotrichosis 
               
               
                   
               
               
                 154774633 
                 NM_017882.2(CLN6):c.200T&gt;C 
                 AGCYGGTATTCCCTCTCGAGTGG 
                 Adult neuronal ceroid lipofuscinosis 
               
               
                   
                 (p.Leu67Pro) 
                   
                   
               
               
                   
               
               
                 111033700 
                 NM_000155.3(GALT):c.482T&gt;C 
                 AGCYGGGTGCCCAGTACCCTTGG 
                 Deficiency of UDPglucose-hexose-1-phosphate 
               
               
                   
                 (p.Leu161Pro) 
                   
                 uridylyltransferase 
               
               
                   
               
               
                 128621198 
                 NM_000061.2(BTK):c.1223T&gt;C 
                 GAGCYGGGGACTGGACAATTTGG, 
                 X-linked agammaglobulinemia 
               
               
                   
                 (p.Leu408Pro) 
                 AGCYGGGGACTGGACAATTTGGG 
                   
               
               
                   
               
               
                 137852611 
                 NM_000211.4(ITGB2):c.446T&gt;C 
                 AGCYAGGTGGCGACCTGCTCCGG 
                 Leukocyte adhesion deficiency 
               
               
                   
                 (p.Leu149Pro) 
                   
                   
               
               
                   
               
               
                 121908838 
                 NM_003722.4(TP63):c.697A&gt;G 
                 AGCTTYTTTGTAGACAGGCATGG 
                 Split-hand/foot malformation 4 
               
               
                   
                 (p.Lys233Glu) 
                   
                   
               
               
                   
               
               
                 397515869 
                 NM_000169.2(GLA):c.1153A&gt;G 
                 AGCTGTGYGATGAAGCAGGCAGG 
                 not specified 
               
               
                   
                 (p.Thr385Ala) 
                   
                   
               
               
                   
               
               
                 118204064 
                 NM_000237.2(LPL):c.548A&gt;G 
                 GCTGGAYCGAGGCCTTAAAAGGG, 
                 Hyperlipoproteinemia, type I 
               
               
                   
                 (p.Asp183Gly) 
                 AGCTGGAYCGAGGCCTTAAAAGG 
                   
               
               
                   
               
               
                 128620186 
                 NM_000061.2(BTK):c.2T&gt;C 
                 AGCTAYGGCCGCAGTGATTCTGG 
                 X-linked agammaglobulinemia 
               
               
                   
                 (p.Met1Thr) 
                   
                   
               
               
                   
               
               
                 786204132 
                 NM_014946.3(SPAST):c.1165A&gt;G 
                 ATTGYCTTCCCATTCCCAGGTGG, 
                 Spastic paraplegia 4, autosomal dominant 
               
               
                   
                 (p.Thr389Ala) 
                 AGCATTGYCTTCCCATTCCCAGG 
                   
               
               
                   
               
               
                 199473661 
                 NM_000218.2(KCNQ1):c.550T&gt;C 
                 CAGCAAGBACGTGGGCCTCTGGG, 
                 Congenital long QT syndrome, Cardiac 
               
               
                   
                 (p.Tyr184His) 
                 AGCAAGBACGTGGGCCTCTGGGG, 
                 arrhythmia 
               
               
                   
                   
                 GCAAGBACGTGGGCCTCTGGGGG 
                   
               
               
                   
               
               
                 387907129 
                 NM_024599.5(RHBDF2):c.557T&gt;C 
                 AGAYTGTGGATCCGCTGGCCCGG 
                 Howel-Evans syndrome 
               
               
                   
                 (p.Ile186Thr) 
                   
                   
               
               
                   
               
               
                 387906702 
                 NM_006306.3(SMC1A):c.2351T&gt;C 
                 AGAYTGGTGTGCGCAACATCCGG 
                 Congenital muscular hypertrophy-cerebral 
               
               
                   
                 (p.Ile784Thr) 
                   
                 syndrome 
               
               
                   
               
               
                 193929348 
                 NM_000525.3(KCNJ11):c.544A&gt;G 
                 AGAYGAGGGTCTCAGCCCTGCGG 
                 Permanent neonatal diabetes mellitus 
               
               
                   
                 (p.Ile182Val) 
                   
                   
               
               
                   
               
               
                 121908934 
                 NM_004086.2(COCH):c.1535T&gt;C 
                 AGATAYGGCTTCTAAACCGAAGG 
                 Deafness, autosomal dominant 9 
               
               
                   
                 (p.Met512Thr) 
                   
                   
               
               
                   
               
               
                 397514377 
                 NM_000060.3(BTD):c.641A&gt;G 
                 AGAGGYTGTGTTTACGGTAGCGG 
                 Biotinidase deficiency 
               
               
                   
                 (p.Asn214Ser) 
                   
                   
               
               
                   
               
               
                 72552295 
                 NM_000531.5(OTC):c.2T&gt;C 
                 AGAAGAYGCTGTTTAATCTGAGG 
                 not provided 
               
               
                   
                 (p.Met1Thr) 
                   
                   
               
               
                   
               
               
                 201893545 
                 NM_016247.3(IMPG2):c.370T&gt;C 
                 ACTYTTTGGGATCGACTTCCTGG 
                 Macular dystrophy, vitelliform, 5 
               
               
                   
                 (p.Phe124Leu) 
                   
                   
               
               
                   
               
               
                 121434469 
                 m.4290T&gt;C 
                 ACTYTGATAGAGTAAATAATAGG 
                   
               
               
                   
               
               
                 121918733 
                 NM_006920.4(SCN1A):c.269T&gt;C 
                 ACTTYTATAGTATTGAATAAAGG, 
                 Severe myoclonic epilepsy in infancy 
               
               
                   
                 (p.Phe90Ser) 
                 CTTYTATAGTATTGAATAAAGG 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 121434471 
                 m.4291T&gt;C 
                 ACTTYGATAGAGTAAATAATAGG 
                 Hypertension, hypercholesterolemia, and 
               
               
                   
                   
                   
                 hypomagnesemia, mitochondrial 
               
               
                   
               
               
                 606231289 
                 NM_001302946.1(TRNT1):c.497T&gt;C 
                 ACTTYATTTGACTACTTTAATGG 
                 Sideroblastic anemia with B-cell 
               
               
                   
                 (p.Leu166Ser) 
                   
                 immunodeficiency, periodic fevers, 
               
               
                   
                   
                   
                 and developmental delay 
               
               
                   
               
               
                 63750067 
                 NM_000517.4(HBA2):c.*92A&gt;G 
                 CTTYATTCAAAGACCAGGAAGGG, 
                 Hemoglobin H disease, nondeletional 
               
               
                   
                   
                 ACTTYATTCAAAGACCAGGAAG 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 121918734 
                 NM_006920.4(SCN1A):c.272T&gt;C 
                 ACTTTTAYAGTATTGAATAAAGG, 
                 Severe myoclonic epilepsy in infancy 
               
               
                   
                 (p.Ile91Thr) 
                 CTTTTAYAGTATTGAATAAAGG 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 137854557 
                 NM_000267.3(NF1):c.1466A&gt;G 
                 ACTTAYAGCTTCTTGTCTCCAGG 
                 Neurofibromatosis, type 1 
               
               
                   
                 (p.Tyr489Cys) 
                   
                   
               
               
                   
               
               
                 397514626 
                 NM_018344.5(SLC29A3):c.607T&gt;C 
                 ACTGATAYCAGGTGAGAGCCAGG, 
                 Histiocytosis-lymphadenopathy plus syndrome 
               
               
                   
                 (p.Ser203Pro) 
                 CTGATAYCAGGTGAGAGCCAGGG 
                   
               
               
                   
               
               
                 118204440 
                 NM_000512.4(GALNS):c.1460A&gt;G 
                 ACGYTGAGCTGGGGCTGCGCGGG, 
                 Mucopolysaccharidosis, MPS-IV-A 
               
               
                   
                 (p.Asn487Ser) 
                 CACGYTGAGCTGGGGCTGCGCGG 
                   
               
               
                   
               
               
                 587776843 
                 NG_012088.1:g.2209A&gt;G 
                 ACCYTATGATCCGCCCGCCTTGG 
                   
               
               
                   
               
               
                 137853033 
                 NM_001080463.1(DYNC2H1):c.4610A&gt;G 
                 ACCYGTGAAGGGAACAGAGATGG 
                 Short-rib thoracic dysplasia 3 with or 
               
               
                   
                 (p.Gln1537Arg) 
                   
                 without polydactyly 
               
               
                   
               
               
                 28933698 
                 NM_000435.2(NOTCH3):c.1363T&gt;C 
                 TTCACCYGTATCTGTATGGCAGG, 
                 Cerebral autosomal dominant arteriopathy with 
               
               
                   
                 (p.Cys455Arg) 
                 ACCYGTATCTGTATGGCAGGTGG 
                 subcortical infarcts and leukoencephalopathy 
               
               
                   
                   
                 ACCYGAGATGCAAAATAGGGAGG, 
                   
               
               
                   
               
               
                 587776766 
                 NM_000463.2(UGT1A1):c.1085-2A&gt;G 
                 GTGACCYGAGATGCAAAATAGGG, 
                 Crigler Najjar syndrome, type 1 
               
               
                   
                   
                 GGTGACCYGAGATGCAAAATAGG 
                   
               
               
                   
               
               
                 587781628 
                 NM_001128425.1(MUTYH):c.1187-2A&gt;G 
                 ACCYGAGAGGGAGGGCAGCCAGG 
                 Hereditary cancer-predisposing syndrome, 
               
               
                   
                   
                   
                 Carcinoma of colon 
               
               
                   
               
               
                 61755817 
                 NM_000322.4(PRPH2):c.736T&gt;C 
                 ACCTGYGGGTGCGTGGCTGCAGG, 
                 Retinitis pigmentosa 
               
               
                   
                 (p.Trp246Arg) 
                 CCTGYGGGTGCGTGGCTGCAGGG 
                   
               
               
                   
               
               
                 121909184 
                 NM_001089.2(ABCA3):c.1702A&gt;G 
                 ACCGTYGTGGCCCAGCAGGACGG 
                 Surfactant metabolism dysfunction, pulmonary, 
               
               
                   
                 (p.Asn568Asp) 
                   
                 3 
               
               
                   
               
               
                 121434466 
                 m.4269A&gt;G 
                 ACAYATTTCTTAGGTTTGAGGGG, 
                   
               
               
                   
                   
                 GACAYATTTCTTAGGTTTGAGGG, 
                   
               
               
                   
                   
                 AGACAYATTTCTTAGGTTTGAGG 
                   
               
               
                   
               
               
                 794726768 
                 NM_001165963.1(SCN1A):c.1048A&gt;G 
                 ACAYATATCCCTCTGGACATTGG 
                 Severe myoclonic epilepsy in infancy 
               
               
                   
                 (p.Met350Val) 
                   
                   
               
               
                   
               
               
                 28934876 
                 NM_001382.3(DPAGT1):c.509A&gt;G 
                 ACAYAGTACAGGATTCCTGCGGG, 
                 Congenital disorder of glycosylation type 1J 
               
               
                   
                 (p.Tyr170Cys) 
                 GACAYAGTACAGGATTCCTGCGG 
                   
               
               
                   
               
               
                 104894749 
                 NM_000054.4(AVPR2):c.614A&gt;G 
                 ACAYAGGTGCGACGGCCCCAGGG, 
                 Nephrogenic diabetes insipidus, Nephrogenic 
               
               
                   
                 (p.Tyr205Cys) 
                 GACAYAGGTGCGACGGCCCCAGG 
                 diabetes insipidus, X-linked 
               
               
                   
               
               
                 128621205 
                 NM_000061.2(BTK):c.1741T&gt;C 
                 ACATTYGGGCTTTTGGTAAGTGG 
                 X-linked agammaglobulinemia 
               
               
                   
                 (p.Trp581Arg) 
                   
                   
               
               
                   
               
               
                 28940892 
                 NM_000529.2(MC2R):c.761A&gt;G 
                 ACATGYAGCAGGCGCAGTAGGGG, 
                 ACTH resistance 
               
               
                   
                 (p.Tyr254Cys) 
                 GACATGYAGCAGGCGCAGTAGGG, 
                   
               
               
                   
                   
                 AGACATGYAGCAGGCGCAGTAGG 
                   
               
               
                   
               
               
                 794726844 
                 NM_001165963.1(SCN1A):c.1046A&gt;G 
                 ACATAYATCCCTCTGGACATTGG 
                 Severe myoclonic epilepsy in infancy 
               
               
                   
                 (p.Tyr349Cys) 
                   
                   
               
               
                   
               
               
                 587783083 
                 NM_003159.2(CDKL5):c.449A&gt;G 
                 ACAGTYTTAGGACATCATTGTGG 
                 not provided 
               
               
                   
                 (p.Lys150Arg) 
                   
                   
               
               
                   
               
               
                 397514651 
                 NM_000108.4(DLD):c.140T&gt;C 
                 ACAGTTAYAGGTTCTGGTCCTGG, 
                 Maple syrup urine disease, type 3 
               
               
                   
                 (p.Ile47Thr) 
                 GTTAYAGGTTCTGGTCCTGGAGG 
                   
               
               
                   
               
               
                 794727060 
                 NM_001848.2(COL6A1):c.957+2T&gt;C 
                 ACAAGGYGAGCGTGGGCTGCTGG, 
                 Ullrich congenital muscular dystrophy, Bethlem 
               
               
                   
                   
                 CAAGGYGAGCGTGGGCTGCTGGG 
                 myopathy 
               
               
                   
               
               
                 72554346 
                 NM_000531.5(OTC):c.284T&gt;C 
                 ACAAGATYGTCTACAGAAACAGG 
                 not provided 
               
               
                   
                 (p.Leu95Ser) 
                   
                   
               
               
                   
               
               
                 483353031 
                 NM_002136.2(HNRNPA1):c.841T&gt;C 
                 AATYTTGGAGGCAGAAGCTCTGG 
                 Chronic progressive multiple sclerosis 
               
               
                   
                 (p.Phe281Leu) 
                   
                   
               
               
                   
               
               
                 104894271 
                 NM_000315.2(PTH):c.52T&gt;C 
                 AATTYGTTTTCTTACAAAATCGG 
                 Hypoparathyroidism familial isolated 
               
               
                   
                 (p.Cys18Arg) 
                   
                   
               
               
                   
               
               
                 267608260 
                 NM_015599.2(PGM3):c.248T&gt;C 
                 AATGTYGGCACCATCCTGGGAGG 
                 Immunodeficiency 23 
               
               
                   
                 (p.Leu83Ser) 
                   
                   
               
               
                   
               
               
                 267606900 
                 NM_018109.3(MTPAP):c.1432A&gt;G 
                 AATGGATYCTGAATGTACAGAGG 
                 Ataxia, spastic, 4, autosomal recessive 
               
               
                   
                 (p.Asn478Asp) 
                   
                   
               
               
                   
               
               
                 796053169 
                 NM_021007.2(SCN2A):c.387- 
                 AATAAAGYAGAATATCGTCAAGG 
                 not provided 
               
               
                   
                 2A&gt;G 
                   
                   
               
               
                   
               
               
                 104894937 
                 NM_000116.4(TAZ):c.352T&gt;C 
                 AAGYGTGTGCCTGTGTGCCGAGG 
                 3-Methylglutaconic aciduria type 2 
               
               
                   
                 (p.Cys118Arg) 
                   
                   
               
               
                   
               
               
                 104893911 
                 NM_001018077.1(NR3C1):c.1712T&gt;C 
                 AAGYGATTGCAGCAGTGAAATGG 
                 Pseudohermaphroditism, female, with 
               
               
                   
                 (p.Val571Ala) 
                   
                 hypokalemia, due to glucocorticoid resistance 
               
               
                   
               
               
                 397514472 
                 NM_004813.2(PEX16):c.992A&gt;G 
                 AAGYAGATTTTCTGCCAGGTGGG, 
                 Peroxisome biogenesis disorder 8B 
               
               
                   
                 (p.Tyr331Cys) 
                 GAAGYAGATTTTCTGCCAGGTGG, 
                   
               
               
                   
                   
                 GTAGAAGYAGATTTTCTGCCAGG 
                   
               
               
                   
               
               
                 121918407 
                 NM_001083112.2(GPD2):c.1904T&gt;C 
                 AAGTYTGATGCAGACCAGAAAGG 
                 Diabetes mellitus type 2 
               
               
                   
                 (p.Phe635Ser) 
                   
                   
               
               
                   
               
               
                 63751110 
                 NM_000251.2(MSH2):c.595T&gt;C 
                 AAGGAAYGTGTTTTACCCGGAGG 
                 Hereditary Nonpolyposis Colorectal Neoplasms 
               
               
                   
                 (p.Cys199Arg) 
                   
                   
               
               
                   
               
               
                 119450945 
                 NM_000026.2(ADSL):c.674T&gt;C 
                 AAGAYGGTGACAGAAAAGGCAGG 
                 Adenylosuccinate lyase deficiency 
               
               
                   
                 (p.Met225Thr) 
                   
                   
               
               
                   
               
               
                 113993988 
                 NM_002863.4(PYGL):c.2461T&gt;C 
                 AAGAAYATGCCCAAAACATCTGG 
                 Glycogen storage disease, type VI 
               
               
                   
                 (p.Tyr821His) 
                   
                   
               
               
                   
               
               
                 119485091 
                 NM_022041.3(GAN):c.1268T&gt;C 
                 AAGAAAAYCTACGCCATGGGTGG, 
                 Giant axonal neuropathy 
               
               
                   
                 (p.Ile423Thr) 
                 AAAAYCTACGCCATGGGTGGAGG 
                   
               
               
                   
               
               
                 137852419 
                 NM_000132.3(F8):c.1660A&gt;G 
                 AACYAGAGTAATAGCGGGTCAGG 
                 Hereditary factor VIII deficiency disease 
               
               
                   
                 (p.Ser554Gly) 
                   
                   
               
               
                   
               
               
                 121964967 
                 NM_000071.2(CBS):c.1150A&gt;G 
                 AACTYGGTCCTGCGGGATGGGGG, 
                 Homocystinuria,pyridox ne-respons ve 
               
               
                   
                 (p.Lys384Glu) 
                 GAACTYGGTCCTGCGGGATGGGG, 
                   
               
               
                   
                   
                 GGAACTYGGTCCTGCGGGATGGG, 
                   
               
               
                   
                   
                 AGGAACTYGGTCCTGCGGGATGG 
                   
               
               
                   
               
               
                 137852376 
                 NM_000132.3(F8):c.1754T&gt;C 
                 AACAGAYAATGTCAGACAAGAGG 
                 Hereditary factor VIII deficiency disease 
               
               
                   
                 (p.Ile585Thr) 
                   
                   
               
               
                   
               
               
                 121917930 
                 NM_006920.4(SCN1A):c.3577T&gt;C 
                 AACAAYGGTGGAACCTGAGAAGG 
                 Generalized epilepsy with febrile seizures plus, 
               
               
                   
                 (p.Trp1193Arg) 
                   
                 type 1, Generalized epilepsy with febrile 
               
               
                   
                   
                   
                 seizures plus, type 2 
               
               
                   
               
               
                 28939717 
                 NM_003907.2(EIF2B5):c.271A&gt;G 
                 AAATGYTTCCTGTACACCTGTGG 
                 Leukoencephalopathy with vanishing white 
               
               
                   
                 (p.Thr91Ala) 
                   
                 matter 
               
               
                   
               
               
                 80357276 
                 NM_007294.3(BRCA1):c.122A&gt;G 
                 AAATATGYGGTCACACTTTGTGG 
                 Familial cancer of breast, Breast-ovarian 
               
               
                   
                 (p.His41Arg) 
                   
                 cancer, familial 1 
               
               
                   
               
               
                 397515897 
                 NM_000256.3(MYBPC3):c.1351+2T&gt;C 
                 AAAGGYGGGCCTGGGACCTGAGG 
                 Familial hypertrophic cardiomyopathy 
               
               
                   
                   
                   
                 4, Cardiomyopathy 
               
               
                   
               
               
                 397514491 
                 NM_005340.6(HINT I):c.152A&gt;G 
                 AAAAYGTGTTGGTGCTTGAGGGG, 
                 Gamstorp-Wohlfart syndrome 
               
               
                   
                 (p.His5lArg) 
                 GAAAAYGTGTTGGTGCTTGAGGG, 
                   
               
               
                   
                   
                 AGAAAAYGTGTTGGTGCTTGAGG 
                   
               
               
                   
               
               
                 387907164 
                 NM_020894.2(UVSSA):c.94T&gt;C 
                 AAAATTYGCAAGTATGTCTTAGG, 
                 UV-sensitive syndrome 3 
               
               
                   
                 (p.Cys32Arg) 
                 AAATTYGCAAGTATGTCTTAGG 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 118161496 
                 NM_025152.2(NUBPL):c.815- 
                 TGGTTCYAATGGATGTCTGCTGG, 
                 Mitochondrial complex I deficiency 
               
               
                   
                 27T&gt;C 
                 GGTTCYAATGGATGTCTGCTGGG 
                   
               
               
                   
               
               
                 764313717 
                 NM_005609.2(PYGM):c.425_528del 
                 TGGCTGYCAGGGACCCAGCAAGG, 
                   
               
               
                   
                   
                 CTGYCAGGGACCCAGCAAGGAGG 
                   
               
               
                   
               
               
                 28934568 
                 NM_003242.5(TGFBR2):c.923T&gt;C 
                 AGTTCCYGACGGCTGAGGAGCGG 
                 Loeys-Dietz syndrome 2 
               
               
                   
                 (p.Leu308Pro) 
                   
                   
               
               
                   
               
               
                 121913461 
                 NM_007313.2(ABL1):c.814T&gt;C 
                 CCAGYACGGGGAGGTGTACGAGG, 
                   
               
               
                   
                 (p.Tyr272His) 
                 CAGYACGGGGAGGTGTACGAGGG 
                   
               
               
                   
               
               
                 377750405 
                 NM_173551.4(ANKS6):c.1322A&gt;G 
                 AGGGCYGTCGGACCTTCGAGTGG, 
                 Nephronophthisis 16 
               
               
                   
                 (p.Gln441Arg) 
                 GGGCYGTCGGACCTTCGAGTGGG, 
                   
               
               
                   
                   
                 GGCYGTCGGACCTTCGAGTGGGG 
                   
               
               
                   
               
               
                 57639980 
                 NM_001927.3(DES):c.1034T&gt;C 
                 ATTCCCYGATGAGGCAGATGCGG, 
                 Myofibrillar myopathy 1 
               
               
                   
                 (p.Leu345Pro) 
                 TTCCCYGATGAGGCAGATGCGGG 
                   
               
               
                   
               
               
                 147391618 
                 NM_020320.3(RARS2):c.35A&gt;G 
                 ATACCYGGCAAGCAATAGCGCGG 
                 Pontocerebellar hypoplasia type 6 
               
               
                   
                 (p.Gln12Arg) 
                   
                   
               
               
                   
               
               
                 182650126 
                 NM_002977.3(SCN9A):c.2215A&gt;G 
                 GTAAYTGCAAGATCTACAAAAGG 
                 Small fiber neuropathy 
               
               
                   
                 (p.Ile739Val) 
                   
                   
               
               
                   
               
               
                 80358278 
                 NM_004700.3(KCNQ4):c.842T&gt;C 
                 ACATYGACAACCATCGGCTATGG 
                 DFNA 2 Nonsyndromic Hearing Loss 
               
               
                   
                 (p.Leu281Ser) 
                   
                   
               
               
                   
               
               
                 786204012 
                 NM_005957.4(MTHFR):c.388T&gt;C 
                 GACCYGCTGCCGTCAGCGCCTGG 
                 Homocysteinemia due to MTHFR deficiency 
               
               
                   
                 (p.Cys130Arg) 
                   
                   
               
               
                   
               
               
                 786204037 
                 NM_005957.4(MTHFR):c.1883T&gt;C 
                 TCCCACYGGACAACTGCCTCTGG 
                 Homocysteinemia due to MTHFR deficiency 
               
               
                   
                 (p.Leu628Pro) 
                   
                   
               
               
                   
               
               
                 202147607 
                 NM_000140.3(FECH):c.1137+3A&gt;G 
                 GTAGAYACCTTAGAGAACAATGG 
                 Erythropoieticprotoporphyria 
               
               
                   
               
               
                 122456136 
                 NM_005183.3(CACNA1F):c.2267T&gt;C 
                 TGCCAYTGCTGTGGACAACCTGG 
                   
               
               
                   
                 (p.Ile756Thr) 
                   
                   
               
               
                   
               
               
                 786204851 
                 NM_007374.2(SIX6):c.110T&gt;C 
                 GTCGCYGCCCGTGGCCCCTGCGG 
                 Cataract, microphthalmia and nystagmus 
               
               
                   
                 (p.Leu37Pro) 
                   
                   
               
               
                   
               
               
                 794728167 
                 NM_000138.4(FBN1):c.1468+2T&gt;C 
                 ATTGGYACGTGATCCATCCTAGG 
                 Thoracic aortic aneurysms and aortic 
               
               
                   
                   
                   
                 dissections 
               
               
                   
               
               
                 121964909 
                 NM_000027.3(AGA):c.214T&gt;C 
                 GACGGCYCTGTAGGCTTTGGAGG 
                 Aspartylglycosaminuria 
               
               
                   
                 (p.Ser72Pro) 
                   
                   
               
               
                   
               
               
                 121964978 
                 NM_000170.2(GLDC):c.2T&gt;C 
                 CGGCCAYGCAGTCCTGTGCCAGG, 
                 Non-ketotichyperglycinemia 
               
               
                   
                 (p.Met1Thr) 
                 GGCCAYGCAGTCCTGTGCCAGGG 
                   
               
               
                   
               
               
                 121965008 
                 NM_000398.6(CYB5R3):c.446T&gt;C 
                 CTGCYGGTCTACCAGGGCAAAGG 
                 METHEMOGLOBINEMIA, TYPE I 
               
               
                   
                 (p.Leu149Pro) 
                   
                   
               
               
                   
               
               
                 121965064 
                 NM_000128.3(F1 1 ):c.90IT&gt;C 
                 TGATYTCTTGGGAGAAGAACTGG 
                 Hereditary factor XI deficiency disease 
               
               
                   
                 (p.Phe301Leu) 
                   
                   
               
               
                   
               
               
                 45517398 
                 NM_000548.3(TSC2):c.5150T&gt;C 
                 GCCCYGCACGCAAATGTGAGTGG, 
                 Tuberous sclerosis syndrome 
               
               
                   
                 (p.Leu1717Pro) 
                 CCCYGCACGCAAATGTGAGTGGG 
                   
               
               
                   
               
               
                 786205857 
                 NM_015662.2(IFT172):c.770T&gt;C 
                 TTGTGCYAGGAAGTTATGACAGG 
                 RETINITIS PIGMENTOSA 71 
               
               
                   
                 (p.Leu257Pro) 
                   
                   
               
               
                   
               
               
                 786205904 
                 NM_001135669.1(XPR1):c.653T&gt;C 
                 GCGTTYACGTGTCCCCCCTTTGG, 
                 BASAL GANGLIA 
               
               
                   
                 (p.Leu218Ser) 
                 CGTTYACGTGTCCCCCCTTTGGG 
                 CALCIFICATION, 
               
               
                   
               
               
                 104893704 
                 NM_000388.3(CASR):c.2641T&gt;C 
                 ACGCTYTCAAGGTGGCTGCCCGG, 
                 Hypercalciuric hypercalcemia 
               
               
                   
                 (p.Phe881Leu) 
                 CGCTYTCAAGGTGGCTGCCCGGG 
                   
               
               
                   
               
               
                 104893747 
                 NM_I98159.2(MITF):c.1195T&gt;C 
                 ACTTYCCCTTATTCCATCCACGG, 
                 Waardenburg syndrome type 2A 
               
               
                   
                 (p.Ser399Pro) 
                 CTTYCCCTTATTCCATCCACGGG 
                   
               
               
                   
               
               
                 104893770 
                 NM_000539.3(RHO):c.133T&gt;C 
                 CATGYTTCTGCTGATCGTGCTGG, 
                 Retinitis pigmentosa 4 
               
               
                   
                 (p.Phe45Leu) 
                 ATGYTTCTGCTGATCGTGCTGGG 
                   
               
               
                   
               
               
                 28937596 
                 NM_003907.2(EIF2B5):c.1882T&gt;C 
                 AGGCCYGGAGCCCTGTTTTTAGG 
                 Leukoencephalopathy with vanishing white 
               
               
                   
                 (p.Trp628Arg) 
                   
                 matter 
               
               
                   
               
               
                 104893876 
                 NM_001151.3(SLC25A4):c.293T&gt;C 
                 GCAGCYCTTCTTAGGGGGTGTGG 
                 Autosomal dominant progressive external 
               
               
                   
                 (p.Leu98Pro) 
                   
                 ophthalmoplegia with mitochondrial 
               
               
                   
                   
                   
                 DNA deletions 2 
               
               
                   
               
               
                 104893883 
                 NM_006005.3(WFS1):c.2486T&gt;C 
                 ACCATCCYGGAGGGCCGCCTGGG 
                 WFS1-Related Disorders 
               
               
                   
                 (p.Leu829Pro) 
                   
                   
               
               
                   
               
               
                 104893962 
                 NM_000165.4(GJA1):c.52T&gt;C 
                 CTACYCAACTGCTGGAGGGAAGG 
                 Oculodentodigital dysplasia 
               
               
                   
                 (p.Ser18Pro) 
                   
                   
               
               
                   
               
               
                   
               
               
                 104893978 
                 NM_000434.3(NEU1):c.718T&gt;C 
                 GCCTCCYGGCGCTACGGAAGTGG, 
                 Sialidosis, type II 
               
               
                   
                 (p.Trp240Arg) 
                 CCTCCYGGCGCTACGGAAGTGGG, 
                   
               
               
                   
                   
                 CTCCYGGCGCTACGGAAGTGGGG 
                   
               
               
                   
               
               
                 104894092 
                 NM_002546.3(TNFRSF11B):c.349T&gt;C 
                 TAGAGYTCTGCTTGAAACATAGG 
                 Hyperphosphatasemia with bone disease 
               
               
                   
                 (p.Phe117Leu) 
                   
                   
               
               
                   
               
               
                 104894135 
                 NM_000102.3(CYP17A1):c.316T&gt;C 
                 CATCGCGYCCAACAACCGTAAGG, 
                 Complete combined 17-alpha- 
               
               
                   
                 (p.Ser106Pro) 
                 ATCGCGYCCAACAACCGTAAGGG 
                 hydroxylase/17,20-lyase 
               
               
                   
               
               
                 104894151 
                 NM_000102.3(CYP17A1):c.1358T&gt;C 
                 AGCTCTYCCTCATCATGGCCTGG 
                 Combined partial 17-alpha-hydroxylase/17,20- 
               
               
                   
                 (p.Phe453Ser) 
                   
                 lyase deficiency 
               
               
                   
               
               
                 36015961 
                 NM_000518.4(HBB):c.344T&gt;C 
                 TGTGTGCYGGCCCATCACTTTGG 
                 Beta thalassemia intermedia 
               
               
                   
                 (p.Leu115Pro) 
                   
                   
               
               
                   
               
               
                 104894472 
                 NM_152443.2(RDH12):c.523T&gt;C 
                 TCCYCGGTGGCTCACCACATTGG 
                 Leber congenital amaurosis 13 
               
               
                   
                 (p.Ser175Pro) 
                   
                   
               
               
                   
               
               
                 104894587 
                 NM_004870.3(MPDU1):c.356T&gt;C 
                 TTCCYGGTCATGCACTACAGAGG 
                 Congenital disorder of glycosylation type 1F 
               
               
                   
                 (p.Leu119Pro) 
                   
                   
               
               
                   
               
               
                 104894588 
                 NM_004870.3(MPDUI):c.2T&gt;C 
                 AATAYGGCGGCCGAGGCGGACGG 
                 Congenital disorder of glycosylation type 1F 
               
               
                   
                 (p.Met1Thr) 
                   
                   
               
               
                   
               
               
                 104894626 
                 NM_000304.3(PMP22):c.82T&gt;C 
                 TAGCAAYGGATCGTGGGCAATGG 
                 Charcot-Marie-Tooth disease, type IE 
               
               
                   
                 (p.Trp28Arg) 
                   
                   
               
               
                   
               
               
                 104894631 
                 NM_018129.3(PNP0):c.784T&gt;C 
                 ACCTYAACTCTGGGACCTGCTGG 
                 “Pyridoxal 5-phosphate-dependent epilepsy” 
               
               
                   
                 (p.Ter262Gln) 
                   
                   
               
               
                   
               
               
                 104894703 
                 NM_032551.4(KISS1R):c.305T&gt;C 
                 GCCCTGCYGTACCCGCTGCCCGG, 
                   
               
               
                   
                 (p.Leu102Pro) 
                 TGCYGTACCCGCTGCCCGGCTGG 
                   
               
               
                   
               
               
                 104894826 
                 NM_000166.5(GJBI):c.407T&gt;C 
                 ATGYCATCAGCGTGGTGTTCCGG 
                 Dejerine-Sottas disease, X-linked hereditary 
               
               
                   
                 
                   
                 
                   
                 motor and sensory neuropathy 
               
               
                   
               
               
                 104894859 
                 NM_001122606.1(LAIVIP2):c.961T&gt;C 
                 CAGCTACYGGGATGCCCCCCTGG, 
                 Danon disease 
               
               
                   
                 (p.Trp321Arg) 
                 AGCTACYGGGATGCCCCCCTGGG 
                   
               
               
                   
               
               
                 104894931 
                 NM_006517.4(SLC16A2):c.1313T&gt;C 
                 TGAGCYGGTGGGCCCAATGCAGG 
                 Allan-Herndon-Dudley syndrome 
               
               
                   
                 (p.Leu438Pro) 
                   
                   
               
               
                   
               
               
                 104894935 
                 NM_000330.3(RS1):c.38T&gt;C 
                 TTACTTCYCTTTGGCTATGAAGG 
                 Juvenile retinoschisis 
               
               
                   
                 (p.Leu13Pro) 
                   
                   
               
               
                   
               
               
                 104895217 
                 NM_001065.3(TNFRSF1A):c.175T&gt;C 
                 TGCYGTACCAAGTGCCACAAAGG 
                 TNF receptor-associated periodic fever 
               
               
                   
                 (p.Cys59Arg) 
                   
                 syndrome (TRAPS) 
               
               
                   
               
               
                 143889283 
                 NM_003793.3(CTSF):c.692A&gt;G 
                 CTCCAYACTGAGCTGTGCCACGG 
                 Ceroid lipofuscinosis, neuronal, 13 
               
               
                   
                 (p.Tyr231Cys) 
                   
                   
               
               
                   
               
               
                 122459147 
                 NM_001159702.2(FHL1):c.310T&gt;C 
                 GGGGYGCTTCAAGGCCATTGTGG 
                 Myopathy, reducing body, X-linked, 
               
               
                   
                 (p.Cys104Arg) 
                   
                 childhood- onset 
               
               
                   
               
               
                 74552543 
                 NM_020184.3(CNNM4):c.971T&gt;C 
                 AAGCTCCYGGACTTTTTTCTGGG 
                 Cone-rod dystrophy amelogenesis imperfecta 
               
               
                   
                 (p.Leu324Pro) 
                   
                   
               
               
                   
               
               
                 199476117 
                 m.10158T&gt;C 
                 AAAYCCACCCCTTACGAGTGCGG 
                 Leigh disease, Leigh syndrome due to 
               
               
                   
                   
                   
                 mitochondrial complex I deficiency, 
               
               
                   
                   
                   
                 Mitochondrial complex I deficiency 
               
               
                   
               
               
                 794727808 
                 NM_020451.2(SEPN1):c.872+2T&gt;C 
                 TTCCGGYGAGTGGGCCACACTGG 
                 Congenital myopathy with fiber type 
               
               
                   
                   
                   
                 disproportion, Eichsfeld type 
               
               
                   
                   
                   
                 congenital muscular dystrophy 
               
               
                   
               
               
                 140547520 
                 NM_005022.3(PFN1):c.350A&gt;G 
                 CACCTYCTTTGCCCATCAGCAGG 
                 Amyotrophic lateral sclerosis 18 
               
               
                   
                 (p.Glu117Gly) 
                   
                   
               
               
                   
               
               
                 397514359 
                 NM_000060.3(BTD):c.445T&gt;C 
                 TCACCGCYTCAATGACACAGAGG 
                 Biotinidase deficiency 
               
               
                   
                 (p.Phe149Leu) 
                   
                   
               
               
                   
               
               
                 207460001 
                 m.15197T&gt;C 
                 CTAYCCGCCATCCCATACATTGG 
                 Exercise intolerance 
               
               
                   
               
               
                 397514406 
                 NM_000060.3(BTD):c.1214T&gt;C 
                 TTCACCCYGGTCCCTGTCTGGGG 
                 Biotinidase deficiency 
               
               
                   
                 (p.Leu405Pro) 
                   
                   
               
               
                   
               
               
                 397514516 
                 NM_006177.3(NRL):c.287T&gt;C 
                 GAGGCCAYGGAGCTGCTGCAGGG 
                 Retinitis pigmentosa 27 
               
               
                   
                 (p.Met96Thr) 
                   
                   
               
               
                   
               
               
                 72554312 
                 NM_000531.5(OTC):c.134T&gt;C 
                 CTCACTCYAAAAAACTTTACCGG 
                 Ornithine carbamoyltransferase deficiency 
               
               
                   
                 (p.Leu45Pro) 
                   
                   
               
               
                   
               
               
                 397514569 
                 NM_178012.4(TUBB2B):c.350T&gt;C 
                 GGTCCYGGATGTGGTGAGGAAGG 
                 Polymicrogyria, asymmetric 
               
               
                   
                 (p.Leu117Pro) 
                   
                   
               
               
                   
               
               
                 397514571 
                 NM_000431.3(MVK):c.122T&gt;C 
                 CGGCYTCAACCCCACAGCAATGG, 
                 Porokeratosis, disseminated superficial actinic 
               
               
                   
                 (p.Leu41Pro) 
                 GGCYTCAACCCCACAGCAATGGG 
                 1 
               
               
                   
               
               
                 794728390 
                 NM_000238.3(KCNH2):c.2396T&gt;C 
                 GCCATCCYGGGTATGGGGTGGGG, 
                 Cardiac arrhythmia 
               
               
                   
                 (p.Leu799Pro) 
                 CCATCCYGGGTATGGGGTGGGGG, 
                   
               
               
                   
                   
                 CATCCYGGGTATGGGGTGGGGGG 
                   
               
               
                   
               
               
                 397514713 
                 NM_001199107.1(TBC1D24):c.686T&gt;C 
                 GGTCTYTGACGTCTTCCTGGTGG 
                 Early infantile epileptic encephalopathy 16 
               
               
                   
                 (p.Phe229Ser) 
                   
                   
               
               
                   
               
               
                 397514719 
                 NM_080605.3(B3GALT6):c.193A&gt;G 
                 CGCYGGCCACCAGCACTGCCAGG 
                 Spondyloepimetaphyseal dysplasia with joint 
               
               
                   
                 (p.Ser65Gly) 
                   
                 laxity 
               
               
                   
               
               
                 730880608 
                 NM_000256.3(MYBPC3):c.3796T&gt;C 
                 GAGYGCCGCCTGGAGGTGCGAGG 
                 Cardiomyopathy 
               
               
                   
                 (p.Cys1266Arg) 
                   
                   
               
               
                   
               
               
                 397515329 
                 NM_001382.3(DPAGTH:c.503T&gt;C 
                 AATCCYGTACTATGTCTACATGG, 
                 Congenital disorder of glycosylation type 1J 
               
               
                   
                 (p.Leu168Pro) 
                 ATCCYGTACTATGTCTACATGGG, 
                   
               
               
                   
                   
                 TCCYGTACTATGTCTACATGGGG 
                   
               
               
                   
               
               
                 397515465 
                 NM_018127.6(ELAC2):c.460T&gt;C 
                 ATAYTTTCTGGTCCATTGAAAGG 
                 Combined oxidative phosphorylation deficiency 
               
               
                   
                 (p.Phe154Leu) 
                   
                 17 
               
               
                   
               
               
                 397515557 
                 NM_005211.3(CSF1R):c.2483T&gt;C 
                 CATCTYTGACTGTGTCTACACGG 
                 Hereditary diffuse leukoencephalopathy with 
               
               
                   
                 (p.Phe828Ser) 
                   
                 spheroids 
               
               
                   
               
               
                 397515599 
                 NM_194248.2(0T0F):c.3413T&gt;C 
                 AGGTGCYGTTCTGGGGCCTACGG, 
                 Deafness, autosomal recessive 9 
               
               
                   
                 (p.Leu1138Pro) 
                 GGTGCYGTTCTGGGGCCTACGGG 
                   
               
               
                   
               
               
                 397515766 
                 NM_000138.4(FBN1):c.2341T&gt;C 
                 GGACAAYGTAGAAATACTCCTGG 
                 Marfan syndrome 
               
               
                   
                 (p.Cys781Arg) 
                   
                   
               
               
                   
               
               
                 565779970 
                 NM_001429.3(EP300):c.3573T&gt;A 
                 CTTAYTACAGTTACCAGAACAGG 
                 Rubinstein-Taybi syndrome 2 
               
               
                   
                 (p.Tyr1191Ter) 
                   
                   
               
               
                   
               
               
                 786200938 
                 NM_080605.3(B3GALT6):c.1A&gt;G 
                 AGCTTCAYGGCGCCCGCGCCGGG, 
                 Spondyloepimetaphyseal dysplasia with joint 
               
               
                   
                 (p.Met1Val) 
                 TCAYGGCGCCCGCGCCGGGCCGG 
                 laxity 
               
               
                   
               
               
                 28942087 
                 NM_000229.1(LCAT):c.698T&gt;C 
                 ATCTCTCYTGGGGCTCCCTGGGG, 
                 Norum disease 
               
               
                   
                 (p.Leu233Pro) 
                 TCTCYTGGGGCTCCCTGGGGTGG 
                   
               
               
                   
               
               
                 128621203 
                 NM_000061.2(BTK):c.1625T&gt;C 
                 TCGGCCYGTCCAGGTGAGTGTGG 
                 X-linked agammaglobulinemia with 
               
               
                   
                 (p.Leu542Pro) 
                   
                 growth hormone deficiency 
               
               
                   
               
               
                 397515412 
                 NM_006383.3(CIB2):c.368T&gt;C 
                 CTTCAYCTGCAAGGAGGACCTGG 
                 Deafness, autosomal recessive 48 
               
               
                   
                 (p.Ile123Thr) 
                   
                   
               
               
                   
               
               
                 193929364 
                 NM_000352.4(ABCC8):c.404T&gt;C 
                 AAGCYGCTAATTGGTAGGTGAGG 
                 Permanent neonatal diabetes mellitus 
               
               
                   
                 (p.Leu135Pro) 
                   
                   
               
               
                   
               
               
                 730880872 
                 NM_000257.3(MYH7):c.1400T&gt;C 
                 TCGAGAYCTTCGATGTGAGTTGG, 
                 Cardiomyopathy 
               
               
                   
                 
                   
                 
                 CGAGAYCTTCGATGTGAGTTGGG 
                   
               
               
                   
               
               
                 80356474 
                 NM_002977.3(SCN9A):c.2543T&gt;C 
                 AAGATCAYTGGTAACTCAGTAGG, 
                 Primary erythromelalgia 
               
               
                   
                 (p.Ile848Thr) 
                 AGATCAYTGGTAACTCAGTAGGG, 
                   
               
               
                   
                   
                 GATCAYTGGTAACTCAGTAGGGG 
                   
               
               
                   
               
               
                 80356489 
                 NM_001164277.1(SLC37A4):c.352T&gt;C 
                 GGGCYGGCCCCCATGTGGGAAGG 
                 Glucose-6-phosphate transport defect 
               
               
                   
                 (p.Trp118Arg) 
                   
                   
               
               
                   
               
               
                 80356536 
                 NM_152296.4(ATP1A3):c.2338T&gt;C 
                 GCCCYTCCTGCTGTTCATCATGG 
                 Dystonia 12 
               
               
                   
                 (p.Phe780Leu) 
                   
                   
               
               
                 80356596 
                 NM_194248.2(0T0F):c.3032T&gt;C 
                 GATGCYGGTGTTCGACAACCTGG 
                 Deafness, autosomal recessive 9, Auditory 
               
               
                   
                 (p.Leu1011Pro) 
                   
                 neuropathy, autosomal recessive, 1 
               
               
                   
               
               
                 80356689 
                 NM_000083.2(CLCN1):c.857T&gt;C 
                 AGGAGYGCTATTTAGCATCGAGG 
                 Myotonia congenita 
               
               
                   
                 (p.Val286Ala) 
                   
                   
               
               
                   
               
               
                 118203884 
                 m.4409T&gt;C 
                 AGGYCAGCTAAATAAGCTATCGG 
                 Mitochondrial myopathy 
               
               
                   
               
               
                 587777625 
                 NM_173596.2(SLC39A5):c.911T&gt;C 
                 AGAACAYGCTGGGGCTTTTGCGG 
                 Myopia 24, autosomal dominant 
               
               
                   
                 (p.Met304Thr) 
                   
                   
               
               
                   
               
               
                 587783087 
                 NM_003159.2(CDKL5):c.602T&gt;C 
                 ATTCYTGGGGAGCTTAGCGATGG 
                 not provided 
               
               
                   
                 (p.Leu201Pro) 
                   
                   
               
               
                   
               
               
                 118203951 
                 NM_013319.2(UBIAD1):c.511T&gt;C 
                 TCTGGCYCCTTTCTCTACACAGG, 
                 Schnyder crystalline corneal dystrophy 
               
               
                   
                 (p.Ser171Pro) 
                 GGCYCCTTTCTCTACACAGGAGG 
                   
               
               
                   
               
               
                 118204017 
                 NM_000018.3(ACADVL):c.1372T&gt;C 
                 TCGCATCYTCCGGATCTTTGAGG, 
                 Very long chain acyl-CoA dehydrogenase 
               
               
                   
                 (p.Phe458Leu) 
                 CGCATCYTCCGGATCTTTGAGGG, 
                 deficiency 
               
               
                   
                   
                 GCATCYTCCGGATCTTTGAGGGG 
                   
               
               
                   
               
               
                 397518466 
                 NM_000833.4(GRIN2A):c.2T&gt;C 
                 CTAYGGGCAGAGTGGGCTATTGG 
                 Focal epilepsy with speech disorder with or 
               
               
                   
                 (p.Met1Thr) 
                   
                 without mental retardation 
               
               
                   
               
               
                 118204069 
                 NM_000237.2(LPL):c.337T&gt;C 
                 GGACYGGCTGTCACGGGCTCAGG 
                 Hyperlipoproteinemia, type I 
               
               
                   
                 (p.Trp113Arg) 
                   
                   
               
               
                   
               
               
                 118204080 
                 NM_000237.2(LPL):c.755T&gt;C 
                 GTGAYTGCAGAGAGAGGACTTGG 
                 Hyperlipoproteinemia, type I 
               
               
                   
                 (p.Ile252Thr) 
                   
                   
               
               
                   
               
               
                 118204111 
                 NM_000190.3(HMBS):c.739T&gt;C 
                 GCTTCGCYGCATCGCTGAAAGGG 
                 Acute intermittent porphyria 
               
               
                   
                 (p.Cys247Arg) 
                   
                   
               
               
                   
               
               
                 80357438 
                 NM_007294.3(BRCA1):c.65T&gt;C 
                 AAATCTYAGAGTGTCCCATCTGG 
                 Familial cancer of breast, Breast-ovarian 
               
               
                   
                 (p.Leu22Ser) 
                   
                 cancer, familial 1, Hereditary cancer-predisposing syndrome 
               
               
                   
               
               
                 139877390 
                 NM_001040431.2(COA3):c.215A&gt;G 
                 CCAYCTGGGGAGGTAGGTTCAGG 
                   
               
               
                   
                 (p.Tyr72Cys) 
                   
                   
               
               
                   
               
               
                 793888527 
                 NM_005859.4(PURA):c.563T&gt;C 
                 GACCAYTGCGCTGCCCGCGCAGG, 
                 not provided, Mental retardation, autosomal 
               
               
                   
                 (p.Ile188Th) 
                 ACCAYTGCGCTGCCCGCGCAGGG, 
                 dominant 31 
               
               
                   
                   
                 CCAYTGCGCTGCCCGCGCAGGGG 
                   
               
               
                   
               
               
                 561425038 
                 NM_002878.3(RAD51D):c.1A&gt;G 
                 CGCCCAYGTTCCCCGCAGGCCGG 
                 Hereditary cancer-predisposing syndrome 
               
               
                   
                 (p.Met1Val) 
                   
                   
               
               
                   
               
               
                 121907934 
                 NM_024105.3(ALG12):c.473T&gt;C 
                 TCCYGCTGGCCCTCGCGGCCTGG 
                 Congenital disorder of glycosylation type 1G 
               
               
                   
                 (p.Leu158Pro) 
                   
                   
               
               
                   
               
               
                 80358207 
                 NM_153212.2(GM4):c.409T&gt;C 
                 CCTCATCYTCAAGGCCGCCGTGG 
                 Erythrokeratodermia variabilis 
               
               
                   
                 (p.Phe137Leu) 
                   
                   
               
               
                   
               
               
                 80358228 
                 NM_002353.2(TACSTD2):c.557T&gt;C 
                 TCGGCYGCACCCCAAGTTCGTGG 
                 Lattice conical dystrophy Type III 
               
               
                   
                 (p.Leu186Pro) 
                   
                   
               
               
                   
               
               
                 121908076 
                 NM_138691.2(TMC1):c.1543T&gt;C 
                 AGGACCTYGCTGGGAAACAATGG, 
                 Deafness, autosomal recessive 7 
               
               
                   
                 (p.Cys515Arg) 
                 ACCTYGCTGGGAAACAATGGTGG, 
                   
               
               
                   
                   
                 CCTYGCTGGGAAACAATGGTGGG 
                   
               
               
                   
               
               
                 121908089 
                 NM_017838.3(NHP2):c.415T&gt;C 
                 GGAGGCTYACGATGAGTGCCTGG, 
                 Dyskeratosis congenita autosomal recessive 1, 
               
               
                   
                 (p.Tyr139His) 
                 GGCTYACGATGAGTGCCTGGAGG 
                 Dyskeratosis congenita, autosomal recessive 2 
               
               
                   
               
               
                 121908154 
                 NM_001243133.1(NLRP3):c.926T&gt;C 
                 GGTGCCTYTGACGAGCACATAGG 
                 Familial cold urticaria, Chronic infantile 
               
               
                   
                 (p.Phe309Ser) 
                   
                 neurological, cutaneous and articular syndrome 
               
               
                   
               
               
                 121908158 
                 NM_001033855.2(DCLRE1C):c.2T&gt;C 
                 GGCGCTAYGAGTTCTTTCGAGGG, 
                 Histiocytic medullary reticulosis 
               
               
                   
                 (p.Met1Thr) 
                 GCGCTAYGAGTTCTTTCGAGGGG 
                   
               
               
                   
               
               
                 796052870 
                 NM_018129.3(PNP0):c.2T&gt;C 
                 CCCCCAYGACGTGCTGGCTGCGG, 
                 not provided 
               
               
                   
                 (p.Met1Thr) 
                 CCCCAYGACGTGCTGGCTGCGGG, 
                   
               
               
                   
                   
                 CCCAYGACGTGCTGGCTGCGGGG 
                   
               
               
                   
               
               
                 121908318 
                 NM_020427.2(SLURP1):c.43T&gt;C 
                 GCAGCCYGGAGCATGGGCTGTGG 
                 Acroerythrokeratoderma 
               
               
                   
                 (p.Trpl5Arg) 
                   
                   
               
               
                   
               
               
                 121908352 
                 NM_022124.5(CDH23):c.5663T&gt;C 
                 CTCACCTYCAACATCACTGCGGG 
                 Deafness, autosomal recessive 12 
               
               
                   
                 (p.Phe1888Ser) 
                   
                   
               
               
                   
               
               
                 121908520 
                 NM_000030.2(AGXT):c.613T&gt;C 
                 CCTGTACYCGGGCTCCCAGAAGG 
                 Primary hyperoxaluria, type I 
               
               
                   
                 (p.Ser205Pro) 
                   
                   
               
               
                   
               
               
                 121908618 
                 NM_004273.4(CHST3):c.920T&gt;C 
                 CGTGCYGGCCTCGCGCATGGTGG 
                 Spondyloepiphyseal dysplasia with congenital 
               
               
                   
                 (p.Leu307Pro) 
                   
                 joint dislocations 
               
               
                   
               
               
                 11694 
                 NM_006432.3(NPC2):c.199T&gt;C 
                 TATTCAGYCTAAAAGCAGCAAGG 
                 Niemann-Pick disease type C2 
               
               
                   
                 (p.Ser67Pro) 
                   
                   
               
               
                   
               
               
                 121908739 
                 NM_000022.2(ADA):c.320T&gt;C 
                 CCTGCYGGCCAACTCCAAAGTGG 
                 Severe combined immunodeficiency due to 
               
               
                   
                 (p.Leu107Pro) 
                   
                 ADA deficiency 
               
               
                   
               
               
                 80359022 
                 NM_000059.3(BRCA2):c.7958T&gt;C 
                 TGCYTCTTCAACTAAAATACAGG 
                 Familial cancer of breast, Breast-ovarian 
               
               
                   
                 (p.Leu2653Pro) 
                   
                 cancer, familial 2 
               
               
                   
               
               
                 121908902 
                 NM_003880.3(WISP3):c.232T&gt;C 
                 AAAATCYGTGCCAAGCAACCAGG, 
                 Progressive pseudorheumatoid dysplasia 
               
               
                   
                 (p.Cys78Arg) 
                 AAATCYGTGCCAAGCAACCAGGG, 
                   
               
               
                   
                   
                 AATCYGTGCCAAGCAACCAGGGG 
                   
               
               
                   
               
               
                 121908947 
                 NM_006892.3(DNMT3B):c.808T&gt;C 
                 CAAGTTCYCCGAGGTGAGTCCGG, 
                 Centromeric instability of chromosomes 1,9 and 
               
               
                   
                 (p.Ser270Pro) 
                 AAGTTCYCCGAGGTGAGTCCGGG, 
                 16 and immunodeficiency 
               
               
                   
                   
                 AGTTCYCCGAGGTGAGTCCGGGG 
                   
               
               
                   
               
               
                 121909028 
                 NM_000492.3(CFTR):c.3857T&gt;C 
                 AGCCTYTGGAGTGATACCACAGG 
                 Cystic fibrosis 
               
               
                   
                 (p.Phe1286Ser) 
                   
                   
               
               
                   
               
               
                 121909135 
                 NM_000085.4(CLCNKB):c.1294T&gt;C 
                 CTTTGTCYATGGTGAGTCTGGGG 
                 Bartter syndrome type 3 
               
               
                   
                 (p.Tyr432His) 
                   
                   
               
               
                   
               
               
                 121909143 
                 NM_001300.5(KLF6):c.506T&gt;C 
                 GGAGCYGCCCTCGCCAGGGAAGG 
                   
               
               
                   
                 (p.Leu169Pro) 
                   
                   
               
               
                   
               
               
                 121909182 
                 NM_001089.2(ABCA3):c.302T&gt;C 
                 GCACYTGTGATCAACATGCGAGG 
                 Surfactant metabolism dysfunction, pulmonary, 
               
               
                   
                 (p.Leu101Pro) 
                   
                 3 
               
               
                   
               
               
                 121909200 
                 NM_000503.5(EYA1):c.1459T&gt;C 
                 CACTCYCGCTCATTCACTCCCGG 
                 Melnick-Fraser syndrome 
               
               
                   
                 (p.Ser487Pro) 
                   
                   
               
               
                   
               
               
                 121909247 
                 NM_004970.2(IGFALS):c.1618T&gt;C 
                 GGACYGTGGCTGCCCTCTCAAGG 
                 Acid-labile subunit deficiency 
               
               
                   
                 (p.Cys540Arg) 
                   
                   
               
               
                   
               
               
                 121909253 
                 NM_005570.3(LMAN1):c.2T&gt;C 
                 AGAYGGCGGGATCCAGGCAAAGG 
                 Combined deficiency of factor V and factor 
               
               
                   
                 (p.Met1Thr) 
                   
                 VIII, 1 
               
               
                   
               
               
                 121909385 
                 NM_000339.2(SLC12A3):c.1868T&gt;C 
                 CAACCYGGCCCTCAGCTACTCGG 
                 Familial hypokalemia-hypomagnesemia 
               
               
                   
                 (p.Leu623Pro) 
                   
                   
               
               
                   
               
               
                 121909497 
                 NM_002427.3(MMP13):c.224T&gt;C 
                 TTCTYCGGCTTAGAGGTGACTGG 
                 Spondyloepimetaphyseal dysplasia, Missouri 
               
               
                   
                 (p.Phe75Ser) 
                   
                 type 
               
               
                   
               
               
                 121909508 
                 NM_000751.2(CHRND):c.188T&gt;C 
                 AACCYCATCTCCCTGGTGAGAGG 
                 MYASTHENIC SYNDROME, 
               
               
                   
                 (p.Leu63Pro) 
                   
                 CONGENITAL, 3B, FAST- 
               
               
                   
                   
                   
                 CHANNEL 
               
               
                   
               
               
                 121909519 
                 NM_001100.3(ACTA1):c.287T&gt;C 
                 CGAGCYTCGCGTGGCTCCCGAGG 
                 Nemaline myopathy 3 
               
               
                   
                 (p.Leu96Pro) 
                   
                   
               
               
                   
               
               
                 121909572 
                 NM_000488.3(SERPINC1):c.667T&gt;C 
                 TGGGTGYCCAATAAGACCGAAGG 
                 Antithrombin III deficiency 
               
               
                   
                 (p.Ser223Pro) 
                   
                   
               
               
                   
               
               
                 121909677 
                 NM_000821.6(GGCX):c.896T&gt;C 
                 TATGTYCTCCTACGTCATGCTGG 
                 Pseudoxanthoma elasticum-like disorder with 
               
               
                   
                 (p.Phe299Ser) 
                   
                 multiple coagulation factor deficiency 
               
               
                   
               
               
                 121909727 
                 NM_001018077.1(NR3C1):c.2209T&gt;C 
                 CTATTGCYTCCAAACATTTTTGG 
                 Glucocorticoid resistance, generalized 
               
               
                   
                 (p.Phe737Leu) 
                   
                   
               
               
                   
               
               
                 139573311 
                 NM_000492.3(CFTR):c.1400T&gt;C 
                 TTCACYTCTAATGGTGATTATGG, 
                 Cystic fibrosis 
               
               
                   
                 (p.Leu467Pro) 
                 TCACYTCTAATGGTGATTATGGG 
                   
               
               
                   
               
               
                 121912441 
                 NM_000454.4(SOD1):c.341T&gt;C 
                 CATCAYTGGCCGCACACTGGTGG 
                 Amyotrophic lateral sclerosis type 1 
               
               
                   
                 (p.Ile114Thr) 
                   
                   
               
               
                 121912446 
                 NM_000454.4(SOD1):c.434T&gt;C 
                 CGTTYGGCTTGTGGTGTAATTGG, 
                 Amyotrophic lateral sclerosis type 1 
               
               
                   
                 (p.Leu145Ser) 
                 GTTYGGCTTGTGGTGTAATTGGG 
                   
               
               
                   
               
               
                 121912463 
                 NM_000213.3(ITGB4):c.1684T&gt;C 
                 GGCCAGYGTGTGTGTGAGCCTGG 
                 Epidermolysis bullosa with pyloric atresia 
               
               
                   
                 (p.Cys562Arg) 
                   
                   
               
               
                   
               
               
                 121912492 
                 NM_002292.3(LAIVIB2):c.961T&gt;C 
                 CCTCAACYGCGAGCAGTGTCAGG 
                 Nephrotic syndrome, type 5, with or 
               
               
                   
                 (p.Cys321Arg) 
                   
                 without ocular abnormalities 
               
               
                   
               
               
                 397516659 
                 NM_001399.4(EDA):c.2T&gt;C 
                 GGCCAYGGGCTACCCGGAGGTGG 
                 Hypohidrotic X-linked ectodermal dysplasia 
               
               
                   
                 (p.Met1Thr) 
                   
                   
               
               
                   
               
               
                 111033589 
                 NM_021044.2(DHH):c.485T&gt;C 
                 GTTGCYGGCGCGCCTCGCAGTGG 
                 46, XY gonadal dysgenesis, complete, dhh- 
               
               
                   
                 (p.Leu162Pro) 
                   
                 related 
               
               
                   
               
               
                 111033622 
                 NM_000206.2(IL2RG):c.343T&gt;C 
                 TGGCYGTCAGTTGCAAAAAAAGG 
                 X-linked severe combined immunodeficiency 
               
               
                   
                 (p.Cys115Arg) 
                   
                   
               
               
                   
               
               
                 121912613 
                 NM_001041.3(SI):c.1859T&gt;C 
                 ATGCYGGAGTTCAGTTTGTTTGG 
                 Sucrase-isomaltase deficiency 
               
               
                   
                 (p.Leu620Pro) 
                   
                   
               
               
                   
               
               
                 121912619 
                 NM_016180.4(SLC45A2):c.1082T&gt;C 
                 GAGTTTCYCATCTACGAAAGAGG 
                 Oculocutaneous albinism type 4 
               
               
                   
                 (p.Leu361Pro) 
                   
                   
               
               
                   
               
               
                 61750581 
                 NM_000552.3(VWF):c.4837T&gt;C 
                 CTGCCYCTGATGAGATCAAGAGG 
                 von Willebrand disease, type 2a 
               
               
                   
                 (p.Ser1613Pro) 
                   
                   
               
               
                   
               
               
                 121912653 
                 NM_000546.5(TP53):c.755T&gt;C 
                 CATCCYCACCATCATCACACTGG 
                 Li-Fraumeni syndrome 1 
               
               
                   
                 (p.Leu252Pro) 
                   
                   
               
               
                   
               
               
                 111033683 
                 NM_000155.3(GALT):c.386T&gt;C 
                 AGGTCAYGTGCTTCCACCCCTGG 
                 Deficiency of UDPglucose-hexose-1-phosphate 
               
               
                   
                 (p.Met129Thr) 
                   
                 uridylyltransferase 
               
               
                   
               
               
                 111033752 
                 NM_000155.3(GALT):c.677T&gt;C 
                 CAGGAGCYACTCAGGAAGGTGGG 
                 Deficiency of UDPglucose-hexose-1-phosphate 
               
               
                   
                 (p.Leu226Pro) 
                   
                 uridylyltransferase 
               
               
                   
               
               
                 121912729 
                 NM_000039.1(AP0A1):c.593T&gt;C 
                 GCGCTYGGCCGCGCGCCTTGAGG 
                 Familial visceral amyloidosis, Ostertag type 
               
               
                   
                 (p.Leu198Ser) 
                   
                   
               
               
                   
               
               
                 769452 
                 NM_000041.3(APOE):c.137T&gt;C 
                 AACYGGCACTGGGTCGCTTTTGG 
                   
               
               
                   
                 (p.Leu46Pro) 
                   
                   
               
               
                   
               
               
                 121912762 
                 NM_016124.4(RHD):c.329T&gt;C 
                 ACACYGTTCAGGTATTGGGATGG 
                   
               
               
                   
                 (p.Leu110Pro) 
                   
                   
               
               
                   
               
               
                 111033824 
                 NM_000155.3(GALT):c.1138T&gt;C 
                 CGCCYGACCACGCCGACCACAGG, 
                 Deficiency of UDPglucose-hexose-1-phosphate 
               
               
                   
                 (p.Ter380Arg) 
                 GCCYGACCACGCCGACCACAGGG 
                 uridylyltransferase 
               
               
                   
               
               
                 111033832 
                 NM_000155.3(GALT):c.980T&gt;C 
                 TCCYGCGCTCTGCCACTGTCCGG 
                 Deficiency of UDPglucose-hexose-1-phosphate 
               
               
                   
                 (p.Leu327Pro) 
                   
                 uridylyltransferase 
               
               
                   
               
               
                 730881974 
                 NM_000455.4(STK11):c.545T&gt;C 
                 GGGAACCYGCTGCTCACCACCGG, 
                 Hereditary cancer-predisposing syndrome 
               
               
                   
                 (p.Leu182Pro) 
                 AACCYGCTGCTCACCACCGGTGG 
                   
               
               
                   
               
               
                 1064644 
                 NM_000157.3(GBA):c.703T&gt;C 
                 GGGYCACTCAAGGGACAGCCCGG 
                 Gaucher disease 
               
               
                   
                 (p.Ser235Pro) 
                   
                   
               
               
                   
               
               
                 796052090 
                 NM_138413.3(HOGA1):c.533T&gt;C 
                 GGACCYGCCTGTGGATGCAGTGG 
                 Primary hyperoxaluria, type III 
               
               
                   
                 (p.Leu178Pro) 
                   
                   
               
               
                   
               
               
                 121913141 
                 NM_000208.2(INSR):c.779T&gt;C 
                 CTACCYGGACGGCAGGTGTGTGG 
                 Leprechaunism syndrome 
               
               
                   
                 (p.Leu260Pro) 
                   
                   
               
               
                   
               
               
                 121913272 
                 NM_006218.2(PIK3CA):c.1258T&gt;C 
                 GGAACACYGTCCATTGGCATGGG, 
                 Congenital lipomatous overgrowth, vascular 
               
               
                   
                 (p.Cys420Arg) 
                 GAACACYGTCCATTGGCATGGGG 
                 malformations, and epidermal nevi, Neoplasm 
               
               
                   
                   
                   
                 of ovary, PIK3CA Related Overgrowth 
               
               
                   
                   
                   
                 Spectrum 
               
               
                   
               
               
                 61751310 
                 NM_000552.3(VWF):c.8317T&gt;C 
                 GCTCCYGCTGCTCTCCGACACGG 
                 von Willebrand disease, type 2a 
               
               
                   
                 (p.Cys2773Arg) 
                   
                   
               
               
                   
               
               
                 312262799 
                 NM_024408.3(NOTCH2):c.1438T&gt;C 
                 TTCACAYGTCTGTGCATGCCAGG 
                 Alagille syndrome 2 
               
               
                   
                 (p.Cys480Arg) 
                   
                   
               
               
                   
               
               
                 121913570 
                 NM_000426.3(LAIVIA2):c.7691T&gt;C 
                 ATCATTCYTTTGGGAAGTGGAGG, 
                 Merosin deficient congenital 
               
               
                   
                 (p.Leu2564Pro) 
                 TCATTCYTTTGGGAAGTGGAGGG 
                 muscular dystrophy 
               
               
                   
               
               
                 121913640 
                 NM_000257.3(MYH7):c.1046T&gt;C 
                 AACTCCAYGTATAAGCTGACAGG 
                 Familial hypertrophic cardiomyopathy 
               
               
                   
                 (p.Met349Thr) 
                   
                 1, Cardiomyopathy 
               
               
                   
               
               
                 121913642 
                 NM_000257.3(MYH7):c.1594T&gt;C 
                 CATCATGYCCATCCTGGAAGAGG 
                 Dilated cardiomyopathy 1S 
               
               
                   
                 (p.Ser532Pro) 
                   
                   
               
               
                   
               
               
                 119463996 
                 NM_001079802.1(FKTN):c.527T&gt;C 
                 GTAGTCTYTCATGAGAGGAGTGG 
                 Limb-girdle muscular 
               
               
                   
                 (p.Phe176Ser) 
                   
                 dystrophy- 
               
               
                   
               
               
                 587776456 
                 NM_002049.3(GATA1):c.1240T&gt;C 
                 GCTCAYGAGGGCACAGAGCATGG 
                 GATA-1-related thrombocytopenia 
               
               
                   
                 (p.Ter414Arg) 
                   
                 with dyserythropoiesis 
               
               
                   
               
               
                 63750654 
                 NM_000184.2(HBG2):c.-228T&gt;C 
                 ATGCAAAYATCTGTCTGAAACGG 
                 Fetal hemoglobin quantitative trait locus 1 
               
               
                   
               
               
                 587776519 
                 NM_001999.3(FBN2):c.3725-15A&gt;G 
                 AGCAYTGCAACCACATTGTCAGG 
                 Congenital contractural arachnodactyly 
               
               
                   
               
               
                 78365220 
                 NM_000402.4(G6PD):c.473T&gt;C 
                 TGCCCYCCACCTGGGGTCACAGG 
                 Anemia, nonspherocytic hemolytic, due to 
               
               
                   
                 (p.Leu158Pro) 
                   
                 G6PD deficiency 
               
               
                   
               
               
                 63750741 
                 NM_000179.2(MSH6):c.1346T&gt;C 
                 CTGGGGCYGGTATTCATGAAAGG 
                 Hereditary Nonpolyposis Colorectal Neoplasms 
               
               
                   
                 (p.Leu449Pro) 
                   
                   
               
               
                   
               
               
                 587776914 
                 NM_017565.3(FAIVI20A):c.590-2A&gt;G 
                 GTAATCYGCAAAGGAGGAGAAGG, 
                 Enamel-renal syndrome 
               
               
                   
                   
                 TAATCYGCAAAGGAGGAGAAGG 
                   
               
               
                   
               
               
                 5030809 
                 NM_000551.3(VHL):c.292T&gt;C 
                 CCCYACCCAACGCTGCCGCCTGG 
                 Von Hippel-Lindau syndrome, 
               
               
                   
                 (p.Tyr98His) 
                   
                 Hereditary cancer-predisposing 
               
               
                   
                   
                   
                 syndrome 
               
               
                   
               
               
                 199476132 
                 m.5728T&gt;C 
                 CAATCYACTTCTCCCGCCGCCGG, 
                 Cytochrome-c oxidase deficiency, Mitochondrial 
               
               
                   
                   
                 AATCYACTTCTCCCGCCGCCGGG 
                 complex I deficiency 
               
               
                   
               
               
                 62637012 
                 NM_014336.4(AIPL1):c.715T&gt;C 
                 CTGCCAGYGCCTGCTGAAGAAGG, 
                 Leber congenital amaurosis 4 
               
               
                   
                 (p.Cys239Arg) 
                 CCAGYGCCTGCTGAAGAAGGAGG 
                   
               
               
                   
               
               
                 199476199 
                 NM_207352.3(CYP4V2):c.1021T&gt;C 
                 AAACTGGYCCTTATACCTGTTGG, 
                 Bietti crystalline comeoretinal dystrophy 
               
               
                   
                 (p.Ser341Pro) 
                 AACTGGYCCTTATACCTGTTGGG 
                   
               
               
                   
               
               
                 587777183 
                 NM_006702.4(PNPLA6):c.3053T&gt;C 
                 CCTYTAACCGCAGCATCCATCGG 
                 Boucher Neuhauser syndrome 
               
               
                   
                 (p.Phe1018Ser) 
                   
                   
               
               
                   
               
               
                 199476389 
                 NM_000487.5(ARSA):c.899T&gt;C 
                 GGTCTCTYGCGGTGTGGAAAGGG 
                 Metachromatic leukodystrophy 
               
               
                   
                 (p.Leu300Ser) 
                   
                   
               
               
                   
               
               
                 199476398 
                 NM_016599.4(MYOZ2):c.142T&gt;C 
                 TTAYCCCATCTCAGTAACCGTGG 
                 Familial hypertrophic cardiomyopathy 16 
               
               
                   
                 (p.Ser48Pro) 
                   
                   
               
               
                   
               
               
                 119456967 
                 NM_001037633.1(SIL1):c.1370T&gt;C 
                 TTGCYGAAGGAGCTGAGATGAGG 
                 Marinesco-Sj\xc3\xb6gren syndrome 
               
               
                   
                 (p.Leu457Pro) 
                   
                   
               
               
                   
               
               
                 730882253 
                 NM_006888.4(CALM1):c.268T&gt;C 
                 GGCAYTCCGAGTCTTTGACAAGG 
                 Long QT syndrome 14 
               
               
                   
                 (p.Phe90Leu) 
                   
                   
               
               
                   
               
               
                 587777283 
                 NM_012338.3(TSPAN12):c.413A&gt;G 
                 TAATCCAYAATTTGTCATCCTGG 
                 Exudative vitreoretinopathy 5 
               
               
                   
                 (p.Tyr138Cys) 
                   
                   
               
               
                   
               
               
                 587777306 
                 NM_015884.3(MBTPS2):c.1391T&gt;C 
                 GCTYTGCTTTGGATGGACAATGG 
                 Palmoplantar keratoderma, mutilating, with 
               
               
                   
                 (p.Phe464Ser) 
                   
                 periorificial keratotic plaques, X-linked 
               
               
                   
               
               
                 56378716 
                 NM_000250.1(MPO):c.752T&gt;C 
                 TCACTCAYGTTCATGCAATGGGG 
                 Myeloperoxidase deficiency 
               
               
                   
                 (p.Met251Thr) 
                   
                   
               
               
                   
               
               
                 587777390 
                 NM_005026.3(PIK3CD):c.1246T&gt;C 
                 GCAGGACYGCCCCATTGCCTGGG 
                 Activated PI3K-delta syndrome 
               
               
                   
                 (p.Cys416Arg) 
                   
                   
               
               
                   
               
               
                 587777480 
                 NM_003108.3(SOXI 1 ):c.I78T&gt;C 
                 TATGGYCCAAGATCGAACGCAGG 
                 Mental retardation, autosomal dominant 27 
               
               
                   
                 (p.Ser60Pro) 
                   
                   
               
               
                   
               
               
                 587777663 
                 NM_001288767.1(ARMC5):c.1379T&gt;C 
                 GCCCGACYGCGGGATGCTGGTGG 
                 Acth-independent macronodular adrenal 
               
               
                   
                 (p.Leu460Pro) 
                   
                 hyperplasia 2 
               
               
                   
               
               
                 61753033 
                 NM_000350.2(ABCA4):c.5819T&gt;C 
                 AAGGCYACATGAACTAACCAAGG 
                 Stargardt disease, Stargardt disease 1, Cone-rod dystrophy 3 
               
               
                   
                 (p.Leu1940Pro) 
                   
                   
               
               
                   
               
               
                 200488568 
                 NM_002972.3(SBF1):c.4768A&gt;G 
                 CAGGCGYCCTCTTGCTCAGCCGG 
                 Charcot-Marie-Tooth disease, type 4B3 
               
               
                   
                 (p.Thr1590Ala) 
                   
                   
               
               
                   
               
               
                 132630274 
                 NM_000377.2(WAS):c.809T&gt;C 
                 CGGAGTCYGTTCTCCAGGGCAGG 
                 Severe congenital neutropenia X-linked 
               
               
                   
                 (p.Leu270Pro) 
                   
                   
               
               
                   
               
               
                 132630308 
                 NM_001399.4(EDA):c.181T&gt;C 
                 CTGCYACCTAGAGTTGCGCTCGG 
                 Hypohidrotic X-linked ectodermal dysplasia 
               
               
                   
                 (p.Tyr61His) 
                   
                   
               
               
                   
               
               
                 60934003 
                 NM_170707.3(LMNA):c.1589T&gt;C 
                 ACGGCTCYCATCAACTCCACTGG, 
                 Benign scapuloperoneal muscular dystrophy 
               
               
                   
                 (p.Leu530Pro) 
                 CGGCTCYCATCAACTCCACTGGG, 
                 with cardiomyopathy 
               
               
                   
                   
                 GGCTCYCATCAACTCCACTGGGG 
                   
               
               
                   
               
               
                 180177160 
                 NM_000030.2(AGXT):c.1076T&gt;C 
                 GGTGCYGCGGATCGGCCTGCTGG, 
                 Primary hyperoxaluria, type I 
               
               
                   
                 (p.Leu359Pro) 
                 GTGCYGCGGATCGGCCTGCTGGG 
                   
               
               
                   
               
               
                 180177222 
                 NM_000030.2(AGXT):c.449T&gt;C 
                 GTGCYGCTGTTCTTAACCCACGG, 
                 Primary hyperoxaluria, type I 
               
               
                   
                 (p.Leu150Pro) 
                 TGCYGCTGTTCTTAACCCACGGG 
                   
               
               
                   
               
               
                 180177254 
                 NM_000030.2(AGXT):c.661T&gt;C 
                 GCTCATCYCCTTCAGTGACAAGG 
                 Primary hyperoxaluria, type I 
               
               
                   
                 (p.Ser221Pro) 
                   
                   
               
               
                   
               
               
                 180177264 
                 NM_000030.2(AGXT):c.757T&gt;C 
                 GGGGCYGTGACGACCAGCCCAGG 
                 Primary hyperoxaluria, type I 
               
               
                   
                 (p.Cys253Arg) 
                   
                   
               
               
                   
               
               
                 180177293 
                 NM_000030.2(AGXT):c.893T&gt;C 
                 GTATCYGCATGGGCGCCTGCAGG 
                 Primary hyperoxaluria, type I 
               
               
                   
                 (p.Leu298Pro) 
                   
                   
               
               
                   
               
               
                 376785840 
                 NM_001282227.1(CECR1):c.1232A&gt;G 
                 GAAATCAYAGGACAAGCCTTTGG 
                 Polyarteritis nodosa 
               
               
                   
                 (p.Tyr411Cys) 
                   
                   
               
               
                   
               
               
                 587779393 
                 NM_000257.3(MYH7):c.4937T&gt;C 
                 GAGCCYCCAGAGCTTGTTGAAGG 
                 Myopathy, distal, 1 
               
               
                   
                 (p.Leu1646Pro) 
                   
                   
               
               
                   
               
               
                 587779410 
                 NM_012434.4(SLC17A5):c.500T&gt;C 
                 ATTGTACYCAGAGCACTAGAAGG 
                 Sialic acid storage disease, severe infantile 
               
               
                   
                 (p.Leu167Pro) 
                   
                 type 
               
               
                   
               
               
                 587779513 
                 NM_000090.3(COL3A1):c.2337+2T&gt;C 
                 AGGYAACCCTTAATACTACCTGG 
                 Ehlers-Danlos syndrome, type 4 
               
               
                   
                 (p.Gly762_Lys779del) 
                   
                   
               
               
                   
               
               
                 777539013 
                 NM_020376.3(PNPLA2):c.757+2T&gt;C 
                 GAACGGYGCGCGGACCCGGGCGG, 
                 Neutral lipid storage disease with myopathy 
               
               
                   
                   
                 AACGGYGCGCGGACCCGGGCGGG 
                   
               
               
                   
               
               
                 34557412 
                 NM_012452.2(TNFRSF13B):c.310T&gt;C 
                 ACTTCYGTGAGAACAAGCTCAGG 
                 Immunoglobulin A deficiency 2, 
               
               
                   
                 (p.Cys104Arg) 
                   
                 Common variable 
               
               
                   
               
               
                 796052970 
                 NM_001165963.1(SCN1A):c.1094T&gt;C 
                 CAAGCTYTGATACCTTCAGTTGG, 
                 not provided 
               
               
                   
                 (p.Phe365Ser) 
                 AAGCTYTGATACCTTCAGTTGGG 
                   
               
               
                   
               
               
                 724159989 
                 NC_012920.1:m.7505T&gt;C 
                 CCTCCAYGACTTTTTCAAAAAGG 
                 Deafness, nonsyndromic sensorineural, 
               
               
                   
                   
                   
                 mitochondrial 
               
               
                   
               
               
                 796053222 
                 NM_014191.3(SCN8A):c.4889T&gt;C 
                 CGTCYGATCAAAGGCGCCAAAGG, 
                 not provided 
               
               
                   
                 (p.Leu1630Pro) 
                 GTCYGATCAAAGGCGCCAAAGGG 
                   
               
               
                   
               
               
                 118192127 
                 NM_000540.2(RYR1):c.10817T&gt;C 
                 TACTACCYGGACCAGGTGGGTGG, 
                 Central core disease 
               
               
                   
                 (p.Leu3606Pro) 
                 ACTACCYGGACCAGGTGGGTGGG, 
                   
               
               
                   
                   
                 CTACCYGGACCAGGTGGGTGGGG 
                   
               
               
                   
               
               
                 118192170 
                 NM_000540.2(RYR1):c.14693T&gt;C 
                 AGGCAYTGGGGACGAGATCGAGG 
                 Malignant hyperthermia susceptibility type 1, 
               
               
                   
                 (p.Ile4898Thr) 
                   
                 Central core disease 
               
               
                   
               
               
                 121917703 
                 NM_005247.2(FGF3):c.466T&gt;C 
                 GTACGTGYCTGTGAACGGCAAGG, 
                 Deafness with labyrinthine aplasia microtia and 
               
               
                   
                 (p.Ser156Pro) 
                 TACGTGYCTGTGAACGGCAAGGG 
                 microdontia (LAIVIM) 
               
               
                   
               
               
                 690016549 
                 NM_005211.3(CSF1R):c.2450T&gt;C 
                 CCGCCYGCCTGTGAAGTGGATGG 
                 Hereditary diffuse leukoencephalopathy h 
               
               
                   
                 (p.Leu817Pro) 
                   
                 spheroids 
               
               
                   
               
               
                 690016552 
                 NM_005211.3(CSF1R):c.2566T&gt;C 
                 GAATCCCYACCCTGGCATCCTGG 
                 Hereditary diffuse leukoencephalopathy with 
               
               
                   
                 (p.Tyr856His) 
                   
                 spheroids 
               
               
                   
               
               
                 121917738 
                 NM_001098668.2(SFTPA2):c.593T&gt;C 
                 GGAGACTYCCGCTACTCAGATGG, 
                 Idiopathic fibrosing alveolitis, chronic form 
               
               
                   
                 (p.Phe198Ser) 
                 GAGACTYCCGCTACTCAGATGGG 
                   
               
               
                   
               
               
                 690016559 
                 NM_005211.3(CSF1R):c.1957T&gt;C 
                 AGCCYGTACCCATGGAGGTAAGG, 
                 Hereditary diffuse leukoencephalopathy with 
               
               
                   
                 (p.Cys653Arg) 
                 GCCYGTACCCATGGAGGTAAGGG 
                 spheroids 
               
               
                   
               
               
                 690016560 
                 NM_005211.3(CSFIR):c.2717T&gt;C 
                 GCAGAYCTGCTCCTTCCTTCAGG 
                 Hereditary diffuse leukoencephalopathy with 
               
               
                   
                 (p.Ile906Thr) 
                   
                 spheroids 
               
               
                   
               
               
                 121917769 
                 NM_003361.3(UMOD):c.376T&gt;C 
                 GGCCACAYGTGTCAATGTGGTGG, 
                 Familial juvenile gout 
               
               
                   
                 (p.Cys126Arg) 
                 GCCACAYGTGTCAATGTGGTGGG 
                   
               
               
                   
               
               
                 121917773 
                 NM_003361.3(UMOD):c.943T&gt;C 
                 ATGGCACYGCCAGTGCAAACAGG 
                 Glomerulocystic kidney disease 
               
               
                   
                 (p.Cys315Arg) 
                   
                 with hyperuricemia and 
               
               
                   
                   
                   
                 isosthenuria 
               
               
                   
               
               
                 121917818 
                 NM_007255.2(B4GALT7):c.617T&gt;C 
                 TGCYCTCCAAGCAGCACTACCGG 
                 Ehlers-Danlos syndrome progeroid type 
               
               
                   
                 (p.Leu206Pro) 
                   
                   
               
               
                   
               
               
                 121917824 
                 NM_021615.4(CHST6):c.827T&gt;C 
                 GGACCYGGCGCGGGAGCCGCTGG 
                 Macular corneal dystrophy Type I 
               
               
                   
                 (p.Leu276Pro) 
                   
                   
               
               
                   
               
               
                 121917848 
                 NM_000452.2(SLC10A2):c.728T&gt;C 
                 TTTCYTCTGGCTAGAATTGCTGG 
                 Bile acid malabsorption, primary 
               
               
                   
                 (p.Leu243Pro) 
                   
                   
               
               
                   
               
               
                 121918006 
                 NM_000478.4(ALPL):c.1306T&gt;C 
                 TGGACYATGGTGAGACCTCCAGG 
                 Infantile hypophosphatasia 
               
               
                   
                 (p.Tyr436His) 
                   
                   
               
               
                   
               
               
                 121918010 
                 NM_000478.4(ALPL):c.979T&gt;C 
                 CAAAGGCYTCTTCTTGCTGGTGG, 
                 Infantile hypophosphatasia 
               
               
                   
                 (p.Phe327Leu) 
                 GGCYTCTTCTTGCTGGTGGAAGG 
                   
               
               
                   
               
               
                 121918088 
                 NM_000371.3(TTR):c.400T&gt;C 
                 CCCCYACTCCTATTCCACCACGG 
                   
               
               
                   
                 (p.Tyr134His) 
                   
                   
               
               
                   
               
               
                 121918110 
                 NM_001042465.1(PSAP):c.1055T&gt;C 
                 GAAGCYGCCGAAGTCCCTGTCGG 
                 Gaucher disease, atypical, due to saposin C 
               
               
                   
                 (p.Leu352Pro) 
                   
                 deficiency 
               
               
                   
               
               
                 121918137 
                 NM_003730.4(RNASET2):c.550T&gt;C 
                 CCAGYGCCTTCCACCAAGCCAGG 
                 Leukoencephalopathy, cystic, 
               
               
                   
                 (p.Cys184Arg) 
                   
                 without megalencephaly 
               
               
                   
               
               
                 121918191 
                 NM_001127628.1(FBP1):c.581T&gt;C 
                 GGAGTYCATTTTGGTGGACAAGG 
                 Fructose-biphosphatase deficiency 
               
               
                   
                 (p.Phe194Ser) 
                   
                   
               
               
                   
               
               
                 121918306 
                 NM_006946.2(SPTBN2):c.758T&gt;C 
                 ACCAAGCYGCTGGATCCCGAAGG, 
                 Spinocerebellar ataxia 5 
               
               
                   
                 (p.Leu253Pro) 
                 AAGCYGCTGGATCCCGAAGGTGG, 
                   
               
               
                   
                   
                 AGCYGCTGGATCCCGAAGGTGGG 
                   
               
               
                   
               
               
                 121918505 
                 NM_000141.4(FGFR2):c.799T&gt;C 
                 AATGCCYCCACAGTGGTCGGAGG 
                 Pfeiffer syndrome, Neoplasm of stomach 
               
               
                   
                 (p.Ser267Pro) 
                   
                   
               
               
                   
               
               
                 121918643 
                 NM_003126.2(SPTAI):c.620T&gt;C 
                 GTGGAGCYGGTAGCTAAAGAAGG, 
                 Hereditary pyropoikilocytosis, Elliptocytosis 2 
               
               
                   
                 (p.Leu207Pro) 
                 TGGAGCYGGTAGCTAAAGAAGGG 
                   
               
               
                   
               
               
                 121918646 
                 NM_001024858.2(SPTB):c.604T&gt;C 
                 CTCCAGCYGGAAGGATGGCTTGG 
                 Spherocytosis type 2 
               
               
                   
                 (p.Trp202Arg) 
                   
                   
               
               
                   
               
               
                 121918648 
                 NM_001024858.2(SPTB):c.6055T&gt;C 
                 ATGCCYCTGTGGCTGAGGCGTGG 
                   
               
               
                   
                 (p.Ser2019Pro) 
                   
                   
               
               
                   
               
               
                 727504166 
                 NM_000543.4(SMPD1):c.475T&gt;C 
                 TGAGGCCYGTGGCCTGCTCCTGG, 
                 Niemann-Pick disease, type A, Niemann-Pick 
               
               
                   
                 (p.Cys159Arg) 
                 GAGGCCYGTGGCCTGCTCCTGGG 
                 disease, type B 
               
               
                   
               
               
                 193922915 
                 NM_000434.3(NEU1):c.1088T&gt;C 
                 CAGCYATGGCCAGGCCCCAGTGG 
                 Sialidosis, type II 
               
               
                   
                 (p.Leu363Pro) 
                   
                   
               
               
                   
               
               
                 727504419 
                 NM_000501.3(ELN):c.889+2T&gt;C 
                 CAGGYAACATCTGTCCCAGCAGG, 
                 Supravalvar aortic stenosis 
               
               
                   
                   
                 AGGYAACATCTGTCCCAGCAGGG 
                   
               
               
                   
               
               
                 376395543 
                 NM_000256.3(MYBPC3):c.26- 
                 GAGACYGAAGGGCCAGGTGGAGG 
                 Primary familial hypertrophic 
               
               
                   
                 2A&gt;G 
                   
                 cardiomyopathy, Familial hypertrophic 
               
               
                   
                   
                   
                 cardiomyopathy 4, Cardiomyopathy 
               
               
                   
               
               
                 1169305 
                 NM_000545.6(RNIF1A):c.1720G&gt;A 
                 GATGCYGGCAGGGTCCTGGCTGG, 
                 Maturity-onset diabetes of the young, type 3 
               
               
                   
                 (p.Gly574Ser) 
                 ATGCYGGCAGGGTCCTGGCTGGG, 
                   
               
               
                   
                   
                 TGCYGGCAGGGTCCTGGCTGGGG 
                   
               
               
                   
               
               
                 730880130 
                 NM_000527.4(LDLR):c.1468T&gt;C 
                 CTACYGGACCGACTCTGTCCTGG, 
                 Familial hypercholesterolemia 
               
               
                   
                 (p.Trp490Arg) 
                 TACYGGACCGACTCTGTCCTGGG 
                   
               
               
                   
               
               
                 281860286 
                 NM_018713.2(SLC30A10):c.500T&gt;C 
                 GGCGCTTYCGGGGGGCCTCAGGG 
                 Hypermanganesemia with dystonia, 
               
               
                   
                 (p.Phe167Ser) 
                   
                 polycythemia and cirrhosis 
               
               
                   
               
               
                 730880306 
                 NM_145693.2(LPIN1):c.441+2T&gt;C 
                 AAGGYACCGCGGGCCTCGCGCGG, 
                 Myoglobinuria, acute recurrent, autosomal 
               
               
                   
                   
                 AGGYACCGCGGGCCTCGCGCGGG 
                 recessive 
               
               
                   
               
               
                 74315452 
                 NM_000454.4(SOD1):c.338T&gt;C 
                 TTGCAYCATTGGCCGCACACTGG 
                 Amyotrophic lateral sclerosis type 1 
               
               
                   
                 (p.Ile113Thr) 
                   
                   
               
               
                   
               
               
                 730880455 
                 NM_000169.2(GLA):c.4IT&gt;C 
                 CGCGCYTGCGCTTCGCTTCCTGG 
                 not provided 
               
               
                   
                 (p.Leul4Pro) 
                   
                   
               
               
                   
               
               
                 267606656 
                 NM_054027.4(ANKH):c.1015T&gt;C 
                 AGCTCYGTTTCGTGATGTTTTGG 
                 Craniometaphyseal dysplasia, autosomal 
               
               
                   
                 (p.Cys339Arg) 
                   
                 dominant 
               
               
                   
               
               
                 267606687 
                 NM_033409.3(SLC52A3):c.1238T&gt;C 
                 AGTTACGYCAAGGTGATGCTGGG 
                 Brown-Vialetto-Van laere syndrome 
               
               
                   
                 (p.Val413Ala) 
                   
                   
               
               
                   
               
               
                 267606721 
                 NM_001928.2(CFD):c.640T&gt;C 
                 GGTGYGCGGGGGCGTGCTCGAGG, 
                 Complement factor d deficiency 
               
               
                   
                 (p.Cys214Arg) 
                 GTGYGCGGGGGCGTGCTCGAGGG 
                   
               
               
                   
               
               
                 267606747 
                 NM_001849.3(COL6A2):c.2329T&gt;C 
                 CGCCYGCGACAAGCCACAGCAGG 
                 Ullrich congenital muscular dystrophy 
               
               
                   
                 (p.Cys777Arg) 
                   
                   
               
               
                   
               
               
                 431905515 
                 NM_001044.4(SLC6A3):c.671T&gt;C 
                 CTGCACCYCCACCAGAGCCATGG 
                 Infantile Parkinsonism-dystonia 
               
               
                   
                 (p.Leu224Pro) 
                   
                   
               
               
                   
               
               
                 267606857 
                 NM_000180.3(GUCY2D):c.2846T&gt;C 
                 AGAGAYCGCCAACATGTCACTGG 
                 Cone-rod dystrophy 6 
               
               
                   
                 (p.Ile949Thr) 
                   
                   
               
               
                   
               
               
                 267606880 
                 NM_022489.3(INF2):c.125T&gt;C 
                 GCTGCYCCAGATGCCCTCTGTGG 
                 Focal segmental glomerulosclerosis 5 
               
               
                   
                 (p.Leu42Pro) 
                   
                   
               
               
                   
               
               
                 515726191 
                 NM_015713.4(RRM2B):c.581A&gt;G 
                 AACTCCTYCTACAGCAGCAAAGG 
                 RRM2B-related mitochondrial disease 
               
               
                   
                 (p.Glu194Gly) 
                   
                   
               
               
                   
               
               
                 267606917 
                 NM_004646.3(NPHS1):c.793T&gt;C 
                 GCTGCCGYGCGTGGCCCGAGGGG, 
                 Finnish congenital nephrotic syndrome 
               
               
                   
                 (p.Cys265Arg) 
                 CTGCCGYGCGTGGCCCGAGGGGG 
                   
               
               
                   
               
               
                 267607104 
                 NM_001199107.1(TBC1D24):c.751T&gt;C 
                 CAAGTTCYTCCACAAGGTGAGGG, 
                 Myoclonic epilepsy, familial infantile 
               
               
                   
                 (p.Phe251Leu) 
                 TTCYTCCACAAGGTGAGGGCCGG 
                   
               
               
                   
               
               
                 267607182 
                 NM_144631.5(ZNF513):c.1015T&gt;C 
                 TGGGCGCYGCATGCGAGGAGAGG, 
                 Retinitis pigmentosa 58 
               
               
                   
                 (p.Cys339Arg) 
                 CGCYGCATGCGAGGAGAGGCTGG 
                   
               
               
                   
               
               
                 267607211 
                 NM_000229.1(LCAT):c.508T&gt;C 
                 TATGACYGGCGGCTGGAGCCCGG 
                 Norum disease 
               
               
                   
                 (p.Trp170Arg) 
                   
                   
               
               
                   
               
               
                 267607215 
                 NM_016269.4(LEF1):c.181T&gt;C 
                 GAACGAGYCTGAAATCATCCCGG 
                 Sebaceous tumors, somatic 
               
               
                   
                 (p.Ser61Pro} 
                   
                   
               
               
                   
               
               
                 587783580 
                 NM_178151.2(DCX):c.683T&gt;C 
                 AAAAAACYCTACACTCTGGATGG 
                 Heterotopia 
               
               
                   
                 (p.Leu228Pro) 
                   
                   
               
               
                   
               
               
                 587783644 
                 NM_004004.5(GM2):c.107T&gt;C 
                 GATCCYCGTTGTGGCTGCAAAGG 
                 Hearing impairment 
               
               
                   
                 (p.Leu36Pro) 
                   
                   
               
               
                   
               
               
                 587783653 
                 NM_005682.6(ADGRG1):c.1460T&gt;C 
                 CCCTGCYCACCTGCCTTTCCTGG 
                 Polymicrogyria, bilateral frontoparietal 
               
               
                   
                 (p.Leu487Pro) 
                   
                   
               
               
                   
               
               
                 587783863 
                 NM_000252.2(MTM1):c.958T&gt;C 
                 GGAAYCTTTAAAAAAAGTGAAGG 
                 Severe X-linked myotubular myopathy 
               
               
                   
                 (p.Ser320Pro) 
                   
                   
               
               
                   
               
               
                 267607751 
                 NM_000249.3(MLH1):c.453+2T&gt;C 
                 ATCACGGYAAGAATGGTACATGG, 
                 Hereditary Nonpolyposis Colorectal Neoplasms 
               
               
                   
                   
                 TCACGGYAAGAATGGTACATGGG 
                   
               
               
                   
               
               
                 119103227 
                 NM_000411.6(HLCS):c.710T&gt;C 
                 CTATCYTTCTCAGGGAGGGAAGG 
                 Holocarboxylase synthetase deficiency 
               
               
                   
                 (p.Leu237Pro) 
                   
                   
               
               
                   
               
               
                 119103237 
                 NM_005787.5(ALG3):c.211T&gt;C 
                 GATTGACYGGAAGGCCTACATGG 
                 Congenital disorder of glycosylation type 1D 
               
               
                   
                 (p.Trp7lArg) 
                   
                   
               
               
                   
               
               
                 398122806 
                 NM_003172.3(SURF1):c.679T&gt;C 
                 CCACYGGCATTATCGAGACCTGG 
                 Congenital myasthenic syndrome, 
               
               
                   
                 (p.Trp227Arg) 
                   
                 acetazolamide-responsive 
               
               
                   
               
               
                 80338747 
                 NM_004525.2(LRP2):c.7564T&gt;C 
                 GTACCTGYACTGGGCTGACTGGG 
                 Donnai Barrow syndrome 
               
               
                   
                 (p.Tyr2522His) 
                   
                   
               
               
                   
               
               
                 398122838 
                 NM_001271723.1(FBX038):c.616T&gt;C 
                 TTCCTYGTATCCCAATGCTAAGG 
                 Distal hereditary motor neuronopathy 2D 
               
               
                   
                 (p.Cys206Arg) 
                   
                   
               
               
                   
               
               
                 398122989 
                 NM_014495.3(ANGPTL3):c.883T&gt;C 
                 ACAAAACYTCAATGAAACGTGGG 
                 Hypobetalipoproteinemia, familial, 2 
               
               
                   
                 (p.Phe295Leu) 
                   
                   
               
               
                   
               
               
                 80338945 
                 NM_004004.5(GM2):c.269T&gt;C 
                 GCTCCYAGTGGCCATGCACGTGG 
                 Deafness, autosomal recessive 1A, Hearing 
               
               
                   
                 (p.Leu90Pro) 
                   
                 impairment 
               
               
                   
               
               
                 80338956 
                 NM_000334.4(SCN4A):c.2078T&gt;C 
                 AAGATCAYTGGCAATTCAGTGGG, 
                 Hyperkalemic Periodic Paralysis Type 1, 
               
               
                   
                 (p.Ile693Thr) 
                 AGATCAYTGGCAATTCAGTGGGG, 
                 Paramyotonia congenita of von Eulenburg 
               
               
                   
                   
                 GATCAYTGGCAATTCAGTGGGGG 
                   
               
               
                   
               
               
                 267608131 
                 NM_000179.2(MSH6):c.4001+2T&gt;C 
                 CGGYAACTAACTAACTATAATGG 
                 Hereditary Nonpolyposis Colorectal Neoplasms 
               
               
                   
               
               
                 587784573 
                 NM_004963.3(GUCY2C):c.2782T&gt;C 
                 TCCCYGTGCTGCTGGAGTTGTGG, 
                 Meconium ileus 
               
               
                   
                 (p.Cys928Arg) 
                 CCCYGTGCTGCTGGAGTTGTGGG 
                   
               
               
                   
               
               
                 267608511 
                 NM_003159.2(CDKL5):c.659T&gt;C 
                 CCAACYTTTTACTATTCAGAAGG 
                 Early infantile epileptic encephalopathy 2 
               
               
                   
                 (p.Leu220Pro) 
                   
                   
               
               
                   
               
               
                 373842615 
                 NM_000118.3(ENG):c.1273-2A&gt;G 
                 CCGCCYGCGGGGATAAAGCCAGG, 
                 Haemorrhagic telangiectasia 1 
               
               
                   
                   
                 CGCCYGCGGGGATAAAGCCAGGG 
                   
               
               
                   
               
               
                 185492581 
                 NM_000335.4(SCN5A):c.376A&gt;G 
                 GAATCTYCACAGCCGCTCTCCGG 
                 Brugada syndrome 
               
               
                   
                 (p.Lys126Glu) 
                   
                   
               
               
                   
               
               
                 200533370 
                 NM_133499.2(SYN1):c.1699A&gt;G 
                 GATGYCTGACGGGTAGCCTGTGG, 
                 Epilepsy, X-linked, with variable learning 
               
               
                   
                 (p.Thr567Ala) 
                 ATGYCTGACGGGTAGCCTGTGGG 
                 disabilities and behavior disorders, not specified 
               
               
                   
               
               
                 118203981 
                 NM_148960.2(CLDN19):c.269T&gt;C 
                 GCTCCYGGGCTTCGTGGCCATGG 
                 Hypomagnesemia 5, renal, with ocular 
               
               
                   
                 (p.Leu90Pro) 
                   
                 involvement 
               
               
                   
               
               
                 137853892 
                 NM_001235.3(SERPINH1):c.233T&gt;C 
                 GTCGCYAGGGCTCGTGTCGCTGG, 
                 Osteogenesis imperfecta type 10 
               
               
                   
                 (p.Leu78Pro) 
                 TCGCYAGGGCTCGTGTCGCTGGG 
                   
               
               
                   
               
               
                 118204024 
                 NM_000263.3(NAGLU):c.142T&gt;C 
                 GGCCGACYTCTCCGTGTCGGTGG 
                 Mucopolysaccharidosis,MPS-III-B 
               
               
                   
               
               
                 690016563 
                 NM_005211.3(CSF1R):c.1745T&gt;C 
                 CAACCYGCAGTTTGGTGAGATGG 
                 Hereditary diffuse leukoencephalopathy with 
               
               
                   
                 (p.Leu582Pro) 
                   
                 spheroids 
               
               
                   
               
               
                 58380626 
                 NM_000526.4(KRT14):c.1243T&gt;C 
                 CGCCACCYACCGCCGCCTGCTGG, 
                 Epidermolysis bullosa herpetiformis, 
               
               
                   
                 (p.Tyr415His) 
                 CACCYACCGCCGCCTGCTGGAGG, 
                 Dowling- Meara 
               
               
                   
                   
                 ACCYACCGCCGCCTGCTGGAGGG 
                   
               
               
                   
               
               
                 113994151 
                 NM_207346.2(TSEN54):c.277T&gt;C 
                 TTGAAGYCTCCCGCGGTGAGCGG, 
                 Pontocerebellar hypoplasia type 4 
               
               
                   
                 (p.Ser93Pro) 
                 AAGYCTCCCGCGGTGAGCGGCGG 
                   
               
               
                   
               
               
                 113994206 
                 NM_004937.2(CTNS):c.473T&gt;C 
                 TGGTCYGAGCTTCGACTTCGTGG 
                 Cystinosis 
               
               
                   
                 (p.Leu158Pro) 
                   
                   
               
               
                   
               
               
                 62516109 
                 NM_000277.1(PAH):c.638T&gt;C 
                 CCACTTCYTGAAAAGTACTGTGG 
                 Phenylketonuria 
               
               
                   
                 (p.Leu213Pro) 
                   
                   
               
               
                   
               
               
                 370011798 
                 NM_001302946.1(TRNT1):c.668T&gt;C 
                 GCAAYTGCAGAAAATGCAAAAGG 
                 Sideroblastic anemia with B-cell 
               
               
                   
                 (p.Ile223Thr) 
                   
                 immunodeficiency, periodic fevers, 
               
               
                   
                   
                   
                 and developmental delay 
               
               
                   
               
               
                 62517167 
                 NM_000277.1(PAH):c.293T&gt;C 
                 AAGATCTYGAGGCATGACATTGG 
                 Mild non-PKU hyperphenylalanem a 
               
               
                   
                 (p.Leu98Ser) 
                   
                   
               
               
                   
               
               
                 12021720 
                 NM_001918.3(DBT):c.1150G&gt;A 
                 GACYCACAGAGCCCAATTTCTGG 
                 Intermediate maple syrup urine disease type 2 
               
               
                   
                 (p.Gly384Ser) 
                   
                   
               
               
                   
               
               
                 104886289 
                 NM_000495.4(COL4A5):c.4756T&gt;C 
                 TCCCCATYGTCCTCAGGGATGGG 
                 Alport syndrome, X-linked recessive 
               
               
                   
                 (p.Cys1586Arg) 
                   
                   
               
               
                   
               
               
                 370471013 
                 NC_012920.1:m.5559A&gt;G 
                 CAACYTACTGAGGGCTTTGAAGG 
                 Leigh disease 
               
               
                   
               
               
                 121434215 
                 NM_000487.5(ARSA):c.410T&gt;C 
                 GCCTTCCYGCCCCCCCATCAGGG 
                 Metachromatic leukodystrophy, adult type 
               
               
                   
                 (p.Leu137Pro) 
                   
                   
               
               
                   
               
               
                 386134128 
                 NM_000096.3(CP):c.1123T&gt;C 
                 ACACTACYACATTGCCGCTGAGG 
                 Deficiency of ferroxidase 
               
               
                   
                 (p.Tyr375His) 
                   
                   
               
               
                   
               
               
                 121434275 
                 NM_001127328.2(ACADM):c.1136T&gt;C 
                 GTGCAGAYACTTGGAGGCAATGG 
                 Medium-chain acyl-coenzyme 
               
               
                   
                 (p.Ile379Thr) 
                   
                 A dehydrogenase deficiency 
               
               
                   
               
               
                 121434276 
                 NM_001127328.2(ACADM):c.1136T&gt;C 
                 CAGCGAYGTTCAGATACTAGAGG 
                 Medium-chain acyl-coenzyme 
               
               
                   
                 (p.Cys248Arg) 
                   
                 A dehydrogenase deficiency 
               
               
                   
               
               
                 121434284 
                 NM_002225.3(IVD):c.134T&gt;C 
                 ATGGGCYAAGCGAGGAGCAGAGG 
                 ISOVALERIC ACIDEMIA, TYPE I 
               
               
                   
                 (p.Leu45Pro) 
                   
                   
               
               
                   
               
               
                 121434334 
                 NM_005908.3(MANBA):c.1513T&gt;C 
                 ATTACGYCCAGTCCTACAAATGG, 
                 Beta-D-mannosidosis 
               
               
                   
                 (p.Ser505Pro) 
                 TTACGYCCAGTCCTACAAATGGG, 
                   
               
               
                   
                   
                 TACGYCCAGTCCTACAAATGGGG 
                   
               
               
                   
               
               
                 121434366 
                 NM_000159.3(GCDH):c.883T&gt;C 
                 CGCCCGGYACGGCATCGCGTGGG, 
                 Glutaric aciduria, type 1 
               
               
                   
                 (p.Tyr295His) 
                 GCCCGGYACGGCATCGCGTGGGG 
                   
               
               
                   
               
               
                 60715293 
                 NM_000424.3(KRT5):c.541T&gt;C 
                 GTTTGCCYCCTTCATCGACAAGG 
                 Epidermolysis bullosa herpetiformis, 
               
               
                   
                 (p.Ser181Pro) 
                   
                 Dowling-Meara 
               
               
                   
               
               
                 121434409 
                 NM_001003722.1(GLE1):c.2051T&gt;C 
                 AAGGACAYTCCTGTCCCCAAGGG 
                 Lethal arthrogryposis with anterior horn cell 
               
               
                   
                 (p.Ile684Thr) 
                   
                 disease 
               
               
                   
               
               
                 121434434 
                 NM_001287.5(CLCN7):c.2297T&gt;C 
                 GGGCCYGCGGCACCTGGTGGTGG 
                 Osteopetrosis autosomal recessive 4 
               
               
                   
                 (p.Leu766Pro) 
                   
                   
               
               
                   
               
               
                 121434455 
                 NM_000466.2(PEX1):c.1991T&gt;C 
                 GATGACCYTGACCTCATTGCTGG 
                 Zellweger syndrome 
               
               
                   
                 (p.Leu664Pro) 
                   
                   
               
               
                   
               
               
                 199422317 
                 NM_001099274.1(TINF2):c.862T&gt;C 
                 CTGYTTCCCTTTAGGAATCTCGG 
                 Aplastic anemia 
               
               
                   
                 (p.Phe288Leu) 
                   
                   
               
               
                   
               
               
                 104895221 
                 NM_001065.3(TNFRSF1A):c.349T&gt;C 
                 CTCTTCTYGCACAGTGGACCGGG 
                 TNF receptor-associated periodic fever 
               
               
                   
                 (p.Cys117Arg) 
                   
                 syndrome (TRAPS) 
               
               
                   
               
               
                 137854459 
                 NM_000138.4(FBN1):c.4987T&gt;C 
                 GGGACAYGTTACAACACCGTTGG 
                 Marfan syndrome 
               
               
                   
                 (p.Cys1663Arg) 
                   
                   
               
               
                   
               
               
                 387907075 
                 NM_024027.4(COLEC11):c.505T&gt;C 
                 CAGCTGYCCTGCCAGGGCCGCGG, 
                 Carnevale syndrome 
               
               
                   
                 (p.Ser169Pro) 
                 AGCTGYCCTGCCAGGGCCGCGGG, 
                   
               
               
                   
                   
                 GCTGYCCTGCCAGGGCCGCGGGG, 
                   
               
               
                   
                   
                 CTGYCCTGCCAGGGCCGCGGGGG 
                   
               
               
                   
               
               
                 1048095 
                 NM_000352.4(ABCC8):c.674T&gt;C 
                 TGCYGTCCAAAGGCACCTACTGG 
                 Permanent neonatal diabetes mellitus 
               
               
                   
                 (p.Leu225Pro) 
                   
                   
               
               
                   
               
               
                 796065347 
                 NM_019074.3(DLL4):c.1168T&gt;C 
                 GAAYGTCCCCCCAACTTCACCGG 
                 Adams-Oliver syndrome, ADAMS- 
               
               
                   
                 (p.Cys390Arg) 
                   
                 OLIVER SYNDROME 6 
               
               
                   
               
               
                 137852347 
                 NM_000402.4(G6PD):c.1054T&gt;C 
                 AGGGYACCTGGACGACCCCACGG 
                 Anemia, nonspherocytic hemolytic, due to 
               
               
                   
                 (p.Tyr352His) 
                   
                 G6PD deficiency 
               
               
                   
               
               
                 74315327 
                 NM_213653.3(HFE2):c.302T&gt;C 
                 GGACCYCGCCTTCCATTCGGCGG 
                 Hemochromatosis type 2A 
               
               
                   
                 (p.Leu101Pro) 
                   
                   
               
               
                   
               
               
                 137852579 
                 NM_000044.3(AR):c.2033T&gt;C 
                 GTCCYGGAAGCCATTGAGCCAGG 
                   
               
               
                   
                 (p.Leu678Pro) 
                   
                   
               
               
                   
               
               
                 137852636 
                 NM_001166107.1(IIMGCS2):c.520T&gt;C 
                 CCCTCYTCAATGCTGCCAACTGG 
                 mitochondria 3-hydroxy-3-methylglutaryl- 
               
               
                   
                 (p.Phe174Leu) 
                   
                 CoA synthase deficiency 
               
               
                   
               
               
                 137852661 
                 NM_033163.3(FGF8):c.118T&gt;C 
                 TTCCCTGYTCCGGGCTGGCCGGG 
                 Kallmann syndrome 6 
               
               
                   
                 (p.Phe40Leu) 
                   
                   
               
               
                   
               
               
                 121912967 
                 NM_005215.3(DCC):c.503T&gt;C 
                 AGCCCAYGCCAACAATCCACTGG 
                   
               
               
                   
                 (p.Met168Thr) 
                   
                   
               
               
                   
               
               
                 137852806 
                 NM_001039523.2(CHRNA1):c.901T&gt;C 
                 TGTGYTCCTTCTGGTCATCGTGG 
                 Myasthenic syndrome, congenital, fast-channel 
               
               
                   
                 (p.Phe301Leu) 
                   
                   
               
               
                   
               
               
                 137852850 
                 NM_182760.3(SUMF1):c.463T&gt;C 
                 GGCGACYCCTTTGTCTTTGAAGG 
                 Multiple sulfatase deficiency 
               
               
                   
                 (p.Ser155Pro) 
                   
                   
               
               
                   
               
               
                 137852886 
                 NM_000158.3(GBE1):c.671T&gt;C 
                 AATGTACYACCAAGAATCAAAGG 
                 Glycogen storage disease, type IV, 
               
               
                   
                 (p.Leu224Pro) 
                   
                 GLYCOGEN STORAGE DISEASE 
               
               
                   
                   
                   
                 IV, NONPROGRESSIVE HEPATIC 
               
               
                   
               
               
                 137852911 
                 NM_000419.3(ITGA2B):c.641T&gt;C 
                 CTGGTGCYTGGGGCTCCTGGCGG 
                 Glanzmann thrombasthenia 
               
               
                   
                 (p.Leu214Pro) 
                   
                   
               
               
                   
               
               
                 137852948 
                 NM_138694.3(PKHD1):c.10658T&gt;C 
                 GAGCCCAYTGAAATACGCTCAGG 
                 Polycystic kidney disease, infantile type 
               
               
                   
                 (p.Ile3553Thr) 
                   
                   
               
               
                   
               
               
                 137852964 
                 NM_024960.4(PANK2):c.178T&gt;C 
                 ATTGACYCAGTCGGATTCAATGG 
                   
               
               
                   
                 (p.Ser60Pro) 
                   
                   
               
               
                   
               
               
                 137853020 
                 NM_006899.3(IDH3B):c.395T&gt;C 
                 TGCGGCYGAGGTAGGTGGTCTGG, 
                 Retinitis pigmentosa 46 
               
               
                   
                 (p.Leu132Pro) 
                 GCGGCYGAGGTAGGTGGTCTGGG 
                   
               
               
                   
               
               
                 137853249 
                 NM_033500.2(HK1):c.1550T&gt;C 
                 GACTTCTYGGCCCTGGATCTTGG, 
                 Hemolytic anemia due to hexokinase deficiency 
               
               
                   
                 (p.Leu517Ser) 
                 TTCTYGGCCCTGGATCTTGGAGG 
                   
               
               
                   
               
               
                 137853270 
                 NM_000444.5(PHEX):c.1664T&gt;C 
                 AGCYCCAGAAGCCTTTCTTTTGG 
                 Familial X-linked hypophosphatemic vitamin 
               
               
                   
                 (p.Leu555Pro) 
                   
                 D refractory rickets 
               
               
                   
               
               
                 137853325 
                 NM_003639.4(IKBKG):c.1249T&gt;C 
                 TGGAGYGCATTGAGTAGGGCCGG 
                 Hypohidrotic ectodermal dysplasia with immune 
               
               
                   
                 (p.Cys417Arg) 
                   
                 deficiency, Hyper-IgM immunodeficiency, X- 
               
               
                   
                   
                   
                 linked, with hypohidrotic ectodermal dysplasia 
               
               
                   
               
               
                 28932769 
                 NM_002055.4(GFAP):c.1055T&gt;C 
                 GGACCYGCTCAATGTCAAGCTGG 
                 Alexander disease 
               
               
                   
                 (p.Leu352Pro) 
                   
                   
               
               
                   
               
               
                 397507439 
                 NM_002769.4(PRSS1):c.116T&gt;C 
                 TACCAGGYGTCCCTGAATTCTGG 
                 Hereditary pancreatitis 
               
               
                   
                 (p.Val39Ala) 
                   
                   
               
               
                   
               
               
                 387906446 
                 NM_000132.3(F8):c.1729T&gt;C 
                 AAAGAAYCTGTAGATCAAAGAGG 
                 Hereditary factor VIII deficiency disease 
               
               
                   
                 (p.Ser577Pro) 
                   
                   
               
               
                   
               
               
                 387906482 
                 NM_000133.3(F9):c.1031T&gt;C 
                 ACGAACAYCTTCCTCAAATTTGG 
                 Hereditary factor LX deficiency disease 
               
               
                   
                 (p.Ile344Thr) 
                   
                   
               
               
                   
               
               
                 387906508 
                 NM_000131.4(F7):c.983T&gt;C 
                 GACGTYCTCTGAGAGGACGCTGG 
                 Factor VII deficiency 
               
               
                   
                 (p.Phe328Ser) 
                   
                   
               
               
                   
               
               
                 387906532 
                 NM_001040113.1(MYH11):c.3791T&gt;C 
                 GAAGCYGGAGGCGCAGGTGCAGG 
                 Aortic aneurysm, familial thoracic 4 
               
               
                   
                 (p.Leu1264Pro) 
                   
                   
               
               
                   
               
               
                 387906658 
                 NM_002465.3(MYBPC1):c.2566T&gt;C 
                 CAAACCYATATCCGCAGAGTTGG 
                 Distal arthrogryposis type 1B 
               
               
                   
                 (p.Tyr856His) 
                   
                   
               
               
                 387906701 
                 NM_003491.3(NAA10):c.109T&gt;C 
                 TGGCCTTYCCTGGCCCCAGGTGG, 
                 N-terminal acetyltransferase deficiency 
               
               
                   
                   
                 GGCCTTYCCTGGCCCCAGGTGGG 
                   
               
               
                   
                   
                 GACTTCAYTGAGGACCAGGGTGG, 
                   
               
               
                   
               
               
                 387906717 
                 NM_000377.2(WAS):c.881T&gt;C 
                 ACTTCAYTGAGGACCAGGGTGGG 
                 Severe congenital neutropenia X-linked 
               
               
                   
                 (p.Ile294Thr) 
                   
                   
               
               
                   
               
               
                 387906809 
                 NM_000287.3(PEX6):c.1601T&gt;C 
                 CTTCYGGGCCGGGACCGTGATGG, 
                 Peroxisome biogenesis disorder 4B 
               
               
                   
                 (p.Leu534Pro) 
                 TTCYGGGCCGGGACCGTGATGGG 
                   
               
               
                   
               
               
                 387906965 
                 NM_024513.3(FYC01):c.4127T&gt;C 
                 CAGCCYGATCCCCATCACTGTGG 
                 Cataract, autosomal recessive congenital 2 
               
               
                   
                 (p.Leu1376Pro) 
                   
                   
               
               
                   
               
               
                 387906967 
                 NM_006147.3(IRF6):c.65T&gt;C 
                 GCCYCTACCCTGGGCTCATCTGG 
                 Van der Woude syndrome, Popliteal pterygium 
               
               
                   
                 (p.Leu22Pro) 
                   
                 syndrome 
               
               
                   
               
               
                 387906982 
                 NM_025132.3(WDR19):c.20T&gt;C 
                 TCTCACYGCTAGAAAAGACTTGG 
                 Asphyxiating thoracic dystrophy 5 
               
               
                   
                 (p.Leu7Pro) 
                   
                   
               
               
                   
               
               
                 387907072 
                 NM_032446.2(MEGF10):c.2320T&gt;C 
                 GGGCAGYGTACTTGCCGCACTGG 
                 Myopathy, areflexia, respiratory distress, and 
               
               
                   
                 (p.Cys774Arg) 
                   
                 dysphagia, early-onset, Myopathy, areflexia, 
               
               
                   
                   
                   
                 respiratory distress, and dysphagia, early-onset, mild variant 
               
               
                   
               
               
                 137854499 
                 NM_005502.3(ABCA1):c.6026T&gt;C 
                 GAGTYCTTTGCCCTTTTGAGAGG 
                 Familial hypoalphalipoproteinemia 
               
               
                   
                 (p.Phe2009Ser) 
                   
                   
               
               
                   
               
               
                 387907117 
                 NM_000196.3(HSD11B2):c.1012T&gt;C 
                 CCGCCGCYATTACCCCGGCCAGG, 
                 Apparent mineralocorticoid excess 
               
               
                   
                 (p.Tyr338His) 
                 CGCCGCYATTACCCCGGCCAGGG 
                   
               
               
                   
               
               
                 387907170 
                 NM_004453.3(ETFDH):c.1130T&gt;C 
                 CCAAAACYCACCTTTCCTGGTGG 
                   
               
               
                   
                 (p.Leu377Pro) 
                   
                   
               
               
                   
               
               
                 387907205 
                 NM_033360.3(KRAS):c.211T&gt;C 
                 GGACCAGYACATGAGGACTGGGG, 
                 Cardiofaciocutaneous syndrome 2 
               
               
                   
                 (p.Tyr71His) 
                 CCAGYACATGAGGACTGGGGAGG, 
                   
               
               
                   
                   
                 CAGYACATGAGGACTGGGGAGGG 
                   
               
               
                   
               
               
                 387907240 
                 NM_024110.4(CARD14):c.467T&gt;C 
                 CAGCAGCYGCAGGAGCACCTGGG 
                 Pityriasis rubra pilaris 
               
               
                   
                 (p.Leu156Pro) 
                   
                   
               
               
                   
               
               
                 387907282 
                 NM_152296.4(ATP1A3):c.2431T&gt;C 
                 TGCCATCYCACTGGCGTACGAGG 
                 Alternating hemiplegia of childhood 2 
               
               
                   
                 (p.Ser811Pro) 
                   
                   
               
               
                   
               
               
                 387907361 
                 NM_005120.2(MED12):c.3493T&gt;C 
                 AGGACYCTGAGCCAGGGGCCCGG 
                 Ohdo syndrome, X-linked 
               
               
                   
                 (p.Ser1165Pro) 
                   
                   
               
               
                   
               
               
                 28933970 
                 NM_006194.3(PAX9):c.62T&gt;C 
                 GGCCGCYGCCCAACGCCATCCGG 
                 Tooth agenesis, selective, 3 
               
               
                   
                 (p.Leu21Pro) 
                   
                   
               
               
                   
               
               
                 137854472 
                 NM_000138.4(FBN1):c.3128A&gt;G 
                 TGCACYTGCCGTGGGTGCAGAGG 
                 Cardiomyopathy, not specified 
               
               
                   
                 (p.Lys1043Arg) 
                   
                   
               
               
                   
               
               
                 727504261 
                 NM_000257.3(MYH7):c.2708A&gt;G 
                 AGCGCYCCTCAGCATCTGCCAGG 
                   
               
               
                   
                 (p.Glu903Gly) 
                   
                   
               
               
                   
               
               
                 81002853 
                 NM_000059.3(BRCA2):c.476- 
                 ACCACYGGGGGTAAAAAAAGGGG, 
                 Familial cancer of breast, Breast-ovarian 
               
               
                   
                 2A&gt;G 
                 TACCACYGGGGGTAAAAAAAGGG, 
                 cancer, familial 2, Hereditary cancer-predisposing syndrome 
               
               
                   
               
               
                 119473032 
                 NM_021020.3(LZTS1):c.355A&gt;G 
                 CCCTYCTCGGAGCCCTGTAGAGG 
                   
               
               
                   
                 (p.Lys119Glu) 
                   
                   
               
               
                   
               
               
                 193922801 
                 NM_000540.2(RYR1):c.7043A&gt;G 
                 TTCYCCTCCACGCTCTCGCCTGG 
                 not provided 
               
               
                   
                 (p.Glu2348Gly) 
                   
                   
               
               
                   
               
               
                 36210419 
                 NM_000218.2(KCNQ1):c.652A&gt;G 
                 GCCCCTYGGAGCCCACGCAGAGG 
                 Torsades de pointes, Cardiac arrhythmia 
               
               
                   
                 (p.Lys218Glu) 
                   
                   
               
               
                   
               
               
                 121964989 
                 NM_000108.4(DLD):c.1483A&gt;G 
                 TTCTCYAAAAGCTTCTGATAAGG 
                 Maple syrup urine disease, type 3 
               
               
                   
                 (p.Arg495Gly) 
                   
                   
               
               
                   
               
               
                 28936669 
                 NM_000095.2(COMP):c.1418A&gt;G 
                 ATTGYCGTCGTCGTCGTCGCAGG 
                   
               
               
                   
                 (p.Asp473Gly) 
                   
                   
               
               
                   
               
               
                 28936696 
                 NM_018488.2(TBX4):c.1592A&gt;G 
                 GTACYGTAAGGAAGATTCTCGGG, 
                 Ischiopatellar dysplasia 
               
               
                   
                 (p.Gln531Arg) 
                 GGTACYGTAAGGAAGATTCTCGG 
                   
               
               
                   
               
               
                 121965077 
                 NM_000137.2(FAH):c.1141A&gt;G 
                 TCCYGGTCTGACCATTCCCCAGG 
                 Tyrosinemia type I 
               
               
                   
                 (p.Arg381Gly) 
                   
                   
               
               
                   
               
               
                 794728203 
                 NM_000138.4(FBND:c.3344A&gt;G 
                 ACTCAYCAATATCTGCAAAATGG 
                 Thoracic aortic aneurysms and aortic 
               
               
                   
                 (p.Asp1115Gly) 
                   
                 dissections 
               
               
                   
               
               
                 786205436 
                 NM_003002.3(SDHD):c.275A&gt;G 
                 GAATAGYCCATCGCAGAGCAAGG 
                 Fatal infantile mitochondrial cardiomyopathy 
               
               
                   
                 (p.Asp92Gly) 
                   
                   
               
               
                   
               
               
                 72551317 
                 NM_000784.3(CYP27A1):c.776A&gt;G 
                 AGTCCACYTGGGGAGGAAGGTGG 
                 Cholestanol storage disease 
               
               
                   
                 (p.Lys259Arg) 
                   
                   
               
               
                   
               
               
                 786205687 
                 NM_016218.2(POLK):c.1385A&gt;G 
                 ATTCACAYTCTTCAACTTAATGG 
                 Malignant tumor of prostate 
               
               
                   
                 (p.Asn462Ser) 
                   
                   
               
               
                   
               
               
                 794728280 
                 NM_000138.4(FBN1):c.7916A&gt;G 
                 TGTTCAYACTGGAAGCCGGCGGG, 
                 Thoracic aortic aneurysms and aortic 
               
               
                   
                 (p.Tyr2639Cys) 
                 CTGTTCAYACTGGAAGCCGGCGG 
                 dissections 
               
               
                   
               
               
                 28937317 
                 NM_000335.4(SCN5A):c.3971A&gt;G 
                 GCAYTGACCACCACCTCAAGTGG 
                 Long QT syndrome 3, Congenital long QT 
               
               
                   
                 (p.Asn1324Ser) 
                   
                 syndrome 
               
               
                   
               
               
                 786205854 
                 NM_144499.2(GNAT1):c.386A&gt;G 
                 CGGAGYCCTTCCACAGCCGCTGG 
                 NIGHT BLINDNESS, 
               
               
                   
                 (p.Asp129Gly) 
                   
                 CONGENITAL 
               
               
                   
               
               
                 104893776 
                 NM_000539.3(RHO):c.533A&gt;G 
                 GGATGYACCTGAGGACAGGCAGG 
                 Retinitis pigmentosa 4 
               
               
                   
                 (p.TyrI78Cys) 
                   
                   
               
               
                   
               
               
                 28937590 
                 NM_001257342.1(BCS1L):c.232A&gt;G 
                 GACACYGAGGTGCTGAGTACGGG, 
                 GRACILE syndrome 
               
               
                   
                 (p.Ser78Gly) 
                 CGACACYGAGGTGCTGAGTACGG 
                   
               
               
                   
               
               
                 104893866 
                 NM_000320.2(QDPR):c.449A&gt;G 
                 TGCCGYACCCGATCATACCTGGG, 
                 Dihydropteridine reductase deficiency 
               
               
                   
                 (p.Tyr150Cys) 
                 ATGCCGYACCCGATCATACCTGG 
                   
               
               
                   
               
               
                 587776590 
                 NM_015629.3(PRPF31):c.527+3A&gt;G 
                 GACAYACCCCTGGGTGGTGGAGG, 
                 Retinitis pigmentosa 11 
               
               
                   
                   
                 GCGGACAYACCCCTGGGTGGTGG 
                   
               
               
                   
               
               
                 104894015 
                 NM_000162.3(GCK):c.64IA&gt;G 
                 GTAGYAGCAGGAGATCATCGTGG 
                 Hyperinsulinemic hypoglycemia familial 3 
               
               
                   
                 (p.Tyr214Cys) 
                   
                   
               
               
                   
               
               
                 202247823 
                 NM_000532.4(PCCB):c.1606A&gt;G 
                 ATATYTGCATGTTTTCTCCAAGG 
                 Propionic ac dem a 
               
               
                   
                 (p.Asn536Asp) 
                   
                   
               
               
                   
               
               
                 104894199 
                 NM_000073.2(CD3G):c.IA&gt;G 
                 CCAYGTCAGTCTCTGTCCTCCGG 
                 Immunodeficiency 17 
               
               
                   
                 (p.Met1Val) 
                   
                   
               
               
                   
               
               
                 104894208 
                 NM_001814.4(CTSC):c.857A&gt;G 
                 CTCCYGAGGGCTTAGGATTGGGG, 
                 Papillon-Lef\xc3\xa8vre syndrome, Haim- 
               
               
                   
                 (p.Gln286Arg) 
                 CCTCCYGAGGGCTTAGGATTGGG, 
                 Munk syndrome 
               
               
                   
                   
                 ACCTCCYGAGGGCTTAGGATTGG 
                   
               
               
                   
               
               
                 104894211 
                 NM_001814.4(CTSC):c.1040A&gt;G 
                 TCCTACAYAGTGGTACTCAGAGG 
                 Papillon-Lenxc3\xa8vre 
               
               
                   
                 (p.Tyr347Cys) 
                   
                 syndrome, Periodontitis, 
               
               
                   
               
               
                 104894290 
                 NM_000448.2(RAG1):c.2735A&gt;G 
                 CTGYACTGGCAGAGGGATTCTGG 
                 Histiocytic medullary reticulosis 
               
               
                   
                 (p.Tyr912Cys) 
                   
                   
               
               
                   
               
               
                 104894354 
                 NM_000217.2(KCNA1):c.676A&gt;G 
                 GCGYTTCCACGATGAAGAAGGGG, 
                 Episodic ataxia type 1 
               
               
                   
                 (p.T1u.226Ala) 
                 AGCGYTTCCACGATGAAGAAGGG, 
                   
               
               
                   
                   
                 CAGCGYTTCCACGATGAAGAAGG 
                   
               
               
                   
               
               
                 104894425 
                 NM_014239.3(EIF2B2):c.638A&gt;G 
                 AGTTGTCYCAATACCTGCTTTGG 
                 Leukoencephalopathy with vanishing bite 
               
               
                   
                 (p.Glu213Gly) 
                   
                 matter, Ovarioleukodystrophy 
               
               
                   
               
               
                 104894450 
                 NM_000270.3(PNP):c.383A&gt;G 
                 ATAYCTCCAACCTCAAACTTGGG, 
                 Purine-nucleoside phosphorylase deficiency 
               
               
                   
                 (p.Asp128Gly) 
                 GATAYCTCCAACCTCAAACTTGG 
                   
               
               
                   
               
               
                 147394623 
                 NM_024887.3(DHDDS):c.124A&gt;G 
                 GGCACTYCTTGGCATAGCGACGG 
                 Retinitis pigmentosa 59 
               
               
                   
                 (p.Lys42Glu) 
                   
                   
               
               
                   
               
               
                 60723330 
                 NM_005557.3(KRT16):c.374A&gt;G 
                 GCGGTCAYTGAGGTTCTGCATGG 
                 Pachyonychia congenita, type 1, Palmoplantar 
               
               
                   
                 (p.Asn125Ser) 
                   
                 keratoderma, nonepidermolytic, focal 
               
               
                   
               
               
                 104894634 
                 NM_030665.3(RAI1):c.4685A&gt;G 
                 CTGCTGCYGTCGTCGTCGCTTGG 
                 Smith-Magenis syndrome 
               
               
                   
                 (p.Gln1562Arg) 
                   
                   
               
               
                   
               
               
                 104894730 
                 NM_000363.4(TNNI3):c.532A&gt;G 
                 CCTYCTTCACCTGCTTGAGGTGG, 
                 Familial restrictive cardiomyopathy 1 
               
               
                   
                 (p.Lys178Glu) 
                 CCTCCTYCTTCACCTGCTTGAGG 
                   
               
               
                   
               
               
                 104894816 
                 NM_002049.3(GATA1):c.653A&gt;G 
                 GTCCTGYCCCTCCGCCACAGTGG 
                 GATA-1-related thrombocytopenia 
               
               
                   
                 (p.Asp218Gly) 
                   
                 with dyserythropoiesis 
               
               
                   
               
               
                 794726773 
                 NM_001165963.1(SCN1A):c.1662+3 
                 GTGCCAYACCTGGTGTGGGGAGG 
                 Severe myoclonic epilepsy in infancy 
               
               
                   
                 A&gt;G 
                   
                   
               
               
                   
               
               
                 104894861 
                 NM_000202.6(IDS):c.404A&gt;G 
                 AAAGACTYTTCCCACCGACATGG 
                 Mucopolysaccharidosis, MPS-II 
               
               
                   
                 (p.Lys135Arg) 
                   
                   
               
               
                   
               
               
                 104894874 
                 NM_000266.3(NDP):c.125A&gt;G 
                 TGGYGCCTCATGCAGCGTCGAGG 
                   
               
               
                   
                 (p.His42Arg) 
                   
                   
               
               
                   
               
               
                 191205969 
                 NM_002420.5(TRPM1):c.296T&gt;C 
                 AAGCYCTTAATATCTGTGCATGG 
                 Congenital stationary night blindness, type 1C 
               
               
                   
                 (p.Leu99Pro) 
                   
                   
               
               
                   
               
               
                 794727073 
                 NM_019109.4(ALG1):c.1188- 
                 TAAACYGCAGAGAGAACCAAGGG, 
                 Congenital disorder of glycosylation type 1K 
               
               
                   
                 2A&gt;G 
                 GTAAACYGCAGAGAGAACCAAG 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 281875236 
                 NM_001004334.3(GPR179):c.659A&gt;G 
                 CCCACAYATCCATCTGCCTGCGG 
                 Congenital stationary night blindness, type 1E 
               
               
                   
                 (p.Tyr220Cys) 
                   
                   
               
               
                   
               
               
                 28939094 
                 NM_015915.4(ATL1):c.1222A&gt;G 
                 CACCCAYCTTCTTCACCCCTCGG 
                 Spastic paraplegia 3 
               
               
                   
                 (p.Met408Val) 
                   
                   
               
               
                   
               
               
                 281875324 
                 NM_005359.5(SMAD4):c.989A&gt;G 
                 ATCCATTYCAAAGTAAGCAATGG 
                 Juvenile polyposis syndrome, Hereditary 
               
               
                   
                 (p.Glu330Gly) 
                   
                 cancer-predisposing syndrome 
               
               
                   
               
               
                 77173848 
                 NM_000037.3(ANK1):c.- 
                 GGGCCYGGCCCGCACGTCACAGG 
                 Spherocytosis, type 1, autosomal recessive 
               
               
                   
                 
                   
                 
                   
                   
               
               
                   
               
               
                 150181226 
                 NM_001159772.1(CANT1):c.671T&gt;C 
                 CGTCYGTACGTGGGCGGCCTGGG, 
                 Desbuquois syndrome 
               
               
                   
                 (p.Leu224Pro) 
                 GCGTCYGTACGTGGGCGGCCTGG 
                   
               
               
                   
               
               
                 397514253 
                 NM_000041.3(APOE):c.237- 
                 CGCCCYGCGGCCGAGAGGGCGGG, 
                 Familial type 3 hyperlipoprote nem a 
               
               
                   
                 2A&gt;G 
                 GCGCCCYGCGGCCGAGAGGGCGG 
                   
               
               
                   
               
               
                 397514348 
                 NM_000060.3(BTD):c.278A&gt;G 
                 GTTCAYAGATGTCAAGGTTCTGG 
                 Biotinidase deficiency 
               
               
                   
                 (p.Tyr93Cys) 
                   
                   
               
               
                   
               
               
                 397514415 
                 NM_000060.3(BTD):c.1313A&gt;G 
                 GGCAYACAGCTCTTTGGATAAGG 
                 Biotinidase deficiency 
               
               
                   
                 (p.Tyr438Cys) 
                   
                   
               
               
                   
               
               
                 397514501 
                 NM_007171.3(POMT1):c.430A&gt;G 
                 GAGCATYCTCTGTTTCAAAGAGG 
                 Limb-girdle muscular 
               
               
                   
                 (p.Asn144Asp) 
                   
                 dystrophy- 
               
               
                   
               
               
                 370382601 
                 NM_174917.4(ACSF3):c.IA&gt;G 
                 GGCAGCAYTGCACTGACAGGCGG 
                 not provided 
               
               
                   
                 (P.Met1Val) 
                   
                   
               
               
                   
               
               
                 72554332 
                 NM_000531.5(OTC):c.238A&gt;G 
                 AAGGACTYCCCTTGCAATAAAGG 
                 Ornithine carbamoyltransferase deficiency 
               
               
                   
                 (p.Lys80Glu) 
                   
                   
               
               
                   
               
               
                 397514599 
                 NM_033109.4(PNPT1):c.1424A&gt;G 
                 GACTYCAGATGTAACTCTTATGG 
                 Deafness, autosomal recessive 70 
               
               
                   
                 (p.Glu475Gly) 
                   
                   
               
               
                   
               
               
                 397514650 
                 NM_000108.4(DLD):c.1444A&gt;G 
                 GACTCYAGCTATATCTTCACAGG 
                 Maple syrup urine disease, type 3 
               
               
                   
                 (p.Arg482G1y) 
                   
                   
               
               
                   
               
               
                 397514675 
                 NM_003156.3(STIMI):c.25IA&gt;G 
                 TTCCACAYCCACATCACCATTGG 
                 Myopathy with tubular aggregates 
               
               
                   
                 (p.Asp84G1y) 
                   
                   
               
               
                   
               
               
                 794728378 
                 NM_000238.3(KCNH2):c.1913A&gt;G 
                 ATCYTCTCTGAGTTGGTGTTGGG, 
                 Cardiac arrhythmia 
               
               
                   
                 (p.Lys638Arg) 
                 GATCYTCTCTGAGTTGGTGTTGG 
                   
               
               
                   
               
               
                 397514711 
                 NM_002163.2(IRF8):c.238A&gt;G 
                 AACCTCGYCTTCCAAGTGGCTGG 
                 Autosomal dominant CD11C+/CD1C+dendritic 
               
               
                   
                 (p.Thr80Ala) 
                   
                 cell deficiency 
               
               
                   
               
               
                 397514729 
                 NM_000388.3(CASR):c.85A&gt;G 
                 CCCCCTYCTTTTGGGCTCGCTGG 
                 Hypocalcemia, autosomal dominant 1, with 
               
               
                   
                 (p.Lys29Glu) 
                   
                 bartter syndrome 
               
               
                   
               
               
                 397514743 
                 NM_022114.3(PRDM16):c.2447A&gt;G 
                 GCCGCCGYTTTGGCTGGCACGGG 
                 Left ventricular noncompaction 8 
               
               
                   
                 (p.Asn816Ser) 
                   
                   
               
               
                   
               
               
                 397514757 
                 NM_005689.2(ABCB6):c.508A&gt;G 
                 TGGGCYGTTCCAAGACACCAGGG, 
                 Dyschromatosis universalis hereditaria 3 
               
               
                   
                 (p.Ser170Gly) 
                 GTGGGCYGTTCCAAGACACCAGG 
                   
               
               
                   
               
               
                 28940313 
                 NM_152443.2(RDH12):c.677A&gt;G 
                 CACTGCGYAGGTGGTGACCCCGG 
                 Leber congenital amaurosis 13 
               
               
                   
                 (p.Tyr226Cys) 
                   
                   
               
               
                   
               
               
                 794728538 
                 NM_000218.2(KCNQ1):c.1787A&gt;G 
                 GTCTYCTACTCGGTTCAGGCGGG, 
                 Cardiac arrhythmia 
               
               
                   
                 (p.Glu596Gly) 
                 TGTCTYCTACTCGGTTCAGGCGG 
                   
               
               
                   
               
               
                 794728569 
                 NM_000218.2(KCNQ1):c.605A&gt;G 
                 AGGYCTGTGGAGTGCAGGAGAGG 
                 Cardiac arrhythmia 
               
               
                   
                 (p.Asp202Gly) 
                   
                   
               
               
                   
               
               
                 794728573 
                 NM 000218.2(KCNQ1):c.1515- 
                 GCCYGCAGTGGAGAGAGGAGAGG 
                 Cardiac arrhythmia 
               
               
                   
                 2A&gt;G 
                   
                   
               
               
                   
               
               
                 370874727 
                 NM_003494.3(DYSF):c.3349- 
                 CCGCCCYGGAGACACGAAGCTGG 
                 Limb-girdle muscular dystrophy, type 2B 
               
               
                   
                 2A&gt;G 
                   
                   
               
               
                   
               
               
                 794728859 
                 NM_198056.2(SCN5A):c.2788- 
                 ACCYGTCGAGATAATGGGTCAGG 
                 not provided 
               
               
                   
                 2A&gt;G 
                   
                   
               
               
                   
               
               
                 794728887 
                 NM_198056.2(SCN5A):c.4462A&gt;G 
                 CCTCTGYCATGAAGATGTCCTGG 
                 not provided 
               
               
                   
                 (p.Thr1488Ala) 
                   
                   
               
               
                   
               
               
                 28940878 
                 NM_000372.4(TYR):c.125A&gt;G 
                 CTCCTGYCCCCGCTCCACGGTGG 
                 Tyrosinase-negative oculocutaneous albinism 
               
               
                   
                 (p.Asp42G1y) 
                   
                   
               
               
                   
               
               
                 397515420 
                 NM_172107.2(KCNQ2):c.1636A&gt;G 
                 GCAYGACACTGCAGGGGGGTGGG, 
                 Early infantile epileptic encephalopathy 7 
               
               
                   
                 (p.Met546Val) 
                 CGCAYGACACTGCAGGGGGGTGG, 
                   
               
               
                   
                   
                 AACCGCAYGACACTGCAGGGGGG 
                   
               
               
                   
               
               
                 397515428 
                 NM_001410.2(MEGF8):c.7099A&gt;G 
                 GACYCCCGTGAAATGATTCCCGG 
                 Carpenter syndrome 2 
               
               
                   
                 (p.Ser2367Gly) 
                   
                   
               
               
                   
               
               
                 143601447 
                 NM_201631.3(TGM5):c.122T&gt;C 
                 TCAACCYCACCCTGTACTTCAGG 
                 Peeling skin syndrome, acral type 
               
               
                   
                 (p.Leu41Pro) 
                   
                   
               
               
                   
               
               
                 397515519 
                 NM_000207.2(INS):c.*59A&gt;G 
                 GGGCYTTATTCCATCTCTCTCGG 
                 Permanent neonatal diabetes mellitus 
               
               
                   
               
               
                 397515523 
                 NM_000370.3(TTPA):c.191A&gt;G 
                 CAGGYCCAGATCGAAATCCCGGG, 
                 Ataxia with vitamin E deficiency 
               
               
                   
                 (p.Asp64G1y) 
                 CCAGGYCCAGATCGAAATCCCGG 
                   
               
               
                   
               
               
                 397515891 
                 NM_000256.3(MYBPC3):c.1224- 
                 TACTTGCYGTAGAACAGAAGGGG 
                 Familial hypertrophic cardiomyopathy 4, Cardiomyopathy 
               
               
                   
                 2A&gt;G 
                   
                   
               
               
                   
               
               
                 397516082 
                 NM_000256.3(MYBPC3):c.927- 
                 GTCCCYGTGTCCCGCAGTCTAGG 
                 Familial hypertrophic cardiomyopathy 
               
               
                   
                 2A&gt;G 
                   
                 4, Cardiomyopathy 
               
               
                   
               
               
                 397516138 
                 NM_000257.3(MYH7):c.2206A&gt;G 
                 TATCAAYGAACTGTCCCTCAGGG, 
                 Familial hypertrophic cardiomyopathy 
               
               
                   
                 (p.Ile736Val) 
                 CTATCAAYGAACTGTCCCTCAGG 
                 1, Cardiomyopathy, not specified 
               
               
                   
               
               
                 1154510 
                 NM_002150.2(HPD):c.97G&gt;A 
                 ATGACGYGGCCTGAATCACAGGG, 
                 4-Alpha-hydroxyphenylpyruvate hydroxylase 
               
               
                   
                 (p.Ala33Thr) 
                 AATGACGYGGCCTGAATCACAGG 
                 deficiency 
               
               
                   
               
               
                 397516330 
                 NM_000260.3(MY07A):c.6439- 
                 ATATCCYGGGGGAGCAGAAAGGG, 
                 Usher syndrome, type 1 
               
               
                   
                 2A&gt;G 
                 GATATCCYGGGGGAGCAGAAAGG 
                   
               
               
                   
               
               
                 72556271 
                 NM_000531.5(OTC):c.482A&gt;G 
                 CAGCCCAYTGATAATTGGGATGG 
                 not provided 
               
               
                   
                 (p.Asn161Ser) 
                   
                   
               
               
                   
               
               
                 606231260 
                 NM_023073.3(C5orf42):c.3290- 
                 ATCYATCAAATACAAAAATTTGG 
                 Orofaciodigital syndrome 6 
               
               
                   
                 2A&gt;G 
                   
                   
               
               
                   
               
               
                 587777521 
                 NM_004817.3(TJP2):c.1992- 
                 CAGCTCYGAGAAGAAACCACGGG, 
                 Progressive familial intrahepatic cholestasis 4 
               
               
                   
                 2A&gt;G 
                 TCAGCTCYGAGAAGAAACCACGG 
                   
               
               
                   
               
               
                 730880846 
                 NM_000257.3(MYH7):c.617A&gt;G 
                 CTTCYTGCTGCGGTCCCCAATGG 
                 Cardiomyopathy 
               
               
                   
                 (p.Lys206Arg) 
                   
                   
               
               
                   
               
               
                 397517978 
                 NM_206933.2(USH2A):c.12067- 
                 TTCCCYGTAAGAAAATTAACAGG 
                 Usher syndrome, type 2A, Retinitis pigmentosa 
               
               
                   
                 2A&gt;G 
                   
                 39 
               
               
                   
               
               
                 606231409 
                 NM_000216.2(ANOS1):c.1A&gt;G 
                 GCACCAYGGCTGCGGGTCGAGGG, 
                 Kallmann syndrome 1 
               
               
                   
                 (p.Met1Val) 
                 GGCACCAYGGCTGCGGGTCGAGG 
                   
               
               
                   
               
               
                 80356546 
                 NM_003334.3(UBA1):c.1639A&gt;G 
                 TGGCYTGTCACCCGGATATGTGG 
                 Arthrogryposis multiplex congenita, distal, 
               
               
                   
                 (p.Ser547Gly) 
                   
                 X- linked 
               
               
                   
               
               
                 80356584 
                 NM_194248.2(OTOF):c.766- 
                 GACCYGCAGGCAGGAGAAGGGGG, 
                 Deafness, autosomal recessive 9 
               
               
                   
                 2A&gt;G 
                 TGACCYGCAGGCAGGAGAAGGGG, 
                   
               
               
                   
                   
                 CTGACCYGCAGGCAGGAGAAGGG, 
                   
               
               
                   
                   
                 GCTGACCYGCAGGCAGGAGAAGG 
                   
               
               
                   
               
               
                 730880930 
                 NM_000257.3(MYH7):c.1615A&gt;G 
                 GGAACAYGCACTCCTCTTCCAGG 
                 Cardiomyopathy 
               
               
                   
                 (p.Met539Val) 
                   
                   
               
               
                   
               
               
                 118203947 
                 NM_013319.2(UBIAD1):c.355A&gt;G 
                 TCCYGTCATCACTCTTTTTGTGG 
                 Schnyder crystalline conical dystrophy 
               
               
                   
                 (p.Arg119Gly) 
                   
                   
               
               
                   
               
               
                 60171927 
                 NM_000526.4(KRT14):c.368A&gt;G 
                 GCGGTCAYTGAGGTTCTGCATGG 
                 Epidermolysis bullosa herpetiformis, 
               
               
                   
                 (p.Asn123Ser) 
                   
                 Dowling- Meara 
               
               
                   
               
               
                 199422248 
                 NM_001363.4(DKC1):c.941A&gt;G 
                 AATCYTGGCCCCATAGCAGATGG 
                 Dyskeratosis congenita X-linked 
               
               
                   
                 (p.Lys314Arg) 
                   
                   
               
               
                   
               
               
                 72558467 
                 NM_000531.5(OTC):c.929A&gt;G 
                 TCCACTYCTTCTGGCTTTCTGGG, 
                 not provided 
               
               
                   
                 (p Glu310Gly) 
                 ATCCACTYCTTCTGGCTTTCTGG 
                   
               
               
                   
               
               
                 72558478 
                 NM_000531.5(OTC):c.988A&gt;G 
                 ACTTTCYGTTTTCTGCCTCTGGG, 
                 not provided 
               
               
                   
                 (p.Arg330G1y) 
                 CACTTTCYGTTTTCTGCCTCTGG 
                   
               
               
                   
               
               
                 118204455 
                 NM_000505.3(F12):c.158A&gt;G 
                 GGTGGYACTGGAAGGGGAAGTGG 
                   
               
               
                   
                 (p.Tyr53Cys) 
                   
                   
               
               
                   
               
               
                 80357477 
                 NM_007294.3(BRCA1):c 
                 TTGYCCTCTGTCCAGGCATCTGG 
                 Familial cancer of breast, Breast-ovarian 
               
               
                   
                 (p.Asp1818Gly) 
                   
                 cancer, familial 1 
               
               
                   
               
               
                 121907908 
                 NM_024426.4(WT1):c.1021A&gt;G 
                 CGCYCTCGTACCCTGTGCTGTGG 
                 Mesothelioma 
               
               
                   
                 (p.Ser341Gly) 
                   
                   
               
               
                   
               
               
                 121907926 
                 NM_000280.4(PAX6):c.1171A&gt;G 
                 GTGGYGCCCGAGGTGCCCATTGG 
                 Optic nerve aplasia, bilateral 
               
               
                   
                 (p.Thr391Ala) 
                   
                   
               
               
                   
               
               
                 121908023 
                 NM_024740.2(ALG9):c.860A&gt;G 
                 TTAYACAAAACAATGTTGAGTGG 
                 Congenital disorder of glycosylation type 1L 
               
               
                   
                 (p.Tyr287Cys) 
                   
                   
               
               
                   
               
               
                 121908148 
                 NM_001243133.1(NLRP3):c.1880A&gt;G 
                 ACAATYCCAGCTGGCTGGGCTGG 
                 Familial cold urticaria 
               
               
                   
                 (p.Glu627Gly) 
                   
                   
               
               
                   
               
               
                 121908166 
                 NM_006492.2(ALX3):c.608A&gt;G 
                 CGGYTCTGGAACCAGACCTGGGG, 
                 Frontonasal dysplasia 
               
               
                   
                 (p.Asn203Ser) 
                 GCGGYTCTGGAACCAGACCTGGG, 
                   
               
               
                   
                   
                 TGCGGYTCTGGAACCAGACCTGG 
                   
               
               
                   
               
               
                 121908184 
                 NM_020451.2(SEPN1):c.1A&gt;G 
                 CCCAYGGCTGCGGCTGGCGGCGG, 
                 Eichsfeld type congenital muscular dystrophy 
               
               
                   
                 (p.Met1Val) 
                 CGGCCCAYGGCTGCGGCTGGCGG 
                   
               
               
                   
               
               
                 121908258 
                 NM_130468.3(CHST14):c.878A&gt;G 
                 AAGTCAYAGTGCACGGCACAAGG 
                 Ehlers-Danlos syndrome, musculocontractural 
               
               
                   
                 (p.Tyr293Cys) 
                   
                 type 
               
               
                   
               
               
                 121908383 
                 NM_001128425.1(MUTYH):c.1241A&gt;G 
                 AAGCYGCTCTGAGGGCTCCCAGG 
                 Neoplasm of stomach 
               
               
                   
                 (p.Gln414Arg) 
                   
                   
               
               
                   
               
               
                 121908580 
                 NM_004328.4(BCS1L):c.148A&gt;G 
                 GTGYGATCATGTAATGGCGCCGG 
                 Mitochondrial complex III deficiency 
               
               
                   
                 (p.Thr50Ala) 
                   
                   
               
               
                   
               
               
                 121908584 
                 NM_016417.2(GLRX5):c.294A&gt;G 
                 CCTGACCYTGTCGGAGCTCCGGG 
                 Anemia, sideroblastic, pyridoxine-refractory, 
               
               
                   
                 (p.Gln98=) 
                   
                 autosomal recessive 
               
               
                   
               
               
                 121908635 
                 NM_022817.2(PER2):c.1984A&gt;G 
                 GCCACACYCTCTGCCTTGCCCGG 
                 Advanced sleep phase syndrome, familial 
               
               
                   
                 (p.Ser662Gly) 
                   
                   
               
               
                   
               
               
                 121908655 
                 NM_003839.3(TNERSF11A):c.508A&gt;G 
                 GGGTCYGCATTTGTCCGTGGAGG 
                 Osteopetrosis autosomal recessive 7 
               
               
                   
                 (p.Arg170Gly) 
                   
                   
               
               
                 29001653 
                 NM_000539.3(RHO):c.886A&gt;G 
                 CGCTCTYGGCAAAGAACGCTGGG, 
                 Retinitis pigmentosa 4 
               
               
                   
                 (p.Lys296Glu) 
                 GCGCTCTYGGCAAAGAACGCTGG 
                   
               
               
                   
               
               
                 56307355 
                 NM_006502.2(POLH):c.1603A&gt;G 
                 AGACTTTYCTGCTTAAAGAAGGG 
                 Xeroderma pigmentosum, variant type 
               
               
                   
                 (p.Lys535Glu) 
                   
                   
               
               
                   
               
               
                 121908919 
                 NM_002977.3(SCN9A):c.1964A&gt;G 
                 CCTTTTCYTGTGTATTTGATTGG 
                 Generalized epilepsy with febrile seizures plus, 
               
               
                   
                 (p.Lys655Arg) 
                   
                 type 7 not specified 
               
               
                   
               
               
                 121908939 
                 NM_006892.3(DNMT3B):c.2450A&gt;G 
                 GACACGYCTGTGTAGTGCACAGG 
                 Centromeric instability of chromosomes 1,9 and 
               
               
                   
                 (p.Asp817Gly) 
                   
                 16 and immunodeficiency 
               
               
                   
               
               
                 121909088 
                 NM_001005360.2(DNM2):c.1684A&gt;G 
                 ACTYCTTCTCTTTCTCCTGAGGG, 
                 Charcot-Marie-Tooth disease, dominant 
               
               
                   
                 (p.Lys562Glu) 
                 TACTYCTTCTCTTTCTCCTGAGG 
                 intermediate b, with neutropenia 
               
               
                   
               
               
                 120074112 
                 NM_000483.4(APOC2):c.1A&gt;G 
                 GCCCAYAGTGTCCAGAGACCTGG 
                 Apolipoprotein C2 deficiency 
               
               
                   
                 (p.Met1Val) 
                   
                   
               
               
                   
               
               
                 121909239 
                 NM_000314.6(PTEN):c.755A&gt;G 
                 ATAYCACCACACACAGGTAACGG 
                 Macrocephaly/autism syndrome 
               
               
                   
                 (p.Asp252G1y) 
                   
                   
               
               
                   
               
               
                 121909251 
                 NM_198217.2(ING1):c.515A&gt;G 
                 TGGYTGCACAGACAGTACGTGGG, 
                 Squamous cell carcinoma of the head and neck 
               
               
                   
                 (p.Asn172Ser) 
                 CTGGYTGCACAGACAGTACGTGG 
                   
               
               
                   
               
               
                 121909396 
                 NM_001174089.1(SLC4A11):c.2518A&gt;G 
                 GATCAYCTTCATGTAGGGCAGGG, 
                 Corneal dystrophy and perceptive deafness 
               
               
                   
                 (p.Met840Val) 
                 AGATCAYCTTCATGTAGGGCAGG 
                   
               
               
                   
               
               
                 121909533 
                 NM_000034.3(ALD0A):c.386A&gt;G 
                 CCAYCCAACCCTAAGAGAAGAGG 
                 HNSHA due to aldolase A deficiency 
               
               
                   
                 (p.Asp129Gly) 
                   
                   
               
               
                   
               
               
                 128627255 
                 NM_004006.2(DMD):c.835A&gt;G 
                 TGACCGYGATCTGCAGAGAAGGG, 
                 Dilated cardiomyopathy 3B 
               
               
                   
                 (p.Thr279Ala) 
                 CTGACCGYGATCTGCAGAGAAGG 
                   
               
               
                   
               
               
                 116929575 
                 NM_001085.4(SERPINA3):c.1240A&gt;G 
                 GCTCAYGAAGAAGATGTTCTGGG, 
                   
               
               
                   
                 (p.Met414Val) 
                 TGCTCAYGAAGAAGATGTTCTGG 
                   
               
               
                   
               
               
                 61748392 
                 NM_004992.3(MECP2):c.410A&gt;G 
                 CAACYCCACTTTAGAGCGAAAGG 
                 Mental retardation, X-linked, syndromic 13 
               
               
                   
                 (p.Glu137Gly) 
                   
                   
               
               
                   
               
               
                 61748906 
                 NM_001005741.2(GBA):c.667T&gt;C 
                 CCCACTYGGCTCAAGACCAATGG 
                 Gaucher disease, type 1 
               
               
                   
                 (p.Trp223Arg) 
                   
                   
               
               
                   
               
               
                 199473024 
                 NM_000238.3(KCNH2):c.3118A&gt;G 
                 CTGCYCTCCACGTCGCCCCGGGG, 
                 Sudden infant death syndrome 
               
               
                   
                 (p.Ser1040Gly) 
                 CCTGCYCTCCACGTCGCCCCGGG, 
                   
               
               
                   
                   
                 GCCTGCYCTCCACGTCGCCCCGG 
                   
               
               
                   
               
               
                 794728365 
                 NM_000238.3(KCNH2):c.1129- 
                 GGACCYGCACCCGGGGAAGGCGG 
                 Cardiac arrhythmia 
               
               
                   
                 
                   
                 
                   
                   
               
               
                   
               
               
                   
               
               
                 72556293 
                 NM_000531.5(OTC):c.548A&gt;G 
                 AGAGCTAYAGTGTTCCTAAAAGG 
                 not provided 
               
               
                   
                 (p.Tyr183Cys) 
                   
                   
               
               
                   
               
               
                 111033244 
                 NM_000441.1(SLC26A4):c.1151A&gt;G 
                 TGAATYCCTAAGGAAGAGACTGG 
                 Pendred syndrome, Enlarged vestibular 
               
               
                   
                 (p.Glu384Gly) 
                   
                 aqueduct syndrome 
               
               
                   
               
               
                 111033415 
                 NM_000260.3(MY07A):c.1344- 
                 AGCYGCAGGGGCACAGGGATGGG, 
                 Usher syndrome, type 1 
               
               
                   
                 2A&gt;G 
                 AAGCYGCAGGGGCACAGGGATGG 
                   
               
               
                   
               
               
                 121912439 
                 NM_000454.4(SOD1):c.302A&gt;G 
                 AGAATCTYCAATAGACACATCGG 
                 Amyotrophic lateral sclerosis type 1 
               
               
                   
                 (p.Glu101Gly) 
                   
                   
               
               
                   
               
               
                 111033567 
                 NM_002769.4(PRSS1):c.68A&gt;G 
                 ATCYTGTCATCATCATCAAAGGG, 
                 Hereditarypancreatitis 
               
               
                   
                 (p.Lys23Arg) 
                 GATCYTGTCATCATCATCAAAG 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 121912565 
                 NM_000901.4(NR3C2):c.2327A&gt;G 
                 TCATCYGTTTGCCTGCTAAGCGG 
                 Pseudohypoaldosteronism type 1 autosomal 
               
               
                   
                 (p.Gln776Arg) 
                   
                 dominant 
               
               
                   
               
               
                 121912574 
                 NM_000901.4(NR3C2):c.2915A&gt;G 
                 CCGACYCCACCTTGGGCAGCTGG 
                 Pseudohypoaldosteronism type 1 autosomal 
               
               
                   
                 (p.Glu972Gly) 
                   
                 dominant 
               
               
                   
               
               
                 121912589 
                 NM_001173464.1(KIF21A):c.2839A&gt;G 
                 ATTCAYATCTGCCTCCATGTTGG 
                 Fibrosis of extraocular muscles, congenital, 1 
               
               
                   
                 (p.Met947Val) 
                   
                   
               
               
                   
               
               
                 111033661 
                 NM_000155.3(GALT):c.253- 
                 ATTCACCYACCGACAAGGATAGG 
                 Deficiency of UDPglucose-hexose-1-phosphate 
               
               
                   
                 2A&gt;G 
                   
                 uridylyltransferase 
               
               
                   
               
               
                 111033669 
                 NM_000155.3(GALT):c.290A&gt;G 
                 GAAGTCGYTGTCAAACAGGAAGG 
                 Deficiency of UDPglucose-hexose-1-phosphate 
               
               
                   
                 (p.Asn97Ser) 
                   
                 uridylyltransferase 
               
               
                   
               
               
                 111033682 
                 NM_000155.3(GALT):c.379A&gt;G 
                 TGACCTYACTGGGTGGTGACGGG, 
                 Deficiency of UDPglucose-hexose-1-phosphate 
               
               
                   
                 (p.Lys127Glu) 
                 ATGACCTYACTGGGTGGTGACGG 
                 uridylyltransferase 
               
               
                   
               
               
                 111033786 
                 NM_000155.3(GALT):c.950A&gt;G 
                 CAGCYGCCAATGGTTCCAGTTGG 
                 Deficiency of UDPglucose-hexose-1-phosphate 
               
               
                   
                 (p.Gln317Arg) 
                   
                 uridylyltransferase 
               
               
                   
               
               
                 121912765 
                 NM_001202.3(BMP4):c.278A&gt;G 
                 CCTCCYCCCCAGACTGAAGCCGG 
                 Microphthalmia syndromic 6 
               
               
                   
                 (p.Glu93Gly) 
                   
                   
               
               
                   
               
               
                 121912856 
                 NM_000094.3(COL7A1):c.425A&gt;G 
                 CACCYTGGGGACACCAGGTCGGG, 
                 Epidermolysis bullosa dystrophica inversa, 
               
               
                   
                 (p.Lys142Arg) 
                 TCACCYTGGGGACACCAGGTCGG 
                 autosomal recessive 
               
               
                   
               
               
                 199474715 
                 NM_152263.3(TPM3):c.505A&gt;G 
                 CCAACTYACGAGCCACCTACAGG 
                 Congenital myopathy with fiber 
               
               
                   
                 (p.Lys169Glu) 
                   
                 type disproportion 
               
               
                   
               
               
                 199474718 
                 NM_152263.3(TPM3):c.733A&gt;G 
                 ATCYCTCAGCAAACTCAGCACGG 
                 Congenital myopathy with fiber 
               
               
                   
                 (p.Arg245Gly) 
                   
                 type disproportion 
               
               
                   
               
               
                 121912895 
                 NM_001844.4(COL2A1):c.2974A&gt;G 
                 CCTCYCTCACCACGTTGCCCAGG 
                 Spondyloepimetaphyseal dysplasia Strudwick 
               
               
                   
                 (p.Arg992Gly) 
                   
                 type 
               
               
                   
               
               
                 121913074 
                 NM_000129.3(F13A1):c.851A&gt;G 
                 ATAGGCAYAGATATTGTCCCAGG 
                 Factor xiii, a subunit, deficiency of 
               
               
                   
                 (p.Tyr284Cys) 
                   
                   
               
               
                   
               
               
                 121913145 
                 NM_000208.2(INSR):c.707A&gt;G 
                 GCTGYGGCAACAGAGGCCTTCGG 
                 Leprechaunism syndrome 
               
               
                   
                 (p.His236Arg) 
                   
                   
               
               
                   
               
               
                 312262745 
                 NM_025137.3(SPG11):c.2608A&gt;G 
                 ACTTAYCCTGGGGAGAAGGATGG 
                 Spastic paraplegia 11, autosomal recessive 
               
               
                   
                 (p.Ile870Val) 
                   
                   
               
               
                   
               
               
                 121913682 
                 NM_000222.2(KIT):c.2459A&gt;G 
                 AGAAYCATTCTTGATGTCTCTGG 
                 Mast cell disease, systemic 
               
               
                   
                 (p.Asp820Gly) 
                   
                   
               
               
                   
               
               
                 587776757 
                 NM_000151.3(G6PC):c.230+4A&gt;G 
                 GTTCYTACCACTTAAAGACGAGG 
                 Glycogen storage disease type 1A 
               
               
                   
               
               
                 61752063 
                 NM_000330.3(RS1):c.286T&gt;C 
                 TTCTTCGYGGACTGCAAACAAGG 
                 Juvenile retinoschisis 
               
               
                   
                 (p.Trp96Arg) 
                   
                   
               
               
                   
               
               
                 367543065 
                 NM_024549.5(TCTN1):c.221- 
                 AGCAACYGCAGAAAAAAGAGGGG, 
                 Joubert syndrome 13 
               
               
                   
                 2A&gt;G 
                 CAGCAACYGCAGAAAAAAGAGG 
                   
               
               
                   
                   
                 G 
                   
               
               
                   
               
               
                 5030773 
                 NM_000894.2(LHB):c.221A&gt;G 
                 CCACCYGAGGCAGGGGCGGCAGG 
                 Isolated lutropin deficiency 
               
               
                   
                 (p.Gln74Arg) 
                   
                   
               
               
                   
               
               
                 199476092 
                 NM_000264.3(PTCH1):c.2479A&gt;G 
                 CGTTACYGAAACTCCTGTGTAGG 
                 Gorlin syndrome, Holoprosencephaly 7, not 
               
               
                   
                 (p.Ser827Gly) 
                   
                 specified 
               
               
                   
               
               
                 398123158 
                 NM_000117.2(EMD):c.450- 
                 CGTTCCCYGAGGCAAAAGAGGGG 
                 not provided 
               
               
                   
                 2A&gt;G 
                   
                   
               
               
                   
               
               
                 199476103 
                 RMRP:n.71A&gt;G 
                 ACTTYCCCCTAGGCGGAAAGGGG, 
                 Metaphyseal chondrodysplasia, McKusick type, 
               
               
                   
                   
                 GACTTYCCCCTAGGCGGAAAGGG, 
                 Metaphyseal dysplasia without hypotrichosis 
               
               
                   
                   
                 GGACTTYCCCCTAGGCGGAAAGG 
                   
               
               
                   
               
               
                 5030856 
                 NM_000277.1(PAH):c.1169A&gt;G 
                 CTCYCTGCCACGTAATACAGGGG, 
                 Phenylketonuria, Hyperphenylalaninemia, non- 
               
               
                   
                 (p.Glu390Gly) 
                 ACTCYCTGCCACGTAATACAGGG, 
                 Pku 
               
               
                   
                   
                 AACTCYCTGCCACGTAATACAGG 
                   
               
               
                   
               
               
                 5030860 
                 NM_000277.1(PAH):c.1241A&gt;G 
                 GGGTCGYAGCGAACTGAGAAGGG, 
                 Phenylketonuria, Hyperphenylalaninem a non- 
               
               
                   
                 (p.Tyr414Cys) 
                 TGGGTCGYAGCGAACTGAGAAGG 
                 Pku 
               
               
                   
               
               
                 587777055 
                 NM_020988.2(GNAO1):c.521A&gt;G 
                 GGATGYCCTGCTCGGTGGGCTGG 
                 Early infantile epileptic encephalopathy 17 
               
               
                   
                 (p.Asp174Gly) 
                   
                   
               
               
                   
               
               
                 587777223 
                 NM_024301.4(FKRP):c.1A&gt;G 
                 CCGCAYGGGGCCGAAGTCTGGGG, 
                 Congenital muscular dystrophy-dystroglycanopathy 
               
               
                   
                 (p.Met1Val) 
                 GCCGCAYGGGGCCGAAGTCTGGG, 
                 with brain and eye anomalies type AS 
               
               
                   
                   
                 AGCCGCAYGGGGCCGAAGTCTGG 
                   
               
               
                   
               
               
                 587777479 
                 NM_003108.3(S0X11):c.347A&gt;G 
                 GTACTTGYAGTCGGGGTAGTCGG 
                 Mental retardation, autosomal dominant 27 
               
               
                   
                 (p.Tyr116Cys) 
                   
                   
               
               
                   
               
               
                 587777496 
                 NM_020435.3(GJC2):c.-170A&gt;G 
                 TTGYTCCCCCCTCGGCCTCAGGG, 
                 Leukodystrophy, hypomyelinating, 2 
               
               
                   
                   
                 ATTGYTCCCCCCTCGGCCTCAGG 
                   
               
               
                   
               
               
                 587777507 
                 NM_022552.4(DNMT3A):c.1943T&gt;C 
                 CTCCYGGTGCTGAAGGACTTGGG, 
                 Tatton-Brown-rahman syndrome 
               
               
                   
                 (p.Leu648Pro) 
                 GCTCCYGGTGCTGAAGGACTTGG 
                   
               
               
                   
               
               
                 587777557 
                 NM_018400.3(SCN3B):c.482T&gt;C 
                 AATCAYGATGTACATCCTTCTGG 
                 Atrial fibrillation, familial, 16 
               
               
                   
                 (p.Met161Thr) 
                   
                   
               
               
                   
               
               
                 587777569 
                 NM_001030001.2(RPS29):c.149T&gt;C 
                 GATAYCGGTTTCATTAAGGTAGG 
                 Diamond-Blackfan anemia 13 
               
               
                   
                 (p.Ile50Thr) 
                   
                   
               
               
                   
               
               
                 587777657 
                 NM_153334.6(SCARF2):c.190T&gt;C 
                 CCACGYGCTGCGCTGGCTGGAGG 
                 Marden Walker like syndrome 
               
               
                   
                 (p.Cys64Arg) 
                   
                   
               
               
                   
               
               
                 587777689 
                 NM_005726.5(TSFM):c.57+4A&gt;G 
                 ACTTCYCACCGGGTAGCTCCCGG 
                 Combined oxidative phosphorylation deficiency 3 
               
               
                   
               
               
                 796052005 
                 NM_000255.3(MUT):c.329A&gt;G 
                 GCAYACTGGCGGATGGTCCAGGG, 
                 not provided 
               
               
                   
                 (p.Tyr110Cys) 
                 AGCAYACTGGCGGATGGTCCAGG 
                   
               
               
                   
               
               
                 587777809 
                 NM_144596.3(TTC8):c.115- 
                 GTTCCYGGAAAGCATTAAGAAGG 
                 Retinitis pigmentosa 51 
               
               
                   
                 2A&gt;G 
                   
                   
               
               
                   
               
               
                 587777878 
                 NM_000166.5(GJB1):c.580A&gt;G 
                 TAGCAYGAAGACGGTGAAGACGG 
                 X-linked hereditary motor and sensory 
               
               
                   
                 (p.Met194Val) 
                   
                 neuropathy 
               
               
                   
               
               
                 74315420 
                 NM_001029871.3(RSPO4):c.194A&gt;G 
                 CGTACYGGCGGATGCCTTCCCGG 
                 Anonychia 
               
               
                   
                 (p.Gln65Arg) 
                   
                   
               
               
                   
               
               
                 180177219 
                 NM_000030.2(AGXT):c.424-2A&gt;G 
                 AGGCCCYGAGGAAGCAGGGACGG 
                 Primary hyperoxaluria, type I 
               
               
                   
                 (p.Gly_142Gln145del) 
                   
                   
               
               
                   
               
               
                 367610201 
                 NM_002693.2(POLG):c.1808T&gt;C 
                 CTCAYGGCACTTACCTGGGATGG 
                 not provided 
               
               
                   
                 (p.Met603Thr) 
                   
                   
               
               
                   
               
               
                 180177319 
                 NM_012203.1(GRHPR):c.84- 
                 TCACAGCYGCGGGGAAAGGGAGG 
                 Primary hyperoxaluria, type II 
               
               
                   
                 2A&gt;G 
                   
                   
               
               
                   
               
               
                 796052068 
                 NM_000030.2(AGXT):c.777- 
                 GGTACCYGGAAGACACGAGGGGG, 
                 Primary hyperoxaluria, type I 
               
               
                   
                 2A&gt;G 
                 TGGTACCYGGAAGACACGAGGGG 
                   
               
               
                   
               
               
                 61754010 
                 NM_000552.3(VWF):c.1583A&gt;G 
                 TGCCAYTGTAATTCCCACACAGG 
                 von Willebrand disease, type 2a 
               
               
                   
                 (p.Asn528Ser) 
                   
                   
               
               
                   
               
               
                 587778866 
                 NM_000321.2(RB1):c.1927A&gt;G 
                 ATTYCAATGGCTTCTGGGTCTGG 
                 Retinoblastoma 
               
               
                   
                 (p.Lys643Glu) 
                   
                   
               
               
                   
               
               
                 74435397 
                 NM_006331.7(EMG1):c.257A&gt;G 
                 ATAYCTGGCCGCGCTTCCCCAGG 
                 Bowen-Conradi syndrome 
               
               
                   
                 (p.Asp86G1y) 
                   
                   
               
               
                   
               
               
                 796052527 
                 NM_000156.5(GAMT):c.1A&gt;G 
                 CGCTCAYGCTGCAGGCTGGACGG 
                 not provided 
               
               
                   
                 (p.Met1Val) 
                   
                   
               
               
                   
               
               
                 796052637 
                 NM_172107.2(KCNQ2):c.848A&gt;G 
                 GTACYTGTCCCCGTAGCCAATGG 
                 not provided 
               
               
                   
                 (p.Lys283Arg) 
                   
                   
               
               
                   
               
               
                 724159963 
                 NM_032228.5(FARH:c.1094A&gt;G 
                 GATAYCATACAGGAATGCTGGGG, 
                 Peroxisomal fatty acyl-coa reductase 1 disorder 
               
               
                   
                 (p.Asp365Gly) 
                 AGATAYCATACAGGAATGCTGGG, 
                   
               
               
                   
                   
                 TAGATAYCATACAGGAATGCTGG 
                   
               
               
                   
               
               
                 587779722 
                 NM_000090.3(COL3A1):c.1762-2A&gt;G 
                 CACCCYAAAGAAGAAGTGGTCGG 
                 Ehlers-Danlos syndrome, type 4 
               
               
                   
                 (p.Gly588_Gln605del) 
                   
                   
               
               
                   
               
               
                 118192102 
                 m.8296A&gt;G 
                 TTTACAGYGGGCTCTAGAGGGGG 
                 Diabetes-deafness syndrome maternally 
               
               
                   
                   
                   
                 transmitted 
               
               
                   
               
               
                 727502787 
                 NM_001077494.3(NEKB2):c.2594A&gt;G 
                 CTGYCTTCCTTCACCTCTGCTGG 
                 Common variable immunodeficiency 10 
               
               
                   
                 (p.Asp865Gly) 
                   
                   
               
               
                   
               
               
                 727503036 
                 NM_000117.2(EMD):c.266- 
                 AGCCYTGGGAAGGGGGGCAGCGG 
                 Emery-Dreifuss muscular dystrophy 1, X-linked 
               
               
                   
                 2A&gt;G 
                   
                   
               
               
                   
               
               
                 690016544 
                 NM_005861.3(STUB1):c.194A&gt;G 
                 GGCCCGGYTGGTGTAATACACGG 
                 Spinocerebellar ataxia, autosomal recessive 16 
               
               
                   
                 (p.Asn65Ser) 
                   
                   
               
               
                   
               
               
                 690016554 
                 NM_005211.3(CSF1R):c.2655- 
                 GTATCYGGGAGATAGGACAGAGG 
                 Hereditary diffuse leukoencephalopathy with 
               
               
                   
                 2A&gt;G 
                   
                 spheroids 
               
               
                   
               
               
                 118192185 
                 NM_172107.2(KCNQ2):c.1A&gt;G 
                 GCACCAYGGTGCCTGGCGGGAGG 
                 Benign familial neonatal seizures 1 
               
               
                   
                 (p.Met1Val) 
                   
                   
               
               
                   
               
               
                 121917869 
                 NM_012064.3(MIP):c.401A&gt;G 
                 AGATCYCCACTGTGGTTGCCTGG 
                 Cataract 15, multiple types 
               
               
                   
                 (p.Glu134Gly) 
                   
                   
               
               
                   
               
               
                 121918014 
                 NM_000478.4(ALPL):c.1250A&gt;G 
                 AGGCCCAYTGCCATACAGGATGG 
                 Infantile hypophosphatasia 
               
               
                   
                 (p.Asn417Ser) 
                   
                   
               
               
                   
               
               
                 121918036 
                 NM_000174.4(GP9):c.110A&gt;G 
                 GCAGYCCACCCACAGCCCCATGG 
                 Bernard-Soulier syndrome type C 
               
               
                   
                 (p.Asp37G1y) 
                   
                   
               
               
                   
               
               
                 121918089 
                 NM_000371.3(TTR):c.379A&gt;G 
                 CGGCAAYGGTGTAGCGGCGGGGG, 
                 Amyloidogenic transthyretin amyloidosis 
               
               
                   
                 (p.Ile127Val) 
                 GCGGCAAYGGTGTAGCGGCGGGG 
                   
               
               
                   
               
               
                 121918121 
                 NM_000823.3(GHRHR):c.985A&gt;G 
                 CGACTYGGAGAGACGCCTGCAGG 
                 Isolated growth hormone deficiency type 1B 
               
               
                   
                 (p.Lys329Glu) 
                   
                   
               
               
                   
               
               
                 121918333 
                 NM_015335.4(MED13L):c.6068A&gt;G 
                 ATATCAYCTAGAGGGAAGGGGGG, 
                 Transposition of great arteries 
               
               
                   
                 (p.Asp2023Gly) 
                 CATATCAYCTAGAGGGAAGGGGG 
                   
               
               
                   
               
               
                 121918605 
                 NM_001035.2(RYR2):c.12602A&gt;G 
                 CGCCAGCYGCATTTCAAAGATGG 
                 Catecholaminergic polymorphic 
               
               
                   
                 (p.Gln4201Arg) 
                   
                 ventricular tachycardia 
               
               
                   
               
               
                 587781262 
                 NM_002764.3(PRPS1):c.343A&gt;G 
                 TAGCAYATTTGCAACAAGCTTGG 
                 Charcot-Marie-Tooth disease, X-linked 
               
               
                   
                 (p.Met115Val) 
                   
                 recessive, type 5, Deafness, high-frequency 
               
               
                   
                   
                   
                 sensorineural, X-linked 
               
               
                   
               
               
                 121918608 
                 NM_001161766.1(AHCY):c.344A&gt;G 
                 GCGGGYACTTGGTGTGGATGAGG 
                 Hypermethioninemia with s-adenosylhomocysteine 
               
               
                   
                 (p.Tyr115Cys) 
                   
                 hydrolase deficiency 
               
               
                   
               
               
                 121918613 
                 NM_000702.3(ATP1A2):c.1033A&gt;G 
                 CTGYCAGGGTCAGGCACACCTGG 
                 Familial hemiplegic migraine type 2 
               
               
                   
                 (p.Thr345Ala) 
                   
                   
               
               
                   
               
               
                 587781339 
                 NM_000535.5(PMS2):c.904- 
                 GCAGACCYGCACAAAATACAAGG 
                 Hereditary cancer-predisposing syndrome 
               
               
                   
                 2A&gt;G 
                   
                   
               
               
                   
               
               
                 121918691 
                 NM_001128177.1(THRB):c.1324A&gt;G 
                 CTTCAYGTGCAGGAAGCGGCTGG 
                 Thyroid hormone resistance, generalized, 
               
               
                   
                 (p.Met442Val) 
                   
                 autosomal dominant 
               
               
                   
               
               
                 121918692 
                 NM_001128177.1(THRB):c.1327A&gt;G 
                 CCACCTYCATGTGCAGGAAGCGG 
                 Thyroid hormone resistance, generalized, 
               
               
                   
                 (p.Lys443Glu) 
                   
                 autosomal dominant 
               
               
                   
               
               
                 727504333 
                 NM_000256.3(MYBPC3):c.2906- 
                 CCGTTCYGTGGGTATAGAGTGGG, 
                 Familial hypertrophic cardiomyopathy 4 
               
               
                   
                 2A&gt;G 
                 GCCGTTCYGTGGGTATAGAGTGG 
                   
               
               
                   
               
               
                 730880805 
                 NM_006204.3(PDE6C):c.1483- 
                 CTTTCYGTTGAAATAAGGATGGG, 
                 Achromatopsia 5 
               
               
                   
                 2A&gt;G 
                 TCTTTCYGTTGAAATAAGGATGG 
                   
               
               
                   
               
               
                 281860296 
                 NM_000551.3(VHL):c.586A&gt;T 
                 GGTCTTYCTGCACATTTGGGTGG 
                 Von Hippel-Lindau syndrome 
               
               
                   
                 (p.Lys196Ter) 
                   
                   
               
               
                   
               
               
                 730880444 
                 NM_000169.2(GLA):c.370- 
                 GTGAACCYGAAATGAGAGGGAGG 
                 not provided 
               
               
                   
                 2A&gt;G 
                   
                   
               
               
                   
               
               
                 756328339 
                 NM_000256.3(MYBPC3):c.1227- 
                 GTACCYGGGTGGGGGCCGCAGGG, 
                 Familial hypertrophic cardiomyopathy 
               
               
                   
                 2A&gt;G 
                 TGTACCYGGGTGGGGGCCGCAGG 
                 4, Cardiomyopathy 
               
               
                   
               
               
                 267606643 
                 NM_013411.4(AK2):c.494A&gt;G 
                 TCAYCTTTCATGGGCTCTTTTGG 
                 Reticular dysgenesis 
               
               
                   
                 (p.Asp165Gly) 
                   
                   
               
               
                   
               
               
                 267606705 
                 NM_005188.3(CBL):c.1144A&gt;G 
                 TATTTYACATAGTTGGAATGTGG 
                 Noonan syndrome-like disorder with or without 
               
               
                   
                 (p.Lys382Glu) 
                   
                 juvenile myelomonocytic leukemia 
               
               
                   
               
               
                 62642934 
                 NM_000277.1(PAH):c.916A&gt;G 
                 GGCCAAYTTCCTGTAATTGGGGG, 
                 Phenylketonuria, Hyperphenylalaninemia, non- 
               
               
                   
                 (p.Ile306Val) 
                 AGGCCAAYTTCCTGTAATTGGGG 
                 Pku 
               
               
                   
               
               
                 267606782 
                 NM_000117.2(EMD):c.1A&gt;G 
                 TCCAYGGCGGGTGCGGGCTCAGG 
                 Emery-Dreifuss muscular dystrophy, X-linked 
               
               
                   
                 (p.Met1Val) 
                   
                   
               
               
                   
               
               
                 267606820 
                 NM_014053.3(FLVCR1):c.361A&gt;G 
                 AGGCGTYGACCAGCGAGTACAGG 
                 Posterior column ataxia with 
               
               
                   
                 (p.Asn121Asp) 
                   
                 retinitis pigmentosa 
               
               
                   
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     In some embodiments, any of the base editors provided herein may be used to treat a disease or disorder. For example, any base editors provided herein may be used to correct one or more mutations associated with any of the diseases or disorders provided herein. Exemplary diseases or disorders that may be treated include, without limitation, 3-Methylglutaconic aciduria type 2, 46, XY gonadal dysgenesis, 4-Alpha-hydroxyphenylpyruvate hydroxylase deficiency, 6-pyruvoyl-tetrahydropterin synthase deficiency, achromatopsia, Acid-labile subunit deficiency, Acrodysostosis, acroerythrokeratoderma, ACTH resistance, ACTH-independent macronodular adrenal hyperplasia, Activated PI3K-delta syndrome, Acute intermittent porphyria, Acute myeloid leukemia, Adams-Oliver syndrome 1/5/6, Adenylosuccinate lyase deficiency, Adrenoleukodystrophy, Adult neuronal ceroid lipofuscinosis, Adult onset ataxia with oculomotor apraxia, Advanced sleep phase syndrome, Age-related macular degeneration, Alagille syndrome, Alexander disease, Allan-Herndon-Dudley syndrome, Alport syndrome, X-linked recessive, Alternating hemiplegia of childhood, Alveolar capillary dysplasia with misalignment of pulmonary veins, Amelogenesis imperfecta, Amyloidogenic transthyretin amyloidosis, Amyotrophic lateral sclerosis, Anemia (nonspherocytic hemolytic, due to G6PD deficiency), Anemia (sideroblastic, pyridoxine-refractory, autosomal recessive), Anonychia, Antithrombin III deficiency, Aortic aneurysm, Aplastic anemia, Apolipoprotein C2 deficiency, Apparent mineralocorticoid excess, Aromatase deficiency, Arrhythmogenic right ventricular cardiomyopathy, Familial hypertrophic cardiomyopathy, Hypertrophic cardiomyopathy, Arthrogryposis multiplex congenital, Aspartylglycosaminuria, Asphyxiating thoracic dystrophy, Ataxia with vitamin E deficiency, Ataxia (spastic), Atrial fibrillation, Atrial septal defect, atypical hemolytic-uremic syndrome, autosomal dominant CD11C+/CD1C+ dendritic cell deficiency, Autosomal dominant progressive external ophthalmoplegia with mitochondrial DNA deletions, Baraitser-Winter syndrome, Bartter syndrome, Basa ganglia calcification, Beckwith-Wiedemann syndrome, Benign familial neonatal seizures, Benign scapuloperoneal muscular dystrophy, Bernard Soulier syndrome, Beta thalassemia intermedia, Beta-D-mannosidosis, Bietti crystalline corneoretinal dystrophy, Bile acid malabsorption, Biotinidase deficiency, Borjeson-Forssman-Lehmann syndrome, Boucher Neuhauser syndrome, Bowen-Conradi syndrome, Brachydactyly, Brown-Vialetto-Van laere syndrome, Brugada syndrome, Cardiac arrhythmia, Cardiofaciocutaneous syndrome, Cardiomyopathy, Carnevale syndrome, Carnitine palmitoyltransferase II deficiency, Carpenter syndrome, Cataract, Catecholaminergic polymorphic ventricular tachycardia, Central core disease, Centromeric instability of chromosomes 1,9 and 16 and immunodeficiency, Cerebral autosomal dominant arteriopathy, Cerebro-oculo-facio-skeletal syndrome, Ceroid lipofuscinosis, Charcot-Marie-Tooth disease, Cholestanol storage disease, Chondrocalcinosis, Chondrodysplasia, Chronic progressive multiple sclerosis, Coenzyme Q10 deficiency, Cohen syndrome, Combined deficiency of factor V and factor VIII, Combined immunodeficiency, Combined oxidative phosphorylation deficiency, Combined partial 17-alpha-hydroxylase/17,20-lyase deficiency, Complement factor d deficiency, Complete combined 17-alpha-hydroxylase/17,20-lyase deficiency, Cone-rod dystrophy, Congenital contractural arachnodactyly, Congenital disorder of glycosylation, Congenital lipomatous overgrowth, Neoplasm of ovary, PIK3CA Related Overgrowth Spectrum, Congenital long QT syndrome, Congenital muscular dystrophy, Congenital muscular hypertrophy-cerebral syndrome, Congenital myasthenic syndrome, Congenital myopathy with fiber type disproportion, Eichsfeld type congenital muscular dystrophy, Congenital stationary night blindness, Corneal dystrophy, Cornelia de Lange syndrome, Craniometaphyseal dysplasia, Crigler Najjar syndrome, Crouzon syndrome, Cutis laxa with osteodystrophy, Cyanosis, Cystic fibrosis, Cystinosis, Cytochrome-c oxidase deficiency, Mitochondrial complex I deficiency, D-2-hydroxyglutaric aciduria, Danon disease, Deafness with labyrinthine aplasia microtia and microdontia (LAMM), Deafness, Deficiency of acetyl-CoA acetyltransferase, Deficiency of ferroxidase, Deficiency of UDPglucose-hexose-1-phosphate uridylyltransferase, Dejerine-Sottas disease, Desbuquois syndrome, DFNA, Diabetes mellitus type 2, Diabetes-deafness syndrome, Diamond-Blackfan anemia, Diastrophic dysplasia, Dihydropteridine reductase deficiency, Dihydropyrimidinase deficiency, Dilated cardiomyopathy, Disseminated atypical mycobacterial infection, Distal arthrogryposis, Distal hereditary motor neuronopathy, Donnai Barrow syndrome, Duchenne muscular dystrophy, Becker muscular dystrophy, Dyschromatosis universalis hereditaria, Dyskeratosis congenital, Dystonia, Early infantile epileptic encephalopathy, Ehlers-Danlos syndrome, Eichsfeld type congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy, Enamel-renal syndrome, Epidermolysis bullosa dystrophica inversa, Epidermolysis bullosa herpetiformis, Epilepsy, Episodic ataxia, Erythrokeratodermia variabilis, Erythropoietic protoporphyria, Exercise intolerance, Exudative vitreoretinopathy, Fabry disease, Factor V deficiency, Factor VII deficiency, Factor xiii deficiency, Familial adenomatous polyposis, breast cancer, ovarian cancer, cold urticarial, chronic infantile neurological, cutaneous and articular syndrome, hemiplegic migraine, hypercholesterolemia, hypertrophic cardiomyopathy, hypoalphalipoproteinemia, hypokalemia-hypomagnesemia, juvenile gout, hyperlipoproteinemia, visceral amyloidosis, hypophosphatemic vitamin D refractory rickets, FG syndrome, Fibrosis of extraocular muscles, Finnish congenital nephrotic syndrome, focal epilepsy, Focal segmental glomerulosclerosis, Frontonasal dysplasia, Frontotemporal dementia, Fructose-biphosphatase deficiency, Gamstorp-Wohlfart syndrome, Ganglioside sialidase deficiency, GATA-1-related thrombocytopenia, Gaucher disease, Giant axonal neuropathy, Glanzmann thrombasthenia, Glomerulocystic kidney disease, Glomerulopathy, Glucocorticoid resistance, Glucose-6-phosphate transport defect, Glutaric aciduria, Glycogen storage disease, Gorlin syndrome, Holoprosencephaly, GRACILE syndrome, Haemorrhagic telangiectasia, Hemochromatosis, Hemoglobin H disease, Hemolytic anemia, Hemophagocytic lymphohistiocytosis, Carcinoma of colon, Myhre syndrome, leukoencephalopathy, Hereditary factor IX deficiency disease, Hereditary factor VIII deficiency disease, Hereditary factor XI deficiency disease, Hereditary fructosuria, Hereditary Nonpolyposis Colorectal Neoplasm, Hereditary pancreatitis, Hereditary pyropoikilocytosis, Elliptocytosis, Heterotaxy, Heterotopia, Histiocytic medullary reticulosis, Histiocytosis-lymphadenopathy plus syndrome, HNSHA due to aldolase A deficiency, Holocarboxylase synthetase deficiency, Homocysteinemia, Howel-Evans syndrome, Hydatidiform mole, Hypercalciuric hypercalcemia, Hyperimmunoglobulin D, Mevalonic aciduria, Hyperinsulinemic hypoglycemia, Hyperkalemic Periodic Paralysis, Paramyotonia congenita of von Eulenburg, Hyperlipoproteinemia, Hypermanganesemia, Hypermethioninemia, Hyperphosphatasemia, Hypertension, hypomagnesemia, Hypobetalipoproteinemia, Hypocalcemia, Hypogonadotropic hypogonadism, Hypogonadotropic hypogonadism, Hypohidrotic ectodermal dysplasia, Hyper-IgM immunodeficiency, Hypohidrotic X-linked ectodermal dysplasia, Hypomagnesemia, Hypoparathyroidism, Idiopathic fibrosing alveolitis, Immunodeficiency, Immunoglobulin A deficiency, Infantile hypophosphatasia, Infantile Parkinsonism-dystonia, Insulin-dependent diabetes mellitus, Intermediate maple syrup urine disease, Ischiopatellar dysplasia, Islet cell hyperplasia, Isolated growth hormone deficiency, Isolated lutropin deficiency, Isovaleric acidemia, Joubert syndrome, Juvenile polyposis syndrome, Juvenile retinoschisis, Kallmann syndrome, Kartagener syndrome, Kugelberg-Welander disease, Lattice corneal dystrophy, Leber congenital amaurosis, Leber optic atrophy, Left ventricular noncompaction, Leigh disease, Mitochondrial complex I deficiency, Leprechaunism syndrome, Arthrogryposis, Anterior horn cell disease, Leukocyte adhesion deficiency, Leukodystrophy, Leukoencephalopathy, Ovarioleukodystrophy, L-ferritin deficiency, Li-Fraumeni syndrome, Limb-girdle muscular dystrophy-dystroglycanopathy, Loeys-Dietz syndrome, Long QT syndrome, Macrocephaly/autism syndrome, Macular corneal dystrophy, Macular dystrophy, Malignant hyperthermia susceptibility, Malignant tumor of prostate, Maple syrup urine disease, Marden Walker like syndrome, Marfan syndrome, Marie Unna hereditary hypotrichosis, Mast cell disease, Meconium ileus, Medium-chain acyl-coenzyme A dehydrogenase deficiency, Melnick-Fraser syndrome, Mental retardation, Merosin deficient congenital muscular dystrophy, Mesothelioma, Metachromatic leukodystrophy, Metaphyseal chondrodysplasia, Methemoglobinemia, methylmalonic aciduria, homocystinuria, Microcephaly, chorioretinopathy, lymphedema, Microphthalmia, Mild non-PKU hyperphenylalanemia, Mitchell-Riley syndrome, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency, Mitochondrial complex I deficiency, Mitochondrial complex III deficiency, Mitochondrial myopathy, Mucolipidosis III, Mucopolysaccharidosis, Multiple sulfatase deficiency, Myasthenic syndrome,  Mycobacterium tuberculosis , Myeloperoxidase deficiency, Myhre syndrome, Myoclonic epilepsy, Myofibrillar myopathy, Myoglobinuria, Myopathy, Myopia, Myotonia congenital, Navajo neurohepatopathy, Nemaline myopathy, Neoplasm of stomach, Nephrogenic diabetes insipidus, Nephronophthisis, Nephrotic syndrome, Neurofibromatosis, Neutral lipid storage disease, Niemann-Pick disease, Non-ketotic hyperglycinemia, Noonan syndrome, Noonan syndrome-like disorder, Norum disease, Macular degeneration, N-terminal acetyltransferase deficiency, Oculocutaneous albinism, Oculodentodigital dysplasia, Ohdo syndrome, Optic nerve aplasia, Ornithine carbamoyltransferase deficiency, Orofaciodigital syndrome, Osteogenesis imperfecta, Osteopetrosis, Ovarian dysgenesis, Pachyonychia, Palmoplantar keratoderma, nonepidermolytic, Papillon-Lef\xc3\xa8vre syndrome, Haim-Munk syndrome, Periodontitis, Peeling skin syndrome, Pendred syndrome, Peroxisomal fatty acyl-coa reductase 1 disorder, Peroxisome biogenesis disorder, Pfeiffer syndrome, Phenylketonuria, Phenylketonuria, Hyperphenylalaninemia, non-PKU, Pituitary hormone deficiency,  Pityriasis rubra  pilaris, Polyarteritis nodosa, Polycystic kidney disease, Polycystic lipomembranous osteodysplasia, Polymicrogyria, Pontocerebellar hypoplasia, Porokeratosis, Posterior column ataxia, Primary erythromelalgia, hyperoxaluria, Progressive familial intrahepatic cholestasis, Progressive pseudorheumatoid dysplasia, Propionic acidemia, Pseudohermaphroditism, Pseudohypoaldosteronism, Pseudoxanthoma elasticum-like disorder, Purine-nucleoside phosphorylase deficiency, Pyridoxal 5-phosphate-dependent epilepsy, Renal dysplasia, retinal pigmentary dystrophy, cerebellar ataxia, skeletal dysplasia, Reticular dysgenesis, Retinitis pigmentosa, Usher syndrome, Retinoblastoma, Retinopathy, RRM2B-related mitochondrial disease, Rubinstein-Taybi syndrome, Schnyder crystalline corneal dystrophy, Sebaceous tumor, Severe congenital neutropenia, Severe myoclonic epilepsy in infancy, Severe X-linked myotubular myopathy, onychodysplasia, facial dysmorphism, hypotrichosis, Short-rib thoracic dysplasia, Sialic acid storage disease, Sialidosis, Sideroblastic anemia, Small fiber neuropathy, Smith-Magenis syndrome, Sorsby fundus dystrophy, Spastic ataxia, Spastic paraplegia, Spermatogenic failure, Spherocytosis, Sphingomyelin/cholesterol lipidosis, Spinocerebellar ataxia, Split-hand/foot malformation, Spondyloepimetaphyseal dysplasia, Platyspondylic lethal skeletal dysplasia, Squamous cell carcinoma of the head and neck, Stargardt disease, Sucrase-isomaltase deficiency, Sudden infant death syndrome, Supravalvar aortic stenosis, Surfactant metabolism dysfunction, Tangier disease, Tatton-Brown-rahman syndrome, Thoracic aortic aneurysms and aortic dissections, Thrombophilia, Thyroid hormone resistance, TNF receptor-associated periodic fever syndrome (TRAPS), Tooth agenesis, Torsades de pointes, Transposition of great arteries, Treacher Collins syndrome, Tuberous sclerosis syndrome, Tyrosinase-negative oculocutaneous albinism, Tyrosinase-positive oculocutaneous albinism, Tyrosinemia, UDPglucose-4-epimerase deficiency, Ullrich congenital muscular dystrophy, Bethlem myopathy Usher syndrome, UV-sensitive syndrome, Van der Woude syndrome, popliteal pterygium syndrome, Very long chain acyl-CoA dehydrogenase deficiency, Vesicoureteral reflux, Vitreoretinochoroidopathy, Von Hippel-Lindau syndrome, von Willebrand disease, Waardenburg syndrome, Warsaw breakage syndrome, WFS1-Related Disorders, Wilson disease, Xeroderma pigmentosum, X-linked agammaglobulinemia, X-linked hereditary motor and sensory neuropathy, X-linked severe combined immunodeficiency, and Zellweger syndrome. 
     The development of nucleobase editing advances both the scope and effectiveness of genome editing. The nucleobase editors described here offer researchers a choice of editing with virtually no indel formation (NBE2), or more efficient editing with a low frequency (here, typically ≤1%) of indel formation (NBE3). That the product of base editing is, by definition, no longer a substrate likely contributes to editing efficiency by preventing subsequent product transformation, which can hamper traditional Cas9 applications. By removing the reliance on double-stranded DNA cleavage and stochastic DNA repair processes that vary greatly by cell state and cell type, nucleobase editing has the potential to expand the type of genome modifications that can be cleanly installed, the efficiency of these modifications, and the type of cells that are amenable to editing. It is likely that recent engineered Cas9 variants 69,70, 82  or delivery methods 71  with improved DNA specificity, as well as Cas9 variants with altered PAM specificities, 72  can be integrated into this strategy to provide additional nucleobase editors with improved DNA specificity or that can target an even wider range of disease-associated mutations. These findings also suggest that engineering additional fusions of dCas9 with enzymes that catalyze additional nucleobase transformations will increase the fraction of the possible DNA base changes that can be made through nucleobase editing. These results also suggest architectures for the fusion of other DNA-modifying enzymes, including methylases and demathylases, that mau enable additional types of programmable genome and epigenome base editing. 
     Materials and Methods 
     Cloning. DNA sequences of all constructs and primers used in this paper are listed in the Supplementary Sequences. Plasmids containing genes encoding NBE1, NBE2, and NBE3 will be available from Addgene. PCR was performed using VeraSeq ULtra DNA polymerase (Enzymatics), or Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs). NBE plasmids were constructed using USER cloning (New England Biolabs). Deaminase genes were synthesized as gBlocks Gene Fragments (Integrated DNA Technologies), and Cas9 genes were obtained from previously reported plasmids. 18  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 in the Supplementary Sequences 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 pFYF1320 (EGFP sgRNA expression plasmid) 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 h, 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 Machi competent cells (ThermoFisher Scientific). 
     In vitro deaminase assay on ssDNA. Sequences of all ssDNA substrates are listed in the Supplementary Sequences. 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 Trisacetate, 10 mM magnesium acetate, 100 ug/mL 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 encoding pET28b-His 6 -rAPOBEC-linker-dCas9 with GGS, (GGS) 3 , (SEQ ID NO: 596) XTEN, or (GGS) 7  (SEQ ID NO: 597) linkers. The resulting expression strains were grown overnight in Luria-Bertani (LB) broth containing 100 μg/mL of kanamycin at 37° C. The cells were diluted 1:100 into the same growth medium and grown at 37° C. to OD 600 =˜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 resuspended in lysis buffer (50 mM tris(hydroxymethyl)-aminomethane (Tris)-HCl, pH 7.0, 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 25,00 g for 15 nm. The lysate was incubated with His-Pur nickel-nitriloacetic acid (nickel-NTA) resin (ThermoFisher Scientific) at 4° C. for 1 h 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 on a SDSPAGE gel. 
     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 primers listed in the Supplementary Sequences with 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. Sequences of 80-nucleotide unlabeled strands are listed in the Supplementary Sequences and were ordered as PAGE-purified oligonucleotides from IDT. The 25-nt Cy3-labeled primer listed in the Supplementary Sequences is complementary to the 3′ end of each 80-nt substrate. This primer was ordered as an HPLC-purified oligonucleotide from IDT. To generate the Cy3-labeled dsDNA substrates, the 80-nt strands (5 μL of a 100 μM solution) were combined with the Cy3-labeled primer (5 μL of a 100 M 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./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-labeled 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 (HTS). The oligonucleotides listed in the Supplementary Sequences 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./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 the HTS primer pairs specified in the Supplementary Sequences 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. 73    
     Cell culture. HEK293T (ATCC CRL-3216), U2OS (ATCC-HTB-96) and ST486 cells (ATCC) were maintained in Dulbecco&#39;s Modified Eagle&#39;s Medium plus GlutaMax (ThermoFisher) supplemented with 10% (v/v) fetal bovine serum (FBS) and penicillin/streptomycin (1×, Amresco), at 37° C. with 5% C02. HCC1954 cells (ATCC CRL-2338) were maintained in RPMI-1640 medium (ThermoFisher Scientific) supplemented as described above. Immortalized rat astrocytes containing the ApoE4 isoform of the APOE gene (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 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 NBE 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. Astrocytes, U2OS, HCC1954, HEK293T and ST486 cells were transfected using appropriate AMAXA NUCLEOFECTOR™ II programs according to manufacturer&#39;s instructions. 40 ng of infrared RFP (Addgene plasmid 45457) 74  was added to the nucleofection solution to assess nucleofection efficiencies in these cell lines. For astrocytes, U2OS, and ST486 cells, nucleofection efficiencies were 25%, 74%, and 92%, respectively. For HCC1954 cells, nucleofection efficiency was &lt;10%. Therefore, following trypsinization, the HCC1954 cells were filtered through a 40 micron strainer (Fisher Scientific), and the nucleofected HCC1954 cells were collected on a Beckman Coulter MoFlo XDP Cell Sorter using the iRFP signal (abs 643 nm, em 670 nm). The other cells were used without enrichment of nucleofected cells. 
     High-throughput DNA sequencing of genomic DNA samples. Transfected cells were harvested after 3 d 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 HTS primer pairs listed in the Supplementary Sequences. 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 (30, 28, 28, 28, 32, and 32 cycles for EMX1, FANCF, HEK293 site 2, HEK293 site 3, HEK293 site 4, and RNF2 primers, respectively). 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. 73    
     Data analysis. Sequencing reads were automatically demultiplexed using MiSeq Reporter (Illumina), and individual FASTQ files were analyzed with a custom Matlab script provided in the Supplementary Notes. 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 N&#39;s 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 shown in the Supplementary Notes using previously described criteria 71 . 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. 
     All publications, patents, patent applications, publication, and database entries (e.g., sequence database entries) mentioned herein, e.g., in the Background, Summary, Detailed Description, Examples, and/or References sections, are hereby incorporated by reference in their entirety as if each individual publication, patent, patent application, publication, and database entry was specifically and individually incorporated herein by reference. In case of conflict, the present application, including any definitions herein, will control. 
     Supplementary Sequences 
     Primers used for generating sgRNA transfection plasmids. rev_sgRNA_plasmid was used in all cases. The pFYF1320 plasmid was used as template as noted in Materials and Methods section. SEQ ID NOs: 329-338 appear from top to bottom below, respectively. 
     
       
         
           
               
               
               
            
               
                 rev_sgRNA_plasmid 
                 GGTGTTTCGTCCTTTCCACAAG 
                   
               
               
                   
               
               
                 fwd_p53_Y163C 
                 GCTTGCAGATGGCCATGGCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC 
               
               
                   
               
               
                 fwd_p53_N239D 
                 TGTCACACATGTAGTTGTAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC 
               
               
                   
               
               
                 fwd_AP0E4_C158R 
                 GAAGCGCCTGGCAGTGTACCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC 
               
               
                   
               
               
                 fwd_EMX1 
                 GAGTCCGAGCAGAAGAAGAAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC 
               
               
                   
               
               
                 fwd_FANCF 
                 GGAATCCCTTCTGCAGCACCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC 
               
               
                   
               
               
                 fwd_HEK293_2 
                 GAACACAAAGCATAGACTGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC 
               
               
                   
               
               
                 fwd_HEK293_3 
                 GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC 
               
               
                   
               
               
                 fwd_HEK293_4 
                 GGCACTGCGGCTGGAGGTGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC 
               
               
                   
               
               
                 fwd_RNF2 
                 GTCATCTTAGTCATTACCTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC 
               
            
           
         
       
     
     Sequences of all ssDNA substrates used in in vitro deaminase assays. SEQ ID NOs: 339-341 appear from top to bottom below, respectively. 
     
       
         
           
               
               
               
            
               
                 rAPOBEC1 substrate 
                 Cy3-ATTATTATTATTCCGCGGATTTATTTATTTATTTATTTATTT 
                   
               
               
                   
               
               
                 hAID/pmCDA1 substrate 
                 Cy3-ATTATTATTATTAGCTATTTATTTATTTATTTATTTATTT 
               
               
                   
               
               
                 hAPOBEC3G substrate 
                 Cy3-ATTATTATTATTCCCGGATTTATTTATTTATTTATTTATTT 
               
            
           
         
       
     
     Primers used for generating PCR products to serve as substrates for T7 transcription of sgRNAs for gel-based deaminase assay. rev_gRNA_T7 was used in all cases. The pFYF1320 plasmid was used as template as noted in Materials and Methods section. SEQ ID NOs: 342-365 appear from top to bottom below, respectively. 
     
       
         
           
               
               
               
            
               
                 rev_sgRNA_T7 
                 AAAAAAAGCACCGACTCGGTG 
                   
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA_2 
                 TAATACGACTCACTATAGGCCGCGGATTTATTTATTTAAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA_3 
                 TAATACGACTCACTATAGGTCCGCGGATTTATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA_4 
                 TAATACGACTCACTATAGGTTCCGCGGATTTATTTATTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA_5 
                 TAATACGACTCACTATAGGATTCCGCGGATTTATTTATTGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA_6 
                 TAATACGACTCACTATAGGTATTCCGCGGATTTATTTATGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA_7 
                 TAATACGACTCACTATAGGTTATTCCGCGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA_S 
                 TAATACGACTCACTATAGGATTATTCCGCGGATTTATTTGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA_9 
                 TAATACGACTCACTATAGGTATTATTCCGCGGATTTATTGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA_10 
                 TAATACGACTCACTATAGGATTATTATCCGGGGATTTATGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA_11 
                 TAATACGACTCACTATAGGTATTATATTCCGCGGATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA_12 
                 TAATACGACTCACTATAGGTTATTATATTCCGCGGATTTGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA_13 
                 TAATACGACTCACTATAGGATTATTATATTCCGCGGATTGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA_14 
                 TAATACGACTCACTATAGGTATTATTATATTCCGCGGATGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA_15 
                 TAATACGACTCACTATAGGATTATTATTATTACCGCGGAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA_18 
                 TAATACGACTCACTATAGGATTATTATTATTATTACCGCGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA_noC 
                 TAATACGACTCACTATAGGATATTAATTTATTTATTTAAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA_ 
                 TAATACGACTCACTATAGGGGAGGACGTGCGCGGCCGCCGTTTTAGAGCTAGAAATAGCA 
               
               
                 APOE4_C112R 
                   
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA 
                 TAATACGACTCACTATAGGGAAGCGCCTGGCAGTGTACCGTTTTAGAGCTAGAAATAGCA 
               
               
                 APOE4_C158R 
                   
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA 
                 TAATACGACTCACTATAGGCTGTGGCAGTGGCACCAGAAGTTTTAGAGCTAGAAATAGCA 
               
               
                 CTNNB1_T41A 
                   
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA 
                 TAATACGACTCACTATAGGCCTCCCGGCCGGCGGTATCCGTTTTAGAGCTAGAAATAGCA 
               
               
                 HRAS_081R 
                   
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA 
                 TAATACGACTCACTATAGGGCTTGCAGATGGCCATGGCGGTTTTAGAGCTAGAAATAGCA 
               
               
                 53_Y163C 
                   
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA 
                 TAATACGACTCACTATAGGACACATGCAGTTGTAGTGGAGTTTTAGAGCTAGAAATAGCA 
               
               
                 53_Y236C 
                   
               
               
                   
               
               
                 fwd_sgRNA_T7_dsDNA 
                 TAATACGACTCACTATAGGTGTCACACATGTAGTTGTAGGTTTTAGAGCTAGAAATAGCA 
               
               
                 53_N23SD 
                   
               
            
           
         
       
     
     Sequences of 80-nucleotide unlabeled strands and Cy3-labeled universal primer used in gel-based dsDNA deaminase assays. SEQ ID NOs: 366-390 appear from top to bottom below, respectively. 
     
       
         
           
               
               
               
               
            
               
                   
                 Cy3-primer 
                 Cy3-GTAGGTAGTTAGGATGAATGGAAGGTTGGTA 
                   
               
               
                   
                   
               
               
                   
                 dsDNA_2 
                 GTCCATGGATCCAGAGGTCATCCATTAAATAAATAAATCCGCGGGGCTATACCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                   
               
               
                   
                 dsDNA_3 
                 GTCCATGGATCCAGAGGTCATCCATAAATAAATAAATCCGCGGAAGCTATACCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                   
               
               
                   
                 dsDNA_4 
                 GTCCATGGATCCAGAGGTCATCCATAATAAATAAATCCGCGGAAGGCTATACCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                   
               
               
                   
                 dsSNA_5 
                 GTCCATGGATCCAGAGGTCATCCAAATAAATAAATCGGCGGAATGGCTATACCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                   
               
               
                   
                 dsDNA_5 
                 GTCCATGGATCCAGAGGTCATCCAATAAATAAATCCGCGGAATAGGCTATACCAACCTTCCATTCATCCTAACTACCTAG 
               
               
                   
                   
               
               
                   
                 dsDNA_7 
                 GTCCATGGATCCAGAGGTCATCCATAAATAAATCCGCGGAATAAGGCTATACCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                   
               
               
                   
                 dsDNA_8 
                 GTCCATGGATCCAGAGGTCATCCAAAATAAATCCGCGGAATAATGGCTATACCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                   
               
               
                   
                 dsDMA_3 
                 GTCCATGGATCCAGAGGTCATCCAAATAAATCCGCGGAATAATAGGCTATACCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                   
               
               
                   
                 dsDMA_10 
                 GTCCATGGATCCAGAGGTCATCCAATAAATCCGCGGATAATAATGGCTATACCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                   
               
               
                   
                 dsDNA_11 
                 GTCCATGGATCCAGAGGTCATCCATAAATCCGCGGAATATAATAGGCTATACCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                   
               
               
                   
                 dsDNA_12 
                 GTCCATGGATCCAGAGGTCATCCAAAATCCGCGGAATATAATAAGGCTATACCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                   
               
               
                   
                 dsDNA_13 
                 GTCCATGGATCCAGAGGTCATCCAAATCCGCGGAATATAATAATGGCTATACCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                   
               
               
                   
                 dsDNA_14 
                 GTCCATGGATCCAGAGGTCATCCAATCCGCGGAATATAATAATAGGCTATACCAACCTTCCATTCATCCTAACTACGTAC 
               
               
                   
                   
               
               
                   
                 dsDNA_15 
                 GTCCATGGATCCAGAGGTCATCCAATCCGCGGTAATAATAATAAGGCTATACCAACCTTCCATTCATCCTAACTACGTAC 
               
               
                   
                   
               
               
                   
                 dsDNA_18 
                 GTCCATGGATGCAGAGGTCATCCAGCGGTAATAATAATAATAATGGCTATACCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                   
               
               
                   
                 dsDNA_noC 
                 GTCCATGGATCCAGAGGTCATCCATTAAATAAATAAATTAATATTACTATACCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                   
               
               
                   
                 dsDNA_8U 
                 5Cy3-GTAGGTGTTAGGATGAATGGAAGGTTGGTAGATATTATCUGCGGATTTATTGGATGAGACCTCTGGATCCATGGACAT 
               
               
                   
                   
               
               
                   
                 dsDNA_APOE_ 
                 GCACCTCGCCGCGGTACTGCACCAGGCGGCCGCGCACGTCCTCCATGTCTACCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                 C112R 
                   
               
               
                   
                   
               
               
                   
                 d5DNA_APOE_ 
                 CGGCGCCCTCGCGGGCCCCGGCCTGGTACACTGCCAGGCGCTTCTGCAGTAOCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                 C158R 
                   
               
               
                   
                   
               
               
                   
                 dsDNA_CTNN5 
                 GTCTTACCTGGACTCTGGAATCCATTCTGGTGCCACTGCCACAGCTCCTTACCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                 T41A 
                   
               
               
                   
                   
               
               
                   
                 dsDNA_HRAS_ 
                 GGAGACGTGCCTGTTGGACATCCTGGATAOCGCCGGaCCGGGAGGAGTACTACCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                 Q61R 
                   
               
               
                   
                   
               
               
                   
                 dsDNA_p53 
                 ACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCCATCTGCAAGCAGTCATACCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                 Y163C 
                   
               
               
                   
                   
               
               
                   
                 dsDNA_p53_ 
                 AGGTTGGCTCTGACTGTACCACCATCCACTACAACTGCATGTGTAACAGTACCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                 Y236C 
                   
               
               
                   
                   
               
               
                   
                 dsDNA_p53_ 
                 TGGCTCTGACTGTACCACCATCCACTACAACTACATGTGTGACAGTTCCTACCAACCTTCCATTCATCCTAACTACCTAC 
               
               
                   
                 N239D 
                   
               
            
           
         
       
     
     Primers used for generating PCR products to serve as substrates for T7 transcription of sgRNAs for high-throughput sequencing. rev_gRNA_T7 (above) was used in all cases. The pFYF1320 plasmid was used as template as noted in Materials and Methods section. SEQ ID NOs: 391-442 appear from top to bottom below, respectively. 
     
       
         
           
               
               
               
            
               
                 fwd_sgRNA_T7_HTS_base 
                 TAATACGACTCACTATAGGTTATTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
                   
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_1A 
                 TAATACGACTCACTATAGGATATTTCGTGGATTTATTTA3TTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_1C 
                 TAATACGACTCACTATAGGCTATTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_1G 
                 TAATACGACTCACTATAGGGTATTTCGTGSATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_2A 
                 TAATACGACTCACTATAGGTAATTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_2C 
                 TAATACGACTCACTATAGGTCATTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_2G 
                 TAATACGACTCACTATAGGTGATTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_3T 
                 TAATACGACTCACTATAGGTTTTTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_3C 
                 TAATACGACTCACTATAGGTTCTTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_3G 
                 TAATACGACTCACTATAGGTTGTTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_4A 
                 TAATACGACTCACTATAGGTTAATTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_4C 
                 TAATACGACTCACTATAGGTTACTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_4G 
                 TAATACGACTCACTATAGGTTAGTTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_5A 
                 TAATACGACTCACTATAGGTTATATCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_5C 
                 TAATACGACTCACTATAGGTTATCTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_5G 
                 TAATACGACTCACTATAGGTTATGTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_6A 
                 TAATACGACTCACTATAGGTTATTACGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_5C 
                 TAATACGACTCACTATAGGTTATTCCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_6G 
                 TAATACGACTCACTATAGGTTATTGCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_8A 
                 TAATACGACTCACTATAGGTTATTTCATGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_8T 
                 TAATACGACTCACTATAGGTTATTTCTTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_8C 
                 TAATACGACTCACTATAGGTTATTTCCTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_9A 
                 TAATACGACTCACTATAGGTTATTTCGAGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_9C 
                 TAATACGACTCACTATAGGTTATTTCGCGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_9G 
                 TAATACGACTCACTATAGGTTATTTCGGGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_10A 
                 TAATACGACTCACTATAGGTTATTTCGTAGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_10T 
                 TAATACGACTCACTATAGGTTATTTCGTIGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_10C 
                 TAATACGACTCACTATAGGTTATTTCGTCGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_11A 
                 TAATACGACTCACTATAGGTTATTTCGTGAATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_11T 
                 TAATACGACTCACTATAGGTTATTTCGTGTATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_11C 
                 TAATACGACTCACTATAGGTTATTTCGTGCATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_12T 
                 TAATACGACTCACTATAGGTTATTTCGTGGTTTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_12C 
                 TAATACGACTCACTATAGGTTATTTCGTGGCTTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_12G 
                 TAATACGACTCACTATAGGTTATTTCGTGGGTTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_13A 
                 TAATACGACTCACTATAGGTTATTTCGTGGAATTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_13C 
                 TAATACGACTCACTATAGGTTATTTCGTGGACTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_13G 
                 TAATACGACTCACTATAGGTTATTTCGTGGAGTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_77_HTS_multiC 
                 TAATACGACTCACTATAGGTTCCCCCCCCGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_77_HTS_TCGCACCC_odd 
                 TAATACGACTCACTATAGGCGCACCCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_77_HTS_CCTCGCAC_odd 
                 TAATACGACTCACTATAGGCTCGCACGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_77_HTS_ACCCTCGC_odd 
                 TAATACGACTCACTATAGGCCCTCGCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_77_HTS_GCACCCTC_even 
                 TAATACGACTCACTATAGGCACCCTCGTGGATTTATTTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_TCGCACCC_even 
                 TAATACGACTCACTATAGGTCGCACCCGTG3ATTTATTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_CCTCGCAC_even 
                 TAATACGACTCACTATAGGCCTCGCACGTGGATTTATTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_ACCCTCGC_even 
                 TAATACGACTCACTATAGGACCCTCGCGTG3ATTTATTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_GCACCCTC_even 
                 TAATACGACTCACTATAGGGCACCCTCGTGGATTTATTAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_EMX1 
                 TAATACGACTCACTATAGGGAGTCCGAGCAGAAGAAGAAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_FANCF 
                 TAATACGACTCACTATAGGGGAATCCCTTCTGCAGCACCGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_HEK293_site2 
                 TAATACGACTCACTATAGGGAACACAAAGCATAGACTGCGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_HEK293_site3 
                 TAATACGACTCACTATAGGGGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_HEK293_siie4 
                 TAATACGACTCACTATAGGGGCACTGCGGCTGGAGGTGGGTTTTAGAGCTAGAAATAGCA 
               
               
                   
               
               
                 fwd_sgRNA_T7_HTS_RNF2 
                 TAATACGACTCACTATAGGGTCATCTTAGTCATTACCTGGTTTTAGAGCTAGAAATAGCA 
               
            
           
         
       
     
     Sequences of in vitro-edited dsDNA for high-throughput sequencing (HTS). Shown are the sequences of edited strands. Reverse complements of all sequences shown were also obtained. dsDNA substrates were obtained by annealing complementary strands as described in Materials and Methods. Oligonucleotides representing the EMX1, FANCF, HEK293 site 2, HEK293 site 3, HEK293 site 4, and RNF2 loci were originally designed for use in the gel-based deaminase assay and therefore have the same 25-nt sequence on their 5′-ends (matching that of the Cy3-primer). SEQ ID NOs: 443-494 appear from top to bottom below, respectively. 
     
       
         
           
               
               
               
            
               
                 Base sequence 
                 ACGTAAACGGCCACAAGTTCTTATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
                   
               
               
                   
               
               
                 1A 
                 ACGTAAACGGCCACAAGTTCATATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 1C 
                 ACGTAAACGGCCACAAGTTCCTATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 1G 
                 ACGTAAACGGCCACAAGTTCGTATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 2A 
                 ACGTAAACGGCCACAAGTTCTAATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 2C 
                 ACGTAAACGGCCACAAGTTCTCATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 2G 
                 ACGTAAACGGCCACAAGTTCTGATTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 3T 
                 ACGTAAACGGCCACAAGTTCTTTTTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 3C 
                 ACGTAAACGGCCACAAGTTCTTCTTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 3G 
                 ACGTAAACGGCCACAAGTTCTTGTTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 4A 
                 ACGTAAACGGCCACAAGTTCTTAATTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 4C 
                 ACGTAAACGGCCACAAGTTCTTACTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 4G 
                 ACGTAAACGGCCACAAGTTCTTAGTTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 5A 
                 ACGTAAACGGCCACAAGTTCTTATATCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 5C 
                 ACGTAAACGGCCACAAGTTCTTATCTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 5G 
                 ACGTAAACGGCCACAAGTTCTTATGTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 5A 
                 ACGTAAACGGCCACAAGTTCTTATTACGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 6C 
                 ACGTAAACGGCCACAAGTTCTTATTCCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 5G 
                 ACGTAAACGGCCACAAGTTCTTATTGCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 8A 
                 ACGTAAACGGCCACAAGTTCTTATTTCATGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 8T 
                 ACGTAAACGGCCACAAGTTCTTATTTCTTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 8C 
                 ACGTAAACGGCCACAAGTTCTTATTTCCTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 9A 
                 ACGTAAACGGCCACAAGTTCTTATTTCGAGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 9C 
                 ACGTAAACGGCCACAAGTTCTTATTTCGCGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 9G 
                 ACGTAAACGGCCACAAGTTCTTATTTCGGGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 10A 
                 ACGTAAACGGCCACAAGTTCTTATTTCGTAGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 10T 
                 ACGTAAACGGCCACAAGTTCTTATTTCGTTGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 10C 
                 ACGTAAACGGCCACAAGTTCTTATTTCGTCGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 11A 
                 ACGTAAACGGCCACAAGTTCTTATTTCGTGAATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 11T 
                 ACGTAAACGGCCACAAGTTCTTATTTCGTGTATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 11C 
                 ACGTAAACGGCCACAAGTTCTTATTTCGTGCATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 12T 
                 ACGTAAACGGCCACAAGTTCTTATTTCGTGGTTTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 12C 
                 ACGTAAACGGCCACAAGTTCTTATTTCGTGGCTTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 12G 
                 ACGTAAACGGCCACAAGTTCTTATTTCGTGGGTTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 13A 
                 ACGTAAACGGCCACAAGTTCTTATTTCGTGGAATTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 13C 
                 ACGTAAACGGCCACAAGTTCTTATTTCGTGGACTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 13G 
                 ACGTAAACGGCCACAAGTTCTTATTTCGTGGAGTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 multiC 
                 ACGTAAACGGCCACAAGTTCTTCCCCCCCCGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 TCGCACCC_odd 
                 ACGTAAACGGCCACAAGTTTCGCACCCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 CCTCGCAC_odd 
                 ACGTAAACGGCCACAAGTTCCTCGCACGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 ACCCTCGC_odd 
                 ACGTAAACGGCCACAAGTTACCCTCGCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 GCACCCTC_odd 
                 ACGTAAACGGCCACAAGTTGCACCCTCGTGGATTTATTTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 TCGCACCC_even 
                 ACGTAAACGGCCACAAGTATTCGCACCCGTGGATTTATTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 CCTCGCAC_even 
                 ACGTAAACGGCCACAAGTATCCTCGCACGTGGATTTATTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 ACCCTCGC_even 
                 ACGTAAACGGCCACAAGTATACCCTCGCGTGGATTTATTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 GCACCCTC_even 
                 ACGTAAACGGCCACAAGTATGCACCCTCGTGGATTTATTATGGCATCTTCTTCAAGGACG 
               
               
                   
               
               
                 EMX1_invitro 
                 GTAGGTAGTTAGGATGAATGGAAGGTTGGTAGGCCTGAGTCCGAGCAGAAGAAGAAGGGCTCCCATCACATCAACCGGTG 
               
               
                   
               
               
                 FANCF_invitro 
                 GTAGGTAGTTAGGATGAATGGAAGGTTGGTACTCATGGAATCCCTTCTGCAGCACCTGGATCGCTTTTCCGAGCTTCTGG 
               
               
                   
               
               
                 HEK293_site2_ 
                 GTAGGTAGTTAGGATGAATGGAAGGTTGGTAAACTGGAACACAAAGCATAGACTGCGGGGCGGGCCAGCCTGAATAGCTG 
               
               
                 invitro 
                   
               
               
                   
               
               
                 HEK293_site3_ 
                 GTAGGTAGTTAGGATGAATGGAAGGTTGGTACTTGGGGCCCAGACTGAGCACGTGATGGCAGAGGAAAGGAAGCCCTGCT 
               
               
                 invitro 
                   
               
               
                   
               
               
                 HEK293_site4_ 
                 GTAGGTAGTTAGGATGAATGGAAGGTTGGTACCGGTGGCACTGCGGCTGGAGGTGGGGGTTAAAGCGGAGACTCTGGTGC 
               
               
                 invitro 
                   
               
               
                   
               
               
                 RNF2_invitro 
                 GTAGGTAGTTAGGATGAATGGAAGGTTGGTATGGCAGTCATCTTAGTCATTACCTGAGGTCGTTGTAACTCATATAA 
               
            
           
         
       
     
     Primers for HTS of in vitro edited dsDNA. SEQ ID NOs: 495-503 appear from top to bottom below, respectively. 
     
       
         
           
               
               
               
            
               
                 fwd_invitro_HTS 
                 ACACTCTTTCCCTACACGAGCTCTTCCGATCTNNNNACGTAAACGGCCACAA 
                   
               
               
                   
               
               
                 rev_invitro_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTCGTCCTTGAAGAAGATGC 
               
               
                   
               
               
                 fwd_invitro_HEK_targets 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTAGGTAGTTAGGATAATGGAA 
               
               
                   
               
               
                 rev_EMX1_invitro 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTCACCGGTTGATGTGATGG 
               
               
                   
               
               
                 rev_FANCF_invitro 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTCCAGAAGCTCGGAAAAGC 
               
               
                   
               
               
                 rev_HEK293_site2_invitro 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTCAGCTATTCAGGCTGGC 
               
               
                   
               
               
                 rev_HEK293_site3_invitro 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTAGCAGGGCTTCCTTTC 
               
               
                   
               
               
                 rev_HEK293_site4_invitro 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGCACCAGAGTCTCCG 
               
               
                   
               
               
                 rev_RNF2_invitro 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTTTATATGAGTTACAACGAACACC 
               
            
           
         
       
     
     Primers for HTS of on-target and off-target sites from all mammalian cell culture experiments. SEQ ID NOs: 504-579 and 1869-1900 appear from top to bottom below, respectively. 
     
       
         
           
               
               
               
            
               
                 fwd_EMX1_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCAGCTCAGCCTGACTGTTGA 
                   
               
               
                   
               
               
                 rev_EMX1_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTCTCGTGGGTTTGTGGTTGC 
               
               
                   
               
               
                 fwd_FANCF_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCATTGCAGAGAGGCGTATCA 
               
               
                   
               
               
                 rev_FANCF_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGGGGTCCCAGGTGCTGAC 
               
               
                   
               
               
                 fwd_HEK293_site2_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCAGCCCCATCTGTCAAACT 
               
               
                   
               
               
                 rev_HEK293_site2_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTTGAATGGATTCCTTGGAAACAATGA 
               
               
                   
               
               
                 fwd_HEK293_Site3_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNATGTGGGCTGCCTAGAAAGG 
               
               
                   
               
               
                 rev_HEK293_sit93_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTCCCAGCCAAACTTGTCAACC 
               
               
                   
               
               
                 fwd_HEK293_site4_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGAACCCAGGTAGCCAGAGAC 
               
               
                   
               
               
                 rev_HEK293_site4_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTTCCTTTCAACCCGAACGGAG 
               
               
                   
               
               
                 fwd_RNF2_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCTCTTCTTTATTTCCAGCAATGT 
               
               
                   
               
               
                 rev_RNF2_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGTTTTCATGTTCTAAAAATGTATCCCA 
               
               
                   
               
               
                 fwd_p53_Y163C_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTACAGTACTCCCCTGCCCTC 
               
               
                   
               
               
                 rev_p53_Y163C_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGCTGCTCACCATCGCTATCT 
               
               
                   
               
               
                 fwd_p53_N239D_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCTCATCTTGGGCCTGTGTT 
               
               
                   
               
               
                 rev_p53_N239D_HTS  
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTAAATCGGTAAGAGGTGGGCC 
               
               
                   
               
               
                 fwd_APOE4_C15SR_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCGGACATGGAGGACGTG 
               
               
                   
               
               
                 rev_AFOE4_C15SR_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTCTGTTCCACCAGGGGCCC 
               
               
                   
               
               
                 fwd_EMX1_off1_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGCCCAATCATTGATGCTTTT 
               
               
                   
               
               
                 rev_EMX1_off1_HTS  
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTAGAAACATTTACCATAGACTATCACCT 
               
               
                   
               
               
                 fwd_EMX1_off2_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAGTAGCCTCTTTCTCAATGTGC 
               
               
                   
               
               
                 rev_EWX1_off2_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGCTTTCACAAGGATGCAGTCT 
               
               
                   
               
               
                 fwd_EMX1_off3_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGAGCTAGACTCCGAGGGGA 
               
               
                   
               
               
                 rev_EMX1_off3_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTTCCTCGTCCTGCTCTCACTT 
               
               
                   
               
               
                 fwd_EMX1_off4_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCC3ATCTNNNNAGAGGCTGAAGAGGAAGACCA 
               
               
                   
               
               
                 rev_EMX1_off4_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGGCCCAGCTGTGCATTCTAT 
               
               
                   
               
               
                 fwd_EMX1_off6_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCAAGAGGGCCAAGTCCTG 
               
               
                   
               
               
                 rev_EMX1_off6_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTCAGCGAGGAGTGACAGCC 
               
               
                   
               
               
                 fwd_EMX1_off7_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCACTCCACCTGATCTCGGGG 
               
               
                   
               
               
                 rev_EMX1_off7_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTCGAGGAGGGAGGGAGCAG 
               
               
                   
               
               
                 fwd_EMX1_off8_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNACCACAAATGCCCAAGAGAC 
               
               
                   
               
               
                 rev_EMX1_off8_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGACACAGTCAAGGGCCGG 
               
               
                   
               
               
                 fwd_EMX1_off9_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCCACCTTTGAGGAGGCAAA 
               
               
                   
               
               
                 rev_EMX1_off9_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTTTCCATCTGAGAAGAGAGTGGT 
               
               
                   
               
               
                 fwd_EMX1_off10_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTCATACCTTGGCCCTTCCT 
               
               
                   
               
               
                 rev_EMX1_off10_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTTCCCTAGGCCCACACCAG 
               
               
                   
               
               
                 fwd_FANCF_off1_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAACCCACTGAAGAAGCAGGG 
               
               
                   
               
               
                 rev_FANCF_off1_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGGTGCTTAATCCGGCTCCAT 
               
               
                   
               
               
                 fwd_FANCF_off2_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCAGTGTTTCCATCCCGAA 
               
               
                   
               
               
                 rev_FANCF_off2_HTS 
                 TGGAGTTCAGACGTGTGOTCTTCCGATCTCCTCTGACCTCCACAACTCT 
               
               
                   
               
               
                 fwd_FANCF_off3_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCTGGGTACAGTTCTGCGTGT 
               
               
                   
               
               
                 rev_FANCF_off3_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTTCACTCTGAGCATCGCCAAG 
               
               
                   
               
               
                 fwd_FANCF_off4_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGTTTAGAGCCAGTGAACTAGAG 
               
               
                   
               
               
                 rev_FANCF_off4_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGCAAGACAAAATCCTCTTTATACTTTG 
               
               
                   
               
               
                 fwd_FANCF_off5_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGGAGGGGACGGCCTTAC 
               
               
                   
               
               
                 rev_FANCF_off5_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGCCTCTGGCGAACATGGC 
               
               
                   
               
               
                 fwd_FANCF_off6_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCTGGTTAAGAGCATGGGC 
               
               
                   
               
               
                 rev_FANCF_off6_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGATTGAGTCCCCACAGCACA 
               
               
                   
               
               
                 fwd_FANCF_off7_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCAGTGTTTCCCATCCCCAA 
               
               
                   
               
               
                 rev_FANCF_off7_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTTGACCTCCACAACTGGAAAAT 
               
               
                   
               
               
                 fwd_FANCF_off8_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCTTCCAGACCCACCTGAAG 
               
               
                   
               
               
                 rev_FANCF_off*_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTACCGAGGAAAATTGCTTGTCG 
               
               
                   
               
               
                 fwd_HEK293_site2_off1_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTGTGGAGAGTGAGTAAGCCA 
               
               
                   
               
               
                 rev_HEK293_site2_off1_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTACGGTAGGATGATTTCAGGCA 
               
               
                   
               
               
                 fwd_HEK293_site2_cff2_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCACAAAGCAGTGTAGCTCAGG 
               
               
                   
               
               
                 rev_HEK293_site2_off2_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTTTTTTGGTACTCGAGTGTTATTCAG 
               
               
                   
               
               
                 fwd_HEK293_site3_off1_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCCCTGTTGACCTGGAGAA 
               
               
                   
               
               
                 rev_HEK293_site3_off1_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTCACTGTACTTGCCCTGACCA 
               
               
                   
               
               
                 fwd_HEK293_site3_off2_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTGGTGTTGACAGGGAGCAA 
               
               
                   
               
               
                 rev_HEK293_site3_off2_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTCTGAGATGTGGGCAGAAGGG 
               
               
                   
               
               
                 fwd_HEK293_site3_off3_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGAGAGGGAACAGAAGGGCT 
               
               
                   
               
               
                 rev_HEK293_site3_off3_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGTCCAAAGGCCCAAGAACCT 
               
               
                   
               
               
                 fwd_HEK2S3_site3_off4_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCCTAGCACTTTGGAAGGTCG 
               
               
                   
               
               
                 rev_HEK293_site3_off4_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGCTCATCTTAATCTGCTCAGCC 
               
               
                   
               
               
                 fwd_HEK293_site3_off5_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAAAGGAGCAGCTCTTCCTGG 
               
               
                   
               
               
                 rev_HEK293_site3_off5_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGTCTGCACCATCTCCCACAA 
               
               
                   
               
               
                 fwd_HEK293_site4_off1_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGCATGGCTTCTGAGACTCA 
               
               
                   
               
               
                 rev_HEK293_site4_off1_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGTCTCCCTTGCACTCCCTGTCTTT 
               
               
                   
               
               
                 fwd_HEK293_site4_off2_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTTGGCAATGGAGGCATTGG 
               
               
                   
               
               
                 rev_HEK293_site4_off2_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGAAGAGGCTGCCCATGAGAG 
               
               
                   
               
               
                 fwd_HEK293_site4_off3_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGTCTGAGGCTCGAATCCTG 
               
               
                   
               
               
                 rev_HEK293_site4_off3_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTCTGTGGCCTCCATATCCCTG 
               
               
                   
               
               
                 fwd_HEK293_site4_off4_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTTTCCACCAGAACTCAGCCC 
               
               
                   
               
               
                 rev_HEK293_site4_off4_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTCCTCGGTTCCTCCACAACAC 
               
               
                   
               
               
                 fwd_HEK293_site4_off5_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCACGGGAAGGACAGGAGAAC 
               
               
                   
               
               
                 rev_HEK293_site4_off5_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGCAGGGGAGGGATAAAGCAG 
               
               
                   
               
               
                 fwd_HEK293_site4_off6_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCACGGGAGATGGCTTATGT 
               
               
                   
               
               
                 rev_HEK293_site4_off6_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTCACATCCTCACTGTGCCACT 
               
               
                   
               
               
                 fwd_HEK293_site4_off7_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTCAGTCTCGGCCCCTCA 
               
               
                   
               
               
                 rev_HEK293_site4_off7_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGCCACTGTAAAGCTCTTGGG 
               
               
                   
               
               
                 fwd_HEK293_site4_off8_HTS 
                 AGACTCTTTCCCTACAC3ACGCTCTTCCGATCTNNNNAGGGTAGAGGGACAGAGCTG 
               
               
                   
               
               
                 rev_HEX293_site4_off8_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGGACCCCACATAGTCAGTGC 
               
               
                   
               
               
                 fwd_HEK293_site4_off9_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCTGTCAGCCCTATCTCCATC 
               
               
                   
               
               
                 rev_HEK293_site4_off9_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTTGGGCAATTAGGACAGGGAC 
               
               
                   
               
               
                 fwd_HEK293_site4_off10_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCAGCGGAGGAGGTAGATTG 
               
               
                   
               
               
                 rev_HEK293_site4_off10_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTCTCAGTACCTGGAGTCCCGA 
               
               
                   
               
               
                 fwd_HEK2_ChIP_off1_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGACAGGCTCAGGAAAGCTGT 
               
               
                   
               
               
                 rev_HEK2_ChIP_off1_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTACACAAGCCTTTCTCCAGGG 
               
               
                   
               
               
                 fwd_HEK2_ChIP_off2_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNAATAGGGGGTGAGACTGGGG 
               
               
                   
               
               
                 rev_HEK2_ChIP_off2_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTGCCTCAGACGAGACTTGAGG 
               
               
                   
               
               
                 fwd_HEK2_ChIP_off3_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGGCCAGCAGGAAAGGAATCT 
               
               
                   
               
               
                 rev_HEK2_ChIp_off3_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTTGACTGCACCTGTAGCCATG 
               
               
                   
               
               
                 fwd_HEK2_ChIP_off4_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTCAAGGAAATCACCCTGCCC 
               
               
                   
               
               
                 rev_HEK2_ChIP_off4_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTAACTTCCTTGGTGTGCAGCT 
               
               
                   
               
               
                 fwd_HEK2_ChIP_off5_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNATOGGCTCAGCTACGTCATG 
               
               
                   
               
               
                 rev_HEK2_ChIP_off5_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTAATAGCAGTGTGGT6GGCAA 
               
               
                   
               
               
                 fWd_HEK3_ChIP_off1_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCGCACATCCCTTGTCTCTCT 
               
               
                   
               
               
                 rev_HEK3_ChIP_off1_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTCTACTGGAGCACACCCCAAG 
               
               
                   
               
               
                 fwd_HEK3_ChIP_off2_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGGGTCACGTAGCTTTGGTC 
               
               
                   
               
               
                 rev_HEK3_ChIP_off2_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTTGGTGGCCATGTGCAACTAA 
               
               
                   
               
               
                 fwd_HEK3_ChIP_off3_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCTACTACGTGCCAGCTCAGG 
               
               
                   
               
               
                 rev_HEK3_ChIP_off3_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTACCTCCCCTCCTCACTAACC 
               
               
                   
               
               
                 fwd_HEK3_ChIP_off4_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGCCTCAGCTCCATTTCCTGT 
               
               
                   
               
               
                 rev_HEK3_ChIP_off4_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTAACCTTTATGGCACCAGGGG 
               
               
                   
               
               
                 fwd_HEK3_ChIP_off5_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGAGCTCAGCATTAGCAGGCT 
               
               
                   
               
               
                 rev_HEK3_ChIP_off5_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTTTCCTGGCTTTCCGATTCCC 
               
               
                   
               
               
                 fwd_HEK4_ChIp_off1_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNGTGCAATTGGAGGAGGAGCT 
               
               
                   
               
               
                 rev_HEK4_ChIp_off1_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTCACCAGCTACAGGCAGAACA 
               
               
                   
               
               
                 fwd_HEK4_ChIP_off3_HTS 
                 ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCTACCCCCAACACAGATGG 
               
               
                   
               
               
                 rev_HEK4_ChIP_off3_HTS 
                 TGGAGTTCAGACGTGTGCTCTTCCGATCTCCACACAACTCAGGTCCTCC 
               
               
                   
               
            
           
         
       
     
     Sequences of single-stranded oligonucleotide donor templates (ssODNs) used in HDR studies. 
     
       
         
           
               
            
               
                 EMX1 sense 
               
               
                 (SEQ ID NO: 580) 
               
               
                 TCATCTGTGCCCCTCCCTCCCTGGCCCAGGTGAAGGTGTGGTTCCAGAAC 
               
               
                   
               
               
                 CGGAGGACAAAGTACAAACGGCAGAAGCTGGAGGAGGAAGGGCCTGAGTT 
               
               
                   
               
               
                 TGAGCAGAAGGAAGGGCTCCCATCACATCAACCGCGGTGGCGCATTGCCA 
               
               
                   
               
               
                 CGAAGCAGGCCAATGGGGAGGACATCGATGTCACCTCCAATGACTAGGGT 
               
               
                   
               
               
                 EMX1 antisense 
               
               
                 (SEQ ID NO: 581) 
               
               
                 ACCCTAGTCATTGGAGGTGACATCGATGTCCTCCCCATTGGCCTGCTTCG 
               
               
                   
               
               
                 TGGCAATGCGCCACCGGTTGATGTGATGGGAGCCCTTCTTCTTCTGCTCA 
               
               
                   
               
               
                 AACTCAGGCCCTTCCTCCTCCAGCTTCTGCCGTTTGTACTTTGTCCTCCG 
               
               
                   
               
               
                 GTTCTGGAACCACACCTTCACCTGGGCCAGGGAGGGAGGGGCACAGATGA 
               
               
                   
               
               
                 HEK293 site 3 sense 
               
               
                 (SEQ ID NO: 582) 
               
               
                 CATGCAATTAGTCTATTTCTGCTGCAAGTAAGGATGCATTTGTAGGCTTG 
               
               
                   
               
               
                 ATGCTTTTTTTCTGCTTCTCCAGCCCTGGCCTGGGTCAATCCTTGGGGCT 
               
               
                   
               
               
                 TAGACTGAGCACGTGATGGCAGAGGAAAGGAAGCCCTGCTTCCTCCAGAG 
               
               
                   
               
               
                 GGCGTCGCAGGACAGCTTTTCCTAGACAGGGGCTAGTATGTGCAGCTCCT 
               
               
                   
               
               
                 HEK293 site 3 antisense 
               
               
                 (SEQ ID NO: 583) 
               
               
                 AGGAGCTGCACATACTAGCCCCTGTCTAGGAAAAGCTGTCCTGCGACGCC 
               
               
                   
               
               
                 CTCTGGAGGAAGCAGGGCTTCCTTTCCTCTGCCATCACGTGCTCAGTCTA 
               
               
                   
               
               
                 AGCCCCAAGGATTGACCCAGGCCAGGGCTGGAGAAGCAGATAAAAAGCAT 
               
               
                   
               
               
                 CAAGCCTACAAATGCATGCTTACTTGCAGCAGAAATAGACTAATTGCATG 
               
               
                   
               
               
                 HEK site 4 sense 
               
               
                 (SEQ ID NO: 584) 
               
               
                 GGCTGACAAAGGCCGGGCTGGGTGGAAGGAAGGGAGGAAGGGCGAGGCAG 
               
               
                   
               
               
                 AGGGTCCAAAGCAGGATGACAGGCAGGGGCACCGCGGCGCCCCGGTGGCA 
               
               
                   
               
               
                 TTGCGGCTGGAGGTGGGGGTTAAAGCGGAGACTCTGGTGCTGTGTGACTA 
               
               
                   
               
               
                 CAGTGGGGGCCGTGCCCTCTCTGAGCCCCCGCCTCCAGGCCTGTGTGTGT 
               
               
                   
               
               
                 HEK site 4 antisense 
               
               
                 (SEQ ID NO: 585) 
               
               
                 ACACACACAGGCCTGGAGGCGGGGGCTCAGAGAGGGCAGGGCCOCCACTG 
               
               
                   
               
               
                 TAGTCACACAGCACCAGAGTCTCCGCTTTAACCCCCACCTCCAGCCGCAA 
               
               
                   
               
               
                 TGCCACCGGGGCGCCGCGGTGCCCCTGCCTGTCATCCTGCTTTGGACCCT 
               
               
                   
               
               
                 CTGCCTCGCCCTTCCTCCCTTCCTTCCACCCAGCCCGGCCTTTGTCAGCC 
               
               
                   
               
               
                 APOE4 sense 
               
               
                 (SEQ ID NO: 743) 
               
               
                 AGCACCGAGGAGCTGCGGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCG 
               
               
                   
               
               
                 TAAGCGGCTCCTCCGCGATGCCGATGACCTGCAGAAGTGCCTGGCAGTGT 
               
               
                   
               
               
                 ACCAGGCCGGGGCCCGCGAGGGCGCCGAGCGCGGCCTCAGCGCCATCCGC 
               
               
                   
               
               
                 GAGCGCCTGGGGCCCCTGGTGGAACAGGGCCGCGTGCGGGCCGCCACTGT 
               
               
                   
               
               
                 APOE4 antisense 
               
               
                 (SEQ ID NO: 744) 
               
               
                 ACAGTGGCGGCCCGCACGCGGCCCTGTTCCACCAGGGGCCCCAGGCGCTC 
               
               
                   
               
               
                 GCGGATGGCGCTGAGGCCGCGCTCGGCGCCCTCGCGGGCCCCGGCCTGGT 
               
               
                   
               
               
                 ACACTGCCAGGCACTTCTGCAGGTCATCGGCATCGCGGAGGAGCCGCTTA 
               
               
                   
               
               
                 CGCAGCTTGCGCAGGTGGGAGGCGAGGCGCACCCGCAGCTCCTCGGTGCT 
               
               
                   
               
               
                 p53 Y163C sense 
               
               
                 (SEQ ID NO: 745) 
               
               
                 ACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGGCCAAGACCTGCCCT 
               
               
                   
               
               
                 GTGCAGCTGTGGGTTGATTCCACACCCCCGCCCGGCACCCGCGTCCGCGC 
               
               
                   
               
               
                 CATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGTTGTGAGGCGCT 
               
               
                   
               
               
                 GCCCCCACCATGAGCGCTGCTCAGATAGCGATGGTGAGCAGCTGGGGCTG 
               
               
                   
               
               
                 p53 Y163C antisense 
               
               
                 (SEQ ID NO: 746) 
               
               
                 CAGCCCCAGCTGCTCACCATCGCTATCTGAGCAGCGCTCATGGTGGGGGC 
               
               
                   
               
               
                 AGCGCCTCACAACCTCCGTCATGTGCTGTGACTGCTTGTAGATGGCCATG 
               
               
                   
               
               
                 GCGCGGACGCGGGTGCCGGGCGGGGGTGTGGAATCAACCCACAGCTGCAC 
               
               
                   
               
               
                 AGGGCAGGTCTTGGCCAGTTGGCAAAACATCTTGTTGAGGGCAGGGGAGT 
               
            
           
         
       
     
     Deaminase Gene gBlocks Gene Fragments 
     
       
         
           
               
            
               
                 hAID 
               
               
                 (SEQ ID NO: 586) 
               
               
                 CATCCTTGGTACCGAGCTCGGATCCAGCCACCATGGATAGCCTCTTGATG 
               
               
                   
               
               
                 AATAGACGCAAGTTCCTGTATCAGTTTAAAAACGTGAGATGGGCAAAAGG 
               
               
                   
               
               
                 CCGACGAGAGACATATCTGTGCTATGTCGTTAAGCGCAGAGATTCAGCCA 
               
               
                   
               
               
                 CCAGTTTCTCTCTCGACTTCGGCTACCTGOGGAACAAGAATGGTTGCCAT 
               
               
                   
               
               
                 GTTGAGCTCCTGTTCCTGAGGTATATCAGCGACTGGGATTTGGACCCAGG 
               
               
                   
               
               
                 GCGGTGCTATAGGGTGACATGGTTTACCTCCTGGTCACCTTGTTATGACT 
               
               
                   
               
               
                 GCGCGCGGCATGTTGCCGATTTTCTGAGAGGGAACCCTAACCTGTCTCTG 
               
               
                   
               
               
                 AGGATCTTCACCGCGCGACTGTACTTCTGTGAGGACCGGAAAGCCGAACC 
               
               
                   
               
               
                 CGAGGGACTGAGACGCCTCCACAGAGCGGGTGTGCAGATTGCCATAATGA 
               
               
                   
               
               
                 CCTTTAAGGACTACTTCTACTGCTGGAACACCTTCGTCGAAAATCACGAG 
               
               
                   
               
               
                 CGGACTTTCAAGGCTTGGGAAGGATTGCATGAAAACAGCGTCAGGCTTTC 
               
               
                   
               
               
                 CAGGCAGCTTCGCCGCATTCTTCTCCCGTTGTACGAGGTTGATGACCTCA 
               
               
                   
               
               
                 GAGATGCCTTTAGAACACTGGGACTGTAGGCGGCCGCTCGATTGGTTTGG 
               
               
                   
               
               
                 TGTGGCTCTAA 
               
               
                   
               
               
                 rAPOBEC1 (mammalian) 
               
               
                 (SEQ ID NO: 587) 
               
               
                 CATCCTTGGTACCGAGCTCGGATCCAGCCACCATGAGCTCAGAGACTGGC 
               
               
                   
               
               
                 CCAGTGGCTGTGGACCCCACATTGAGACGGCGGATCGAGCCCCATGAGTT 
               
               
                   
               
               
                 TGAGGTATTCTTCGATCCGAGAGAGCTCCGCAAGGAGACCTGCCTGCTTT 
               
               
                   
               
               
                 ACGAAATTAATTGGGGGGGCCGGCACTCCATTTGGCGACATACATCACAG 
               
               
                   
               
               
                 AACACTAACAAGCACGTCGAAGTCAACTTCATCGAGAAGTTCACGACAGA 
               
               
                   
               
               
                 AAGATATTTCTGTCCGAACACAAGGTGCAGCATTACCTGGTTTCTCAGCT 
               
               
                   
               
               
                 GGAGCCCATGCGGCGAATGTAGTAGGGCCATCACTGAATTCCTGTCAAGG 
               
               
                   
               
               
                 TATCCCCACGTCACTCTGTTTATTTACATCGCAAGGCTGTACCACCACGC 
               
               
                   
               
               
                 TGACCCCCGCAATCGACAAGGCCTGCGGGATTTGATCTCTTCAGGTGTGA 
               
               
                   
               
               
                 CTATCCAAATTATGACTGAGCAGGAGTCAGGATACTGCTGGAGAAACTTT 
               
               
                   
               
               
                 GTGAATTATAGCCCGAGTAATGAAGCCCACTGGCCTAGGTATCCCCATCT 
               
               
                   
               
               
                 GTGGGTACGACTGTACGTTCTTGAACTGTACTGCATCATACTGGGCCTGC 
               
               
                   
               
               
                 CTCCTTGTCTCAACATTCTGAGAAGGAAGCAGCCACAGCTGACATTCTTT 
               
               
                   
               
               
                 ACCATCGCTCTTCAGTCTTGTCATTACCAGCGACTGCCCCCACACATTCT 
               
               
                   
               
               
                 CTGGGCCACCGGGTTGAAATGAGCGGCCGCTCGATTGGTTTGGTGTGGCT 
               
               
                   
               
               
                 CTAA 
               
               
                   
               
               
                 pmCDA1 
               
               
                 (SEQ ID NO: 588) 
               
               
                 CATCCTTGGTACCGAGCTCGGATCCAGCCACCATGACAGACGCTGAATAT 
               
               
                   
               
               
                 GTTAGGATCCATGAAAAACTGGATATCTATACATTTAAGAAGCAGTTCTT 
               
               
                   
               
               
                 CAATAACAAAAAGTCAGTATCTCACAGATGCTATGTCCTGTTCGAACTCA 
               
               
                   
               
               
                 AGAGAAGAGGAGAAAGGCGGGCCTGTTTCTGGGGGTACGCGGTTAATAAA 
               
               
                   
               
               
                 CCCCAGTCCGGGACCGAGAGGGGGATTCACGCCGAGATCTTTTCAATTAG 
               
               
                   
               
               
                 GAAGGTTGAAGAGTATCTTCGCGACAATCCCGGTCAGTTCACAATTAACT 
               
               
                   
               
               
                 GGTACAGCTCCTGGAGCCCTTGCGCTGATTGCGCCGAGAAAATACTCGAA 
               
               
                   
               
               
                 TGGTACAACCAGGAGTTGAGAGGCAATGGCCACACTCTCAAGATTTGGGC 
               
               
                   
               
               
                 TTGCAAGCTTTACTACGAGAAGAACGCGAGAAATCAGATTGGCTTGTGGA 
               
               
                   
               
               
                 ACCTCAGGGACAACGGGGTCGGGTTGAATGTTATGGTGTCCGAACATTAC 
               
               
                   
               
               
                 CAGTGCTGTAGAAAGATCTTCATTCAGTCCAGTCACAATCAGCTGAACGA 
               
               
                   
               
               
                 GAACAGATGGCTGGAGAAAACACTGAAACGGGCAGAGAAAAGGCGCTCAG 
               
               
                   
               
               
                 AGCTGAGTATCATGATCCAGGTCAAAATCTCTGCATACAACCAAAAGCCC 
               
               
                   
               
               
                 GGCTGTATAAGCGGCCGCTCGATTGGTTTGGTGTGGCTCTAA 
               
               
                   
               
               
                 haPOBEC3G 
               
               
                 (SEQ ID NO: 589) 
               
               
                 CATCCTTGGTACCGAGCTCGGATCCAGCCACCATGGAGCTGAAGTATCAC 
               
               
                   
               
               
                 CCTGAGATGCGGTTTTTCCACTGGTTTAGTAAGTGGCGCAAACTTCATCG 
               
               
                   
               
               
                 GGATCAGGAGTATGAAGTGACCTGGTATATCTCTTGGTCTCCCTGCACAA 
               
               
                   
               
               
                 AATGTACACGCGACATGGCCACATTTCTGGCCGAGGATCCAAAGGTGACG 
               
               
                   
               
               
                 CTCACAATCTTTGTGGCCCGCCTGTATTATTTCTGGGACCCGGATTATCA 
               
               
                   
               
               
                 GGAGGCACTTAGGTCATTGTGCCAAAAGCGCGACGGACCACGGGCGACTA 
               
               
                   
               
               
                 TGAAAATCATGAATTATGACGAATTCCAGCATTGCTGGAGTAAGTTTGTG 
               
               
                   
               
               
                 TACAGCCAGCGGGAGCTGTTCGAGCCCTGGAACAATCTTCCCAAGTACTA 
               
               
                   
               
               
                 CATACTGCTTCACATTATGTTGGGGGAGATCCTTCGGCACTCTATGGATC 
               
               
                   
               
               
                 CTCCTACCTTTACGTTTAACTTTAATAATGAGCCTTGGGTTCGCGGGCGC 
               
               
                   
               
               
                 CATGAAACCTATTTGTGCTACGAGGTCGAGCGGATGCATAATGATACGTG 
               
               
                   
               
               
                 GGTCCTGCTGAATCAGAGGAGGGGGTTTCTGTGTAACCAGGCTCCACATA 
               
               
                   
               
               
                 AACATGGATTTCTCGAGGGGCGGCACGCCGAACTGTGTTTCCTTGATGTG 
               
               
                   
               
               
                 ATACCTTTCTGGAAGCTCGACCTTGATCAAGATTACAGGGTGACGTGTTT 
               
               
                   
               
               
                 CACCTCCTGGTCACCCTGCTTCAGTTGCGCCCAAGAGATGGCTAATTTAT 
               
               
                   
               
               
                 CAGTAAGAACAAGCATGTGTCCCTCTGTATTTTTACAGCCAGAATTTATG 
               
               
                   
               
               
                 ATGACCAGGGCCGGTGCCAGGAGGGGCTGCGGACACTCGCTGAGGCGGGC 
               
               
                   
               
               
                 GCGAAGATCAGCATAATGACATACTCCGAATTCAAACACTGTTGGGACAC 
               
               
                   
               
               
                 TTTTGTGGACCACCAGGGCTGCCCATTTCAGCCGTGGGATGGGCTCGACG 
               
               
                   
               
               
                 AACATAGTCAGGATCTCTCAGGCCGGCTGOGAGCCATATTGCAGAACCAG 
               
               
                   
               
               
                 GAGAATTAGGCGGCCGCTCGATTGGTTTGGTGTGGCTCTAA 
               
               
                   
               
               
                 rAPOBEC1 ( E. Coli ) 
               
               
                 (SEQ ID NO: 590) 
               
               
                 GGCCGGGGATTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATAC 
               
               
                   
               
               
                 CATGGATGTCTTCTGAAACCGGTCCGGTTGCGGTTGACCCGACCCTGCGT 
               
               
                   
               
               
                 CGTCGTATCGAACCGCACGAATTCGAAGTTTTCTTCGACCCGCGTGAACT 
               
               
                   
               
               
                 GCGTAAAGAAACCTGCCTGCTGTACGAAATCAACTGGGGTGGTCGTCACT 
               
               
                   
               
               
                 CTATCTGGCGTCACACCTCTCAGAACACCAACAAACACGTTGAAGTTAAC 
               
               
                   
               
               
                 TTCATCGAAAAATTCACCACCGAACGTTACTTCTGCCCGAACACCCGTTG 
               
               
                   
               
               
                 CTCTATCACCTGGTTCCTGTCTTGGTCTCCGTGCGGTGAATGCTCTCGTG 
               
               
                   
               
               
                 CGATCACCGAATTCCTGTCTCGTTACCCGCACGTTACCCTGTTCATCTAC 
               
               
                   
               
               
                 ATCGCGCGTCTGTACCACCACGCGGACCCGCGTAACCGTCAGGGTCTGCG 
               
               
                   
               
               
                 TGACCTGATCTCTTCTGGTGTTACCATCCAGATCATGACCGAACAGGAAT 
               
               
                   
               
               
                 CTGGTTACTGCTGGCGTAACTTCGTTAACTACTCTCCGTCTAACGAAGCT 
               
               
                   
               
               
                 GCACTGGCCGCGTTACTCCGCACCTGTGGGTTCGTCTGTACGTTCTGGAA 
               
               
                   
               
               
                 CTGTACTGCATCATCCTGGGTCTGCCGCCGTGCCTGAACATCCTGCGTCG 
               
               
                   
               
               
                 TAAACAGCCGCAGCTGACCTTCTTCACCATCGCGCTGCAGTCTTGCCACT 
               
               
                   
               
               
                 ACCAGCGTCTGCCGCCGCACATCCTGTGGGCGACCGGTCTGAAAGGTGGT 
               
               
                   
               
               
                 AGTGGAGGGAGCGGCGGTTCAATGGATAAGAAATAC 
               
            
           
         
       
     
     Amino Acid Sequences of NBE1, NBE2, and NBE3. 
     
       
         
           
               
               
            
               
                 NBE1 for  E. Coli  expression (His 6 -rAPOBEC1-XTEN-dCas9) 
                   
               
               
                 (SEQ ID NO: 591) 
                   
               
               
                 MGSSHHHHHHMSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTN 
                   
               
               
                   
               
               
                 KHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGL 
               
               
                   
               
               
                 RDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQ 
               
               
                   
               
               
                 LTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSK 
               
               
                   
               
               
                 KFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL 
               
               
                   
               
               
                 EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL 
               
               
                   
               
               
                 NPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL 
               
               
                   
               
               
                 GLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSA 
               
               
                   
               
               
                 SMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELL 
               
               
                   
               
               
                 VKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF 
               
               
                   
               
               
                 AWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTE 
               
               
                   
               
               
                 GMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK 
               
               
                   
               
               
                 DFLDNEENEDILEDIVLTLTLFEDREMEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQS 
               
               
                   
               
               
                 GKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDEL 
               
               
                   
               
               
                 VKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ 
               
               
                   
               
               
                 NGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQL 
               
               
                   
               
               
                 LNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK 
               
               
                   
               
               
                 SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG 
               
               
                   
               
               
                 KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQT 
               
               
                   
               
               
                 GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSS 
               
               
                   
               
               
                 FEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKL 
               
               
                   
               
               
                 KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTINL 
               
               
                   
               
               
                 GAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSPKKKRKV 
               
               
                   
               
               
                 NBE1 for Mammalian expression (rAPOBEC1-XTEN-dCas9-NLS) 
               
               
                 (SEQ ID NO: 592) 
                   
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKF 
                   
               
               
                   
               
               
                 TTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQ 
               
               
                   
               
               
                 IMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSC 
               
               
                   
               
               
                 HYQRLPPHILWATGLKSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR 
               
               
                   
               
               
                 HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLIQEIFSNEMAKVDDSFFHRLEESFLVEEDKK 
               
               
                   
               
               
                 HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLF 
               
               
                   
               
               
                 IQLVQTYNQLFEENPINASGVDAKAISARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL 
               
               
                   
               
               
                 AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQ 
               
               
                   
               
               
                 DLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ 
               
               
                   
               
               
                 RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITP 
               
               
                   
               
               
                 WNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNNELTKVKYVTEGMRKPAFLSGEQ 
               
               
                   
               
               
                 KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKHKDKDFLDNEENEDILE 
               
               
                   
               
               
                 DIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFA 
               
               
                   
               
               
                 NRNFIAGLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAKKGILQPVKVVDELVKVMGRHKPENIVI 
               
               
                   
               
               
                 EMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDI 
               
               
                   
               
               
                 NRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF 
               
               
                   
               
               
                 YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRMIAKSEQEIGKATAKYFFYSNIM 
               
               
                   
               
               
                 NFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQNIVKKTEVQTGGFSKESILPKRN 
               
               
                   
               
               
                 SDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG 
               
               
                   
               
               
                 YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL 
               
               
                   
               
               
                 FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTI 
               
               
                   
               
               
                 DRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSPKKKRKV 
               
            
           
         
       
     
     
       
         
           
               
               
            
               
                 Alternative NBE1 for Mammalian expression with human APOBEC1 (hAPOBEC1- 
                   
               
               
                 XTEN-dCas9-NLS) 
               
               
                 (SEQ ID NO: 5737) 
                   
               
               
                 MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTN 
                   
               
               
                   
               
               
                 HVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFW 
               
               
                   
               
               
                 HMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLY 
               
               
                   
               
               
                 ALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWRGSETP 
               
               
                   
               
               
                 GTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL 
               
               
                   
               
               
                 FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK 
               
               
                   
               
               
                 HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGD 
               
               
                   
               
               
                 LNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKK 
               
               
                   
               
               
                 NGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAA 
               
               
                   
               
               
                 KNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQ 
               
               
                   
               
               
                 SKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIH 
               
               
                   
               
               
                 LGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW 
               
               
                   
               
               
                 NFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMR 
               
               
                   
               
               
                 KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDRFNASLGTYH 
               
               
                   
               
               
                 DLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY 
               
               
                   
               
               
                 TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQ 
               
               
                   
               
               
                 GDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQK 
               
               
                   
               
               
                 NSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD 
               
               
                   
               
               
                 YDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ 
               
               
                   
               
               
                 RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK 
               
               
                   
               
               
                 VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY 
               
               
                   
               
               
                 KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVW 
               
               
                   
               
               
                 DKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGG 
               
               
                   
               
               
                 FDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKD 
               
               
                   
               
               
                 LIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNE 
               
               
                   
               
               
                 QKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTL 
               
               
                   
               
               
                 TNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSPKKK 
               
               
                   
               
               
                 RKV 
               
               
                   
               
               
                 NBE2 (rAPOBEC1-XTEN-dCas9-UGI-NLS) 
               
               
                 (SEQ ID NO: 593) 
                   
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDFRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKF 
                   
               
               
                   
               
               
                 TTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQ 
               
               
                   
               
               
                 IMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSC 
               
               
                   
               
               
                 HYQRLPPHILWATGLKSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR 
               
               
                   
               
               
                 HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK 
               
               
                   
               
               
                 HERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLF 
               
               
                   
               
               
                 IQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL 
               
               
                   
               
               
                 AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQ 
               
               
                   
               
               
                 DLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ 
               
               
                   
               
               
                 RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITP 
               
               
                   
               
               
                 WNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ 
               
               
                   
               
               
                 KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILE 
               
               
                   
               
               
                 DIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFA 
               
               
                   
               
               
                 NRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVI 
               
               
                   
               
               
                 EMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDI 
               
               
                   
               
               
                 NRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF 
               
               
                   
               
               
                 YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIM 
               
               
                   
               
               
                 NFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRN 
               
               
                   
               
               
                 SDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG 
               
               
                   
               
               
                 YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL 
               
               
                   
               
               
                 FVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTI 
               
               
                   
               
               
                 DRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIG 
               
               
                   
               
               
                 NKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRKV 
               
               
                   
               
               
                 NBE3 (rAPOBEC1-XTEN-Cas9n-UGI-NLS) 
               
               
                 (SEQ ID NO: 594) 
                   
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKF 
                   
               
               
                   
               
               
                 TTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQ 
               
               
                   
               
               
                 IMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSC 
               
               
                   
               
               
                 HYQRLPPHILWATGLKSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR 
               
               
                   
               
               
                 HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKK 
               
               
                   
               
               
                 HERHPIFGNIVDEVAYHEKYFTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLF 
               
               
                   
               
               
                 IQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL 
               
               
                   
               
               
                 AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQ 
               
               
                   
               
               
                 DLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ 
               
               
                   
               
               
                 RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPVYVGPLARGNSRFAWMTRKSEETITP 
               
               
                   
               
               
                 WNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ 
               
               
                   
               
               
                 KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILE 
               
               
                   
               
               
                 DIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFA 
               
               
                   
               
               
                 NRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVI 
               
               
                   
               
               
                 EMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDI 
               
               
                   
               
               
                 NRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 
               
               
                   
               
               
                 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF 
               
               
                   
               
               
                 YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIM 
               
               
                   
               
               
                 NFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRN 
               
               
                   
               
               
                 SDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG 
               
               
                   
               
               
                 YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQL 
               
               
                   
               
               
                 FVEQHKHYLDENIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTI 
               
               
                   
               
               
                 DRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIG 
               
               
                   
               
               
                 NKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVQDSNGENKIKMLSGGSPKKKRKV 
               
               
                   
               
               
                 pmCDA1-XTEN-dCas9-UGI (bacteria) 
               
               
                 (SEQ ID NO: 5742) 
                   
               
               
                 MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQS 
                   
               
               
                   
               
               
                 GTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTL 
               
               
                   
               
               
                 KIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWL 
               
               
                   
               
               
                 EKTLKRAEKRRSELSIMIQVKILHTTKSPAVSGSETPGTSESATPESDKKYSIGLAIGTNSV 
               
               
                   
               
               
                 GWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRK 
               
               
                   
               
               
                 NRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYH 
               
               
                   
               
               
                 LRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE 
               
               
                   
               
               
                 ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLA 
               
               
                   
               
               
                 EDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS 
               
               
                   
               
               
                 MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILE 
               
               
                   
               
               
                 KMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLP 
               
               
                   
               
               
                 NEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK 
               
               
                   
               
               
                 QLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT 
               
               
                   
               
               
                 LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLK 
               
               
                   
               
               
                 SDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVV 
               
               
                   
               
               
                 DELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT 
               
               
                   
               
               
                 QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKN 
               
               
                   
               
               
                 RGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL 
               
               
                   
               
               
                 VETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY 
               
               
                   
               
               
                 HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS 
               
               
                   
               
               
                 NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ 
               
               
                   
               
               
                 TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV 
               
               
                   
               
               
                 KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQ 
               
               
                   
               
               
                 KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
               
               
                   
               
               
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEV 
               
               
                   
               
               
                 LDATLIHQSITGLYETRIDLSQLGGDSGGSMTNLSDIIEKETGKQLVIQESILMLPEEVEEVI 
               
               
                   
               
               
                 GNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML 
               
               
                   
               
               
                 pmCDA1-XTEN-nCas9-UGI-NLS (mammalian construct) 
               
               
                 (SEQ ID NO: 5743) 
                   
               
               
                 MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQS 
                   
               
               
                   
               
               
                 GTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTL 
               
               
                   
               
               
                 KIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWL 
               
               
                   
               
               
                 EKTLKRAEKRRSELSIMIQVKILHTTKSPAVSGSETPGTSESATPESDKKYSIGLAIGTNSV 
               
               
                   
               
               
                 GWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRK 
               
               
                   
               
               
                 NRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYH 
               
               
                   
               
               
                 LRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE 
               
               
                   
               
               
                 ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLA 
               
               
                   
               
               
                 EDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS 
               
               
                   
               
               
                 MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILE 
               
               
                   
               
               
                 KMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK 
               
               
                   
               
               
                 ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLP 
               
               
                   
               
               
                 NEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK 
               
               
                   
               
               
                 QLKEDYFKKIECFDSVETSGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT 
               
               
                   
               
               
                 LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLK 
               
               
                   
               
               
                 SDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVV 
               
               
                   
               
               
                 DELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENT 
               
               
                   
               
               
                 QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKN 
               
               
                   
               
               
                 RGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL 
               
               
                   
               
               
                 VETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY 
               
               
                   
               
               
                 HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS 
               
               
                   
               
               
                 NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ 
               
               
                   
               
               
                 TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV 
               
               
                   
               
               
                 KELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQ 
               
               
                   
               
               
                 KGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVIL 
               
               
                   
               
               
                 ADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEV 
               
               
                   
               
               
                 LDATLIHQSITGLYETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIG 
               
               
                   
               
               
                 NKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKR 
               
               
                   
               
               
                 KV 
               
               
                   
               
               
                 huAPOBEC3G-XTEN-dCas9-UGI (bacteria) 
               
               
                 (SEQ ID NO: 5744) 
                   
               
               
                 MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLE 
                   
               
               
                   
               
               
                 GRHAELCFLDVIPFWKLDLDDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIY 
               
               
                   
               
               
                 DDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGR 
               
               
                   
               
               
                 LRAILQSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR 
               
               
                   
               
               
                 HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL 
               
               
                   
               
               
                 EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
               
               
                   
               
               
                 KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE 
               
               
                   
               
               
                 NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIG 
               
               
                   
               
               
                 DQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL 
               
               
                   
               
               
                 PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT 
               
               
                   
               
               
                 FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMT 
               
               
                   
               
               
                 RKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTK 
               
               
                   
               
               
                 VKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDR 
               
               
                   
               
               
                 FNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV 
               
               
                   
               
               
                 MKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKED 
               
               
                   
               
               
                 IQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN 
               
               
                   
               
               
                 QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ 
               
               
                   
               
               
                 LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDE 
               
               
                   
               
               
                 NDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLE 
               
               
                   
               
               
                 SEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN 
               
               
                   
               
               
                 GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD 
               
               
                   
               
               
                 WDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK 
               
               
                   
               
               
                 GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKL 
               
               
                   
               
               
                 KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA 
               
               
                   
               
               
                 ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 SGGSMTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLL 
               
               
                   
               
               
                 TSDAPEYKPWALVIQDSNGENKIKML 
               
               
                   
               
               
                 huAPOBEC3G-XTEN-nCas9-UGI-NLS (mammalian construct) 
               
               
                 (SEQ ID NO: 5745) 
                   
               
               
                 MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLE 
                   
               
               
                   
               
               
                 GRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIY 
               
               
                   
               
               
                 DDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGR 
               
               
                   
               
               
                 LRAILQSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR 
               
               
                   
               
               
                 HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL 
               
               
                   
               
               
                 EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
               
               
                   
               
               
                 KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE 
               
               
                   
               
               
                 NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIG 
               
               
                   
               
               
                 DQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL 
               
               
                   
               
               
                 PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT 
               
               
                   
               
               
                 FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMT 
               
               
                   
               
               
                 RKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTK 
               
               
                   
               
               
                 VKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDR 
               
               
                   
               
               
                 FNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV 
               
               
                   
               
               
                 MKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKED 
               
               
                   
               
               
                 IQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN 
               
               
                   
               
               
                 QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ 
               
               
                   
               
               
                 LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDE 
               
               
                   
               
               
                 NDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLE 
               
               
                   
               
               
                 SEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN 
               
               
                   
               
               
                 GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD 
               
               
                   
               
               
                 WDPKKYGGFDSPTVAYS VLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK 
               
               
                   
               
               
                 GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKL 
               
               
                   
               
               
                 KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA 
               
               
                   
               
               
                 ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 SGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLT 
               
               
                   
               
               
                 SDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRKV 
               
               
                   
               
               
                 huAPOBEC3G (D316R_D317R)-XTEN-nCas9-UGI-NLS (mammalian construct) 
               
               
                 (SEQ ID NO: 5746) 
                   
               
               
                 MDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLE 
                   
               
               
                   
               
               
                 GRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIY 
               
               
                   
               
               
                 RRQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGR 
               
               
                   
               
               
                 LRAILQSGSETPGTSESATPESDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDR 
               
               
                   
               
               
                 HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDS FFHRL 
               
               
                   
               
               
                 EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI 
               
               
                   
               
               
                 KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE 
               
               
                   
               
               
                 NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIG 
               
               
                   
               
               
                 DQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL 
               
               
                   
               
               
                 PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT 
               
               
                   
               
               
                 FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMT 
               
               
                   
               
               
                 RKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTK 
               
               
                   
               
               
                 VKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVETSGVEDR 
               
               
                   
               
               
                 FNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV 
               
               
                   
               
               
                 MKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKED 
               
               
                   
               
               
                 IQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN 
               
               
                   
               
               
                 QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ 
               
               
                   
               
               
                 ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ 
               
               
                   
               
               
                 LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDE 
               
               
                   
               
               
                 NDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLE 
               
               
                   
               
               
                 SEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN 
               
               
                   
               
               
                 GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKD 
               
               
                   
               
               
                 WDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK 
               
               
                   
               
               
                 GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKL 
               
               
                   
               
               
                 KGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA 
               
               
                   
               
               
                 ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 
               
               
                   
               
               
                 SGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLT 
               
               
                   
               
               
                 SDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRKV 
               
            
           
         
       
     
     Base Calling Matlab Script 
     
       
         
           
               
            
               
                 (SEQ ID NO: 595) 
               
               
                 WTnuc = GCGGACATGGAGGACGTGCGCGGCCGCCTGGTGCAGTACCGC 
               
               
                   
               
               
                 GGCGAGGTGCAGGCCATGCTCGGCCAGA 
               
               
                   
               
               
                 GCACCGAGGAGCTGCGGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCGT 
               
               
                   
               
               
                 AAGCGGCTCCTCCGCGATGCCGATGAC 
               
               
                   
               
               
                 CTGCAGAAGCGCCTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGA 
               
               
                   
               
               
                 GCGCGGCCTCAGCGCCATCCGCGAGCG CCTGGGGCCCCTGGTGGAACA 
               
               
                   
               
               
                 G′; 
               
            
           
         
       
     
     
       
         
           
               
               
             
               
                   
               
             
            
               
                 %cycle through fastq files for different samples files=dir(‘*.fastq’); 
                   
               
               
                 for d=1:20 
                   
               
               
                 filename=files(d).name; 
                   
               
               
                 %read fastq file 
                   
               
               
                 [header,seqs,qscore]=fastqread(filename); 
                   
               
               
                 seqsLength=length(seqs); 
                    % number of sequences seqsFile= 
               
               
                 strrep(filename,‘.fastq’,‘’); 
                    % trims off .fastq 
               
            
           
           
               
            
               
                 %create a directory with the same name as fastq file ifexist(seqsFile,‘dir’); 
               
               
                  error(‘Directory already exists. Please rename or move it before moving on.’); 
               
            
           
           
               
               
            
               
                 end 
                   
               
               
                 mkdir(seqsFile); 
                    % make directory 
               
               
                 wtLength=length(WTnuc); 
                     % length of wildtype sequence 
               
               
                 %% aligning back to the wildtype nucleotide sequence 
                   
               
               
                 % 
                   
               
               
                 % MN is a matrix of the nucleotide alignment window=1:wtLength; 
                   
               
               
                 sBLength=length(seqs); 
                   % number of sequences 
               
               
                 % counts number of skips nSkips = 0; 
                   
               
               
                 ALN=repmat(‘’,[sBLengthwtLength]); 
                   
               
               
                 % iterate through each sequencing read for i = 1:sBLength 
                   
               
               
                 %If you only have forward read fastq files leave as is 
                   
               
            
           
           
               
            
               
                 %If you have R1 foward and R2 is reverse fastq files uncomment the 
               
            
           
           
               
               
            
               
                 %next four lines of code and the subsequent end statement 
                   
               
               
                 % 
                 ifmod(d,2)==0; 
               
               
                 % 
                  reverse=seqrcomplement(seqs{i}); 
               
               
                 % 
                  [score,alignment,start]= 
               
               
                 swalign(reverse,WTnuc,‘Alphabet’,‘NT’); 
                   
               
               
                 % 
                 else 
               
               
                 [score,alignment,start]=swalign(seqs{i},WTnuc,‘Alphabet’,‘NT’); 
                   
               
               
                 % 
                 end 
               
               
                 % length of the sequencing read len= 
                   
               
               
                 length(alignment(3,:)); 
                   
               
               
                 % if there is a gap in the alignment, skip = 1 and we will 
                   
               
               
                  % throw away the entire read skip = 0; 
                   
               
               
                  for j = 1:len 
                   
               
               
                 if (alignment(3,j) == ‘−’|| alignment(1,j) == ‘−’) skip = 1; 
                   
               
               
                      break; 
                   
               
               
                  end 
                   
               
               
                  %in addition if the qscore for any given base in the read is 
                   
               
            
           
           
               
            
               
                     %below 31 the nucleotide is turned into an N (fastq qscores that are not letters) 
               
            
           
           
               
               
            
               
                  ifisletter(qscore{i}(start(1)+j−1)) else 
                   
               
               
                  alignment(1,j) = ‘N’; 
                   
               
               
                   end 
                   
               
               
                  end 
                   
               
               
                  if skip == 0 &amp;&amp; len&gt;10 
                   
               
               
                  ALN(i, start(2):(start(2)+length(alignment)−1))=alignment(1,:); 
                   
               
               
                    end 
                   
               
               
                  end 
                   
               
            
           
           
               
            
               
                 % with the alignment matrices we can simply tally up the occurrences of 
               
            
           
           
               
               
            
               
                 % each nucleotide at each column in the alignment these 
                   
               
               
                 % tallies ignore bases annotated as N 
                   
               
               
                 % due to low qscores 
                   
               
               
                 TallyNTD=zeros(5,wtLength); for i=1:wtLength 
                   
               
            
           
           
               
            
               
                 TallyNTD(:,i)=[sum(ALN(:,i)==‘A’),sum(ALN(:,i)==‘C’),sum(ALN(:,i)==‘G’), 
               
            
           
           
               
               
            
               
                 sum(ALN(:,i)==‘T’),sum(ALN(:,i)==‘N’)]; 
                   
               
               
                 end 
                   
               
               
                 % we then save these tally matrices in the respective folder for 
                   
               
               
                 % further processing 
                   
               
            
           
           
               
            
               
                 save(strcat(seqsFile,‘/TallyNTD’),‘TallyNTD’); dlmwrite(strcat(seqsFile,‘/TallyNTD.txt’),TallyNTD,‘precision’, 
               
            
           
           
               
               
            
               
                 ‘%.3f’,‘newline’,‘pc’); end 
                   
               
               
                   
               
            
           
         
       
     
     INDEL Detection Matlab Script 
     
       
         
           
               
            
               
                 (SEQ ID NO: 595) 
               
               
                 WTnuc = ′GCGGACATGGAGGACGTGCGCGGCCGCCTGGTGCAGTACCG 
               
               
                   
               
               
                 CGGCGAGGTGCAGGCCATGCTCGGCCAGA 
               
               
                   
               
               
                 GCACCGAGGAGCTGCGGGTGCGCCTCGCCTCCCACCTGCGCAAGCTGCGT 
               
               
                   
               
               
                 AAGCGGCTCCTCCGCGATGCCGATGAC 
               
               
                   
               
               
                 CTGCAGAAGCGCCTGGCAGTGTACCAGGCCGGGGCCCGCGAGGGCGCCGA 
               
               
                   
               
               
                 GCGCGGCCTCAGCGCCATCCGCGAGCG CCTGGGGCCCCTGGTGGAACA 
               
               
                   
               
               
                 G′; 
               
            
           
         
       
     
     
       
         
           
               
             
               
                   
               
             
            
               
                 %cycle through fastq files for different samples files=dir(‘*.fastq’); 
               
               
                 %specify start and width of indel window as well as length of each flank indelstart=154; 
               
            
           
           
               
               
            
               
                 width=30; flank=10; 
                   
               
               
                 for d=1:3 
                   
               
               
                 filename=files(d).name; 
                   
               
               
                 %read fastq file 
                   
               
               
                 [header,seqs,qscore]=fastqread(filename); 
                   
               
               
                 seqsLength=length(seqs); 
                 % number of sequences seqsFile 
               
               
                 =strcat(strrep(filename,‘.fastq’,‘’),‘_INDELS’); 
                   
               
            
           
           
               
            
               
                 %create a directory with the same name as fastq file+_INDELS ifexist(seqsFile,‘dir’); 
               
               
                    error(‘Directory already exists. Please rename or move it before moving on.’); 
               
            
           
           
               
               
            
               
                 end 
                   
               
               
                 mkdir(seqsFile); 
                 % make directory 
               
               
                 wtLength=length(WTnuc); 
                 % length of wildtype sequence sBLength = 
               
               
                 length(seqs); 
                 % number of sequences 
               
               
                 % initialize counters and cell arrays 
                   
               
               
                 nSkips = 0; notINDEL=0; 
                   
               
               
                 ins={}; 
                   
               
               
                 dels={}; NumIns=0; 
                   
               
               
                 NumDels=0; 
                   
               
            
           
           
               
            
               
                 % iterate through each sequencing read for i = 1:sBLength 
               
               
                    %search for 10BP sequences that should flank both sides of the “INDEL WINDOW” 
               
               
                   windowstart=strfind(seqs{i},WTnuc(indelstart-flank:indelstart)); 
               
               
                     windowend=strfind(seqs{i},WTnuc(indelstart+width:indelstart+width+flank 
               
            
           
           
               
               
            
               
                 )); 
                   
               
               
                 %if the flanks are found proceed 
                   
               
               
                 iflength(windowstart)==1&amp;&amp;length(windowend)==1 
                   
               
               
                 %if the sequence length matches the INDEL window length save as 
                   
               
               
                  %not INDEL 
                   
               
               
                 if windowend-windowstart==width+flank notINDEL=notINDEL+1; 
                   
               
               
                  %if the sequence is two or more bases longer than the INDEL 
                   
               
               
                  %window length save as an Insertion 
                   
               
               
                 elseif windowend-windowstart&gt;=width+flank+2 NumIns=NumIns+1; 
                   
               
               
                  ins{NumDels}=seqs{i}; 
                   
               
               
                  %if the sequence is two or more bases shorter than the INDEL 
                   
               
               
                  %window length save as a Deletion 
                   
               
               
                 elseif windowend-windowstart&lt;=width+flank−2 NumDels=NumDels+1; 
                   
               
               
                  dels{NumDels}=seqs{i}; 
                   
               
               
                  %keep track of skipped sequences that are either one base 
                   
               
               
                  %shorter or longer than the INDEL window width else 
                   
               
               
                  nSkips=nSkips+1; 
                   
               
               
                  end 
                   
               
               
                  %keep track of skipped sequences that do not possess matching flank 
                   
               
               
                  %sequences else 
                   
               
               
                  nSkips=nSkips+1; 
                   
               
               
                   end 
                   
               
               
                  end 
                   
               
               
                  fid=fopen(strcat(seqsFile,‘/summary.txt’),‘wt’); 
                   
               
            
           
           
               
            
               
                  fprintf(fid, ‘Skipped reads %i\n not INDEL %i\n Insertions %i\n Deletions 
               
            
           
           
               
               
            
               
                 %i\n’, [nSkips, notINDEL, NumIns, NumDels]); fclose(fid); 
                   
               
            
           
           
               
            
               
                  save(strcat(seqsFile,‘/nSkips’),‘nSkips’); save(strcat(seqsFile,‘/notINDEL’),‘notINDEL’); 
               
               
                  save(strcat(seqsFile,‘/NumIns’),‘NumIns’); save(strcat(seqsFile,‘/NumDels’),‘NumDels’); 
               
            
           
           
               
               
            
               
                  save(strcat(seqsFile,‘/dels’),‘dels’); 
                   
               
               
                  C = dels; 
                   
               
               
                  fid=fopen(strcat(seqsFile,‘/dels.txt’),‘wt’); fprintf(fid,‘“%s”\n’,C{:}); 
                   
               
               
                  fclose(fid); 
                   
               
               
                  save(strcat(seqsFile,‘/ins’),‘ins’); C = ins; 
                   
               
               
                  fid=fopen(strcat(seqsFile,‘/ins.txt’), ‘wt’); fprintf(fid,‘“%s”\n’,C{:}); 
                   
               
               
                  fclose(fid); 
                   
               
               
                  end 
               
               
                   
               
            
           
         
       
     
     Example 5: Cas9 Variant Sequences 
     The disclosure provides Cas9 variants, for example Cas9 proteins from one or more organisms, which may comprise one or more mutations (e.g., to generate dCas9 or Cas9 nickase). In some embodiments, one or more of the amino acid residues, identified below by an asterisk, of a Cas9 protein may be mutated. In some embodiments, the D10 and/or H840 residues of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, are mutated. In some embodiments, the D10 residue of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, is mutated to any amino acid residue, except for D. In some embodiments, the D10 residue of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, is mutated to an A. In some embodiments, the H840 residue of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding residue in any of the amino acid sequences provided in SEQ ID NOs: 11-260, is an H. In some embodiments, the H840 residue of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, is mutated to any amino acid residue, except for H. In some embodiments, the H840 residue of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding mutation in any of the amino acid sequences provided in SEQ ID NOs: 11-260, is mutated to an A. In some embodiments, the D10 residue of the amino acid sequence provided in SEQ ID NO: 10, or a corresponding residue in any of the amino acid sequences provided in SEQ ID NOs: 11-260, is a D. 
     A number of Cas9 sequences from various species were aligned to determine whether corresponding homologous amino acid residues of D10 and H840 of SEQ ID NO: 10 or SEQ ID NO: 11 can be identified in other Cas9 proteins, allowing the generation of Cas9 variants with corresponding mutations of the homologous amino acid residues. The alignment was carried out using the NCBI Constraint-based Multiple Alignment Tool (COBALT(accessible at st-va.ncbi.nlm.nih.gov/tools/cobalt), with the following parameters. Alignment parameters: Gap penalties −11, −1; End-Gap penalties −5, −1. CDD Parameters: Use RPS BLAST on; Blast E-value 0.003; Find Conserved columns and Recompute on. Query Clustering Parameters: Use query clusters on; Word Size 4; Max cluster distance 0.8; Alphabet Regular. 
     An exemplary alignment of four Cas9 sequences is provided below. The Cas9 sequences in the alignment are: Sequence 1 (S1): SEQ ID NO: 11|WP_010922251|gi 499224711|type II CRISPR RNA-guided endonuclease Cas9 [ Streptococcus pyogenes ]; Sequence 2 (S2): SEQ ID NO: 12|WP_039695303|gi 746743737|type II CRISPR RNA-guided endonuclease Cas9 [ Streptococcus gallolyticus ]; Sequence 3 (S3): SEQ ID NO: 13|WP_045635197|gi 782887988|type II CRISPR RNA-guided endonuclease Cas9 [ Streptococcus mitis ]; Sequence 4 (S4): SEQ ID NO: 14|5AXW_A|gi 924443546| Staphylococcus aureus  Cas9. The HNH domain (bold and underlined) and the RuvC domain (boxed) are identified for each of the four sequences. Amino acid residues 10 and 840 in S1 and the homologous amino acids in the aligned sequences are identified with an asterisk following the respective amino acid residue. 
     
       
         
           
               
               
               
               
               
            
               
                 S1 
                 1 
                 --MDKK-   YSIGLD*IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI--GALLEDSG--ET   AEATRLKRTARRRYT 
                 73 
                   
               
               
                   
               
               
                 S2 
                 1 
                 --MTKKN   YSIGLD*IGTNSVGWAVITDDYKVPAKKMKVLGNTDKKYIKKNLL--GALLFDSG--ET   AEATRLKRTARRRYT 
                 74 
               
               
                   
               
               
                 S3 
                 1 
                 --M-KKG   YSIGLD*IGTNSVGFAVITDDYKVPSKKMKVLGNTDKRFIKKNLI--GALLFDEG--TT   AEARRLKRTARRRYT 
                 73 
               
               
                   
               
               
                 S4 
                 1 
                 GSHMKRN   YILGLD*IGITSVGYGII--DYET-----------------RDVIDAGVRLFKEANVEN   NEGRRSKRGARRLKR 
                 61 
               
               
                   
               
               
                 S1 
                 74 
                 RRKNRICYLQEIFSNEMAKVDDSFEHRLEESELVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL 
                 153 
               
               
                   
               
               
                 S2 
                 75 
                 RRKNRLRYLQEIFANETAKVDESFFQRLDESFLTDDDKTEDSHPIFGNKAEEDAYHQKFPTIYHLRKHLADSSEKADLRL 
                 154 
               
               
                   
               
               
                 S3 
                 74 
                 RRKNRLRYLQEIFSEEMSKVDSSFEHRLDDSFLIPEDKRESKYPIFATLTEEKEYHKQFPTIYHLRKQLADSKEKTDLRL 
                 153 
               
               
                   
               
               
                 S4 
                 62 
                 RRRHRIQRVKKLL--------------FDYNLLTD--------------------HSELSGINPYEARVKGLSQKLSEEE 
                 107 
               
               
                   
               
               
                 S1 
                 154 
                 IYLALAHNIKERGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEK 
                 233 
               
               
                   
               
               
                 S2 
                 155 
                 VYLALAHNIKERGHFLIEGELNAENTDVQKIFADFVGVYNRTFDDSHLSEITVDVASILTEKISKSRRLENLIKYYPTEK 
                 234 
               
               
                   
               
               
                 S3 
                 154 
                 TYLALAHNIKYRGHFLYEEAFDIKNNDIQKIFNEFISIYDNTFEGSSLSGQNAQVEAIFTDKISKSAKRERVLKLEPDEK 
                 233 
               
               
                   
               
               
                 S4 
                 108 
                 FSAALLHLAKRRG----------------------VHNVNEVEEDT---------------------------------- 
                 131 
               
               
                   
               
               
                 S1 
                 234 
                 KNGLFGNLIALSLGLTPNEKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEIT 
                 313 
               
               
                   
               
               
                 S2 
                 235 
                 KNTLFGNLIALALGLQPNEKTNFKLSEDAKLQFSKDTYEEDLEELLGKIGDDYADLFTSAKNLYDAILLSGILTVDDNST 
                 314 
               
               
                   
               
               
                 S3 
                 234 
                 STGLFSEFLKLIVGNQADFKKHFDLEDKAPLQFSKDTYDEDLENLLGQIGDDFTDLFVSAKKLYDAILLSGILTVTDPST 
                 313 
               
               
                   
               
               
                 S4 
                 132 
                 -----GNELS------------------TKEQISRN-------------------------------------------- 
                 144 
               
               
                   
               
               
                 S1 
                 314 
                 KAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKM--DGTEELLV 
                 391 
               
               
                   
               
               
                 S2 
                 315 
                 KAPLSASMIKRYVEHHEDLEKLKEFIKANKSELYHDIFKDKNKNGYAGYIENGVKQDEFYKYLKNILSKIKIDGSDYFLD 
                 394 
               
               
                   
               
               
                 S3 
                 314 
                 KAPLSASMIERYENHQNDLAALKQFIKNNLPEKYDEVFSDQSKDGYAGYIDGKTTQETFYKYIKNLLSKF--EGTDYFLD 
                 391 
               
               
                   
               
               
                 S4 
                 145 
                 ----SKALEEKYVAELQ-------------------------------------------------LERLKKDG------ 
                 165 
               
               
                   
               
               
                 S1 
                 392 
                 KLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEE 
                 471 
               
               
                   
               
               
                 S2 
                 395 
                 KIEREDFLRKQRTFDNGSIPHQIHLQEMHAILRRQGDYYPFLKEKQDRIEKILTFRIPYYVGPLVRKDSRFAWAEYRSDE 
                 474 
               
               
                   
               
               
                 S3 
                 392 
                 KIEREDFLRKQRTFDNGSIPHQIHLQEMNAILRRQGEYYPFLKDNKEKIEKILTFRIPYYVGPLARGNRDFAWLTRNSDE 
                 471 
               
               
                   
               
               
                 S4 
                 166 
                 --EVRGSINRFKTSD--------YVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGP--GEGSPFGW------K 
                 227 
               
               
                   
               
               
                 S1 
                 472 
                 TITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL 
                 551 
               
               
                   
               
               
                 S2 
                 475 
                 KITPWNFDKVIDKEKSAEKFITRMTLNDLYLPEEKVLPKHSHVYETYAVYNELTKIKYVNEQGKE-SFFDSNMKQEIFDH 
                 553 
               
               
                   
               
               
                 S3 
                 472 
                 AIRPWNFEEIVDKASSAEDFINKMTNYDLYLPEEKVLPKHSLLYETFAVYNELTKVKFIAEGLRDYQFLDSGQKKQIVNQ 
                 551 
               
               
                   
               
               
                 S4 
                 228 
                 DIKEW---------------YEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEK---LEYYEKFQIIEN 
                 289 
               
               
                   
               
               
                 S1 
                 552 
                 LEKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDR---FNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFED 
                 628 
               
               
                   
               
               
                 S2 
                 554 
                 VFKENRKVTKEKLLNYLNKEFFEYRIKDLIGLDKENKSFNASLGTYHDLKKIL-DKAFLDDKVNEEVIEDIIKTLTLFED 
                 632 
               
               
                   
               
               
                 S3 
                 552 
                 LEKENRKVTEKDIIHYLHN-VDGYDGIELKGIEKQ---FNASLSTYHDLLKIIKDKEENDDAKNEAILENIVHTLTIFED 
                 627 
               
               
                   
               
               
                 S4 
                 290 
                 VFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEF---TNLKVYHDIKDITARKEII---ENAELLDQIAKILTIYQS 
                 363 
               
               
                   
               
               
                 S1 
                 629 
                 REMIEERLKTYAHLFDDKVMKQLKR-RRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKED 
                 707 
               
               
                   
               
               
                 S2 
                 633 
                 KDMIHERLQKYSDIFTANQLKKLER-RHYTGWGRLSYKLINGIRNKENNKTILDYLIDDGSANRNFMQLINDDTLPFKQI 
                 711 
               
               
                   
               
               
                 S3 
                 628 
                 REMIKQRLAQYDSLFDEKVIKALTR-RHYTGWGKLSAKLINGICDKQTGNTILDYLIDDGKINRNFMQLINDDGLSFKEI 
                 706 
               
               
                   
               
               
                 S4 
                 364 
                 SEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDE------LWHTNDNQIAIFNRLKLVP--------- 
                 428 
               
               
                   
               
               
                 S1 
                 708 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 781 
               
               
                   
               
               
                 S2 
                 712 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 784 
               
               
                   
               
               
                 S3 
                 707 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 779 
               
               
                   
               
               
                 S4 
                 429 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 505 
               
               
                   
               
               
                 S1 
                 782 
                 
                   
                     KRIEEGIKELGSQIL-------KEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSD----YDVDH*IVPQSFLKDD 
                   
                 
                 850 
               
               
                   
               
               
                 S2 
                 785 
                 
                   
                     KKLQNSLKELGSNILNEEKPSYIEDKVENSHLQNDQLFLYYIQNGKDMYTGDELDIDHLSD----YDIDH*IIPQAFIKDD 
                   
                 
                 860 
               
               
                   
               
               
                 S3 
                 780 
                 
                   
                     KRIEDSLKILASGL---DSNILKENPTDNNQLQNDRLFLYYLQNGKDMYTGEALDINQLSS----YDIDH*IIPQAFIKDD 
                   
                 
                 852 
               
               
                   
               
               
                 S4 
                 506 
                 
                   
                     ERIEEIIRTTGK---------------ENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDH*IIPRSVSFDN 
                   
                 
                 570 
               
               
                   
               
               
                 S1 
                 851 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 922 
               
               
                   
               
               
                 S2 
                 861 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 932 
               
               
                   
               
               
                 S3 
                 853 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 924 
               
               
                   
               
               
                 S4 
                 571 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 650 
               
               
                   
               
               
                 S1 
                 923 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 1002 
               
               
                   
               
               
                 S2 
                 933 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 1012 
               
               
                   
               
               
                 S3 
                 925 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 1004 
               
               
                   
               
               
                 S4 
                 651 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 712 
               
               
                   
               
               
                 S1 
                 1003 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 1077 
               
               
                   
               
               
                 S2 
                 1013 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 1083 
               
               
                   
               
               
                 S3 
                 1005 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 1081 
               
               
                   
               
               
                 S4 
                 713 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 764 
               
               
                   
               
               
                 S1 
                 1078 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 1149 
               
               
                   
               
               
                 S2 
                 1084 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 1158 
               
               
                   
               
               
                 S3 
                 1082 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 1156 
               
               
                   
               
               
                 S4 
                 765 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 835 
               
               
                   
               
               
                 S1 
                 1150 
                 EKGKSKKLKSVKELLGITIMERSSFEKNPI-DFLEAKG-----YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKG 
                 1223 
               
               
                   
               
               
                 S2 
                 1159 
                 EKGKAKKLKTVKELVGISIMERSFFEENPV-EFLENKG-----YHNIREDKLIKLPKYSLFEFEGGRRRLLASASELQKG 
                 1232 
               
               
                   
               
               
                 S3 
                 1157 
                 EKGKAKKLKTVKTLVGITIMEKAAFEENPI-TFLENKG-----YHNVRKENILCLPKYSLFELENGRRRLLASAKELQKG 
                 1230 
               
               
                   
               
               
                 S4 
                 836 
                 DPQTYQKLK--------LIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKV 
                 907 
               
               
                   
               
               
                 S1 
                 1224 
                 NELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEITEQISEFSKRVILADANLDKVLSAYNKH------ 
                 1297 
               
               
                   
               
               
                 S2 
                 1233 
                 NEMVLPGYLVELLYHAHRADNF-----NSTEYLNYVSEHKKEFEKVLSCVEDFANLYVDVEKNLSKIRAVADSM------ 
                 1301 
               
               
                   
               
               
                 S3 
                 1231 
                 NEIVLPVYLTTLLYHSKNVHKL-----DEPGHLEYIQKHRNEFKDLLNLVSEFSQKYVLADANLEKIKSLYADN------ 
                 1299 
               
               
                   
               
               
                 S4 
                 908 
                 VKLSLKPYRFD-VYLDNGVYKFV-----TVKNLDVIK--KENYYEVNSKAYEEAKKLKKISNQAEFIASFYNNDLIKING 
                 979 
               
               
                   
               
               
                 S1 
                 1298 
                 RDKPIREQAENITHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSIT--------GLYETRI----DLSQL 
                 1365 
               
               
                   
               
               
                 S2 
                 1302 
                 DNFSIEEISNSFINLLTLTALGAPADFNFLGEKIPRKRYTSTKECLNATLIHQSIT--------GLYETRI----DLSKL 
                 1369 
               
               
                   
               
               
                 S3 
                 1300 
                 EQADIEILANSFINLLTFTALGAPAAFKFFGKDIDRKRYTTVSEILNATLIHQSIT--------GLYETWI----DLSKL 
                 1367 
               
               
                   
               
               
                 S4 
                 980 
                 ELYRVIGVNNDLLNRIEVNMIDITYR-EYLENMNDKRPPRIIKTIASKT---QSIKKYSTDILGNLYEVKSKKHPQIIKK 
                 1055 
               
               
                   
               
               
                 S1 
                 1366 
                 GGD 
                 1368 
               
               
                   
               
               
                 S2 
                 1370 
                 GEE 
                 1372 
               
               
                   
               
               
                 S3 
                 1368 
                 GED 
                 1370 
               
               
                   
               
               
                 S4 
                 1056 
                 G-- 
                 1056 
               
            
           
         
       
     
     The alignment demonstrates that amino acid sequences and amino acid residues that are homologous to a reference Cas9 amino acid sequence or amino acid residue can be identified across Cas9 sequence variants, including, but not limited to Cas9 sequences from different species, by identifying the amino acid sequence or residue that aligns with the reference sequence or the reference residue using alignment programs and algorithms known in the art. This disclosure provides Cas9 variants in which one or more of the amino acid residues identified by an asterisk in SEQ ID NOs: 11-14 (e.g., S1, S2, S3, and S4, respectively) are mutated as described herein. The residues D10 and H840 in Cas9 of SEQ ID NO: 10 that correspond to the residues identified in SEQ ID NOs: 11-14 by an asterisk are referred to herein as “homologous” or “corresponding” residues. Such homologous residues can be identified by sequence alignment, e.g., as described above, and by identifying the sequence or residue that aligns with the reference sequence or residue. Similarly, mutations in Cas9 sequences that correspond to mutations identified in SEQ ID NO: 10 herein, e.g., mutations of residues 10, and 840 in SEQ ID NO: 10, are referred to herein as “homologous” or “corresponding” mutations. For example, the mutations corresponding to the D10A mutation in SEQ ID NO: 10 or S1 (SEQ ID NO: 11) for the four aligned sequences above are D11A for S2, D10A for S3, and D13A for S4; the corresponding mutations for H840A in SEQ ID NO: 10 or S1 (SEQ ID NO: 11) are H850A for S2, H842A for S3, and H560A for S4. 
     A total of 250 Cas9 sequences (SEQ ID NOs: 11-260) from different species were aligned using the same algorithm and alignment parameters outlined above. Amino acid residues homologous to residues 10, and 840 of SEQ ID NO: 10 were identified in the same manner as outlined above. The alignments are provided below. The HNH domain (bold and underlined) and the RuvC domain (boxed) are identified for each of the four sequences. Single residues corresponding to amino acid residues 10, and 840 in SEQ ID NO: 10 are boxed in SEQ ID NO: 11 in the alignments, allowing for the identification of the corresponding amino acid residues in the aligned sequences. 
       
     
       
         
           
               
             
               
                 Lengthy table referenced here 
               
             
            
               
                 US20220220462A1-20220714-T00001 
               
               
                 Please refer to the end of the specification for access instructions. 
               
            
           
         
       
     
     EXAMPLE 6: Next Generation C to T Editors 
     Other familes of cytidine deaminases as alterantives to base etitor 3 (BE3) constructs were examined. The different C to T editors were developed to have a narrow or different editing window, alternate sequence specificity to expand targetable substrates, and to have higher activity. 
     Using the methods described in Example 4, the pmCDA1 (cytidine deaminase 1 from  Petromyzon marinus ) activity at the HeK-3 site is evaluated ( FIG. 42 ). The pmCDA1-nCas9-UGI-NLS (nCas9 indicates the Cas9 nickase described herein) construct is active on some sites (e.g., the C bases on the complementary strand at position 9, 5, 4, and 3) that are not accessible with rAPOBEC1 (BE3). 
     The pmCDA1 activity at the HeK-2 site is given in  FIG. 43 . The pmCDA1-XTEN-nCas9-UGI-NLS construct is active on sites adjacent to “G,” while rAPOBEC1 analog (BE3 construct) has low activity on “C”s that are adjacent to “G”s, e.g., the C base at position 11 on the complementary strand. 
     The percent of total sequencing reads with target C converted to T ( FIG. 44 ), C converted to A ( FIG. 45 ), and C converted to G ( FIG. 46 ) are shown for CDA and APOBEC1 (the BE3 construct). 
     The huAPOBEC3G activity at the HeK-2 site is shown in  FIG. 47 . Two constructs were used: huAPOBEC3G-XTEN-nCas9-UGI-NLS and huAPOBEC3G*(D316R_D317R)-XTEN-nCas9-UGI-NLS. The huAPOBEC3G-XTEN-nCas9-UGI-NLS construct has different sequence specificity than rAPOBEC1 (BE3), as shown in  FIG. 47 , the editing window appears narrow, as indicated by APOBEC3G&#39;s descreased activity at position 4 compared to APOBEC1. Mutations made in huAPOBEC3G (D316R and D317R) increased ssDNA binding and resulted in an observable effect on expanding the sites which were edited (compare APOBEC3G with APOBEC3G_RR in  FIG. 47 ). Mutations were chosen based on APOBEC3G crystal structure, see: Holden et al., Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implication.  Nature.  (2008); 121-4, the entire contents of which are incorporated herein by reference. 
     EXAMPLE 7: pmCDA1/huAPOBEC3G/rAPOBEC1 work in  E. coli    
     LacZ selection optimization for the A to I conversion was performed using a bacterial strain with lacZ encoded on the F plasmid. A critical glutamic acid residue was mutated (e.g., GAG to GGG, Glu to Gly mutation) so that G to A by a cytidine deaminase would restore lacZ activity ( FIG. 48 ). Strain CC102 was selected for the selection assay. APOBEC1 and CDA constructs were used in a selection assay to optimize G to A conversion. 
     To evaluate the the effect of copy number of the plasmids encoding the deaminase constructs on lacZ reversion frequency, the CDA and APOBEC1 deaminases were cloned into 4 plasmids with different replication origins (hence different copy numbers). SC 101, CloDF3, RSF1030, and PUC (copy number: PUC&gt;RSF1030&gt;CloDF3&gt;SC101) and placed under an inducible promoter. The plasmids were individually transformed into  E. coli  cells harboring F plasmid containing the mutated LacZ gene. The expression of the deaminases were induced and LacZ activity was detected for each construct ( FIG. 49 ). As shown in  FIG. 49 , CDA exhibited significantly higher activity than APOBEC1 in all instances, regardless of the plasmid copy number the deaminases were cloned in. Further, In terms of the copy number, the deaminase activity was positively correlated with the copy number of the plasmid they are cloned in, i.e., PUC&gt;CloDF3&gt;SC101. 
     LacZ reversions were confirmed by sequencing of the genomic DNA at the lacZ locus. To obtain the genomic DNA containing the corrected LacZ gene, cells were grown media containg X-gal, where cells having LacZ activity form blue colonies. Blue colonies were selected and grown in minimial media containing lactose. The cells were spun down, washed, and re-plated on minimal media plates (lactose). The blue colony at the highest dilution was then selected, and its genomic DNA was sequenced at the lacZ locus ( FIG. 50 ). 
     A chloramphenicol reversion assay was designed to test the activity of different cytidine deaminases (e.g., CDA, and APOBEC1). A plasmid harboring a mutant CAT1 gene which confers chloramphenicol resistance to bacteria is constructed with RSF1030 as the replication origin. The mutant CAT1 gene encodings a CAT1 protein that has a H195R (CAC to CGC) mutation, rendering the protein inactive ( FIG. 51 ). Deamination of the C base-paired to the G base in the CGC codon would convert the codon back to a CAC codon. restoring the activity of the protein. As shown in  FIG. 52 , CDA outperforms rAPOBEC in  E. coli  in restoring the acitivty of the chloramphenicol reisitance gene. The minimum inhibitory concentration (MIC) of chlor in S1030 with the selection plasmid (pNMG_ch_5) was approximately 1 μg/mL. Both rAPOBEC-XTEN-dCas9-UGI and CDA-XTEN-dCas9-UGI induced DNA correction on the selection plasmid ( FIG. 53 ). 
     Next, the huAPOBEC3G-XTEN-dCas9-UGI protein was tested in the same assay. Interestingly, huAPOBEC3G-XTEN-dCas9-UGI exhibited different sequence specificity than the rAPOBEC1-XTEN-dCas9-UGI fusion protein. Only position 8 was edited with APOBEC3G-XTEN-dCas9-UGI fusion, as compared to the rAPOBEC11-XTEN-dCas9-UGIfusion (in which positions 3, 6, and 8 were edited) ( FIG. 54 ). 
     EXAMPLE 8: C to T Base Editors with Less Off Target Editing 
     Current base editing technologies allow for the sequence-specific conversion of a C:G base pair into a T:A base pair in genomic DNA. This is done via the direct catalytic conversion of cytosine to uracil by a cytidine deaminase enzyme and thus, unlike traditional genome editing technologies, does not introduce double-stranded DNA breaks (DSBs) into the DNA as a first step. See, Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A., and Liu, D. R. (2016), “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.” Nature 533, 420-424; the entire contents of which are incorporated by reference herein. Instead, catalytically dead SpCas9 (dCas9) or a SpCas9 nickase (dCas9(A840H)) is tethered to a cytidine deaminase enzyme such as rAPOBEC 1, pmCDA 1, or hAPOBEC3G. The genomic locus of interest is encoded by an sgRNA, and DNA binding and local denaturation is facilitated by the dCas9 portion of the fusion. However, just as wt dCas9 and wt Cas9 exhibit off-target DNA binding and cleavage, current base editors also exhibit C to T editing at Cas9 off-target loci, which limits their therapeutic usefulness. 
     It has been reported that the introduction of just three to four mutations into SpCas9 that neutralize nonspecific electrostatic interactions between the protein and the sugar-phosphate backbone of its target DNA, increases the DNA binding specificity of SpCas9. See. Kleinstiver, B. P., Pattanayak, V., Prew, M. S., Tsai, S. Q., Nguyen, N. T., Zheng, Z., and Joung, J. K. (2016) “High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.” Nature 529, 490-495; and Slaymaker, I. M., Gao, L., Zetsche, B., Scott, D. A., Yan, W. X., and Zhang, F. (2015) “Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84-88: the entire contents of each are hereby incorporated by reference herein. Four reported neutralizing mutations were therefore incorporated into the initially reported base editor BE3 (SEQ ID NO: 285), and found that off-target C to T editing of this enzyme is also drastically reduced ( FIG. 55  ), with no decrease in on-target editing ( FIG. 56 ). 
     As shown in  FIG. 55 , HEK293T cells were transfected with plasmids expressing BE3 or HF-BE3 and a sgRNA matching the EMX1 sequence using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target locus, plus the top ten known Cas9 off-target loci for the EMX1 sgRNA, as previously determined by Joung and coworkers using the GUIDE-seq method. See Tsai, S. Q., Zheng, Z., Nguyen, N. T., Liebers, M., Topkar, V. V., Thapar, V., Wyvekens, N., Khayter, C., Iafrate, A. J., Le, L. P., et al. (2015) “GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.”  Nat Biotech  33, 187-197; the entire contents of which are incorporated by reference herein. EMX1 off-target 5 locus did not amplify and is not shown. Sequences of the on-target and off-target protospacers and protospacer adjacent motifs (PAMs) are displayed ( FIG. 55 ). Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with T at each position of an original C within the protospacer, are shown for BE3 and HF-BE3. 
     In  FIG. 56 , HEK293T cells were transfected with plasmids expressing BE3 or HF-BE3 and sgRNAs matching the genomic loci indicated using Lipofectamine 2000. Three days after transfection, genomic DNA was extracted, amplified by PCR, and analyzed by high-throughput DNA sequencing at the on-target loci. The percentage of total DNA sequencing reads with all four bases at the target Cs within each protospacer are shown for treatment with BE3 or HF-BE3 ( FIG. 56 ). Frequencies of indel formation are shown as well. 
     
       
         
           
               
            
               
                 Primary Protein Sequence  
               
               
                 of HF-BE3 (SEQ ID NO: 285): 
               
               
                 MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSI 
               
               
                   
               
               
                 WRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAI 
               
               
                   
               
               
                 TEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESG 
               
               
                   
               
               
                 YCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQ 
               
               
                   
               
               
                 PQLTFFTIALQSCHYQRLPPHILWATGLKSGSETPGTSESATPESDKKYS 
               
               
                   
               
               
                 IGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG 
               
               
                   
               
               
                 ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFL 
               
               
                   
               
               
                 VEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYL 
               
               
                   
               
               
                 ALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGV 
               
               
                   
               
               
                 DAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF 
               
               
                   
               
               
                 DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDIL 
               
               
                   
               
               
                 RVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSK 
               
               
                   
               
               
                 NGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD 
               
               
                   
               
               
                 NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLA 
               
               
                   
               
               
                 RGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEK 
               
               
                   
               
               
                 VLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTN 
               
               
                   
               
               
                 RKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF 
               
               
                   
               
               
                 LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY 
               
               
                   
               
               
                 TGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMALIHDDSLTFKE 
               
               
                   
               
               
                 DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKP 
               
               
                   
               
               
                 ENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQL 
               
               
                   
               
               
                 QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV 
               
               
                   
               
               
                 LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERG 
               
               
                   
               
               
                 GLSELDKAGFIKRQLVETRAITKHVAQILDSRMNTKYDENDKLIREVKVI 
               
               
                   
               
               
                 TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLES 
               
               
                   
               
               
                 EFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE 
               
               
                   
               
               
                 IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFS 
               
               
                   
               
               
                 KESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKK 
               
               
                   
               
               
                 LKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFEL 
               
               
                   
               
               
                 ENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ 
               
               
                   
               
               
                 LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQA 
               
               
                   
               
               
                 ENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY 
               
               
                   
               
               
                 ETRIDLSQLGGDSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGN 
               
               
                   
               
               
                 KPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLS 
               
               
                   
               
               
                 GGSPKKKRKV 
               
            
           
         
       
     
     EXAMPLE 9: Development of Base Editors that Use Cas9 Variants and Modulation of the Base Editor Processivity to Increase the Target Range and Precision of the Base Editing Technology 
     Unlike iraditional genome edicing platforms, base editing technology allows precise single nucleotide changes in the DNA without inducing double-stranded breaks(DSBs). See, Komor, A. C. et al.  Nature  533, 420-424 (2016). The current generation of base editor uses the NGG PAM exclusively. This limits its ability to edit desired bases within the genome, as the base editor needs to be placed at a precise location where the target base is placed within a 4-base region (the ‘deamination window’), approximately 15 bases upstream of the PAM. See, Komor, A. C. et al.  Nature  533, 420-424 (2016). Moreover, due to the high processivity of cytidine deaminase, the base editor may convert all cytidines within its deamination window into thymidines, which could induce amino acid changes other than the one desired by the researcher. See, Komor, A. C. et al.  Nature  533, 420-424 (2016). 
     Expanding the Scope of Base Editing Through the Development of Base Editors with Cas9 Variants 
     Cas9 homologs and other RNA-guided DNA binders that have different PAM specificities were incorporated into the base editor architecture. See, Kleinstiver, B. P. et al.  Nature  523, 481-485 (2015); Kleinstiver, B. P. et al.  Nature Biotechnology  33, 1293-1298 (2015); and Zetsche, B. et al.  Cell  163, 759-771 (2015); the entire contents of each are incorporated by reference herein. Furthermore, innovations that have broadened the PAM specificities of various Cas9 proteins were also incorporated to expand the target reach of the base editor even more. See, Kleinstiver, B. P. et al.  Nature  523, 481-485 (2015); and Kleinstiver, B. P. et al.  Nature Biotechnology  33, 1293-1298 (2015). The current palette of base editors is summarized in Table 4. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 New base editors made from Cas9 Variants 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Base Editor  
                 Reference for  
               
               
                 Species 
                 PAM 
                 Name 
                 Cas9 variant 
               
               
                   
               
               
                 
                   S. pyogenes 
                 
                 ...NGG 
                 BE3 
                 Wild-type 
               
               
                   
                 ...NGA 
                 VQR BE3 or  
                 Kleinstiver, B. P.  
               
               
                   
                   
                 EQR BE3 
                 et al. 
               
               
                   
                 ...NGCG 
                 VRER BE3 
                 Kleinstiver, B. P.  
               
               
                   
                   
                   
                 et al. 
               
               
                 
                   S. aureus 
                 
                 ...NNGRRT 
                 SaBE3 
                 Wild-type 
               
               
                   
                 ...NNNRRT 
                 SaKKH BE3 
                 Kleinstiver, B. P.  
               
               
                   
                   
                   
                 et al. 
               
               
                 
                   L. bacterium 
                 
                 TTTN... 
                 dCpf1 BE2 
                 Zetsche, B. et al. 
               
               
                   
               
            
           
         
       
     
     Modulating Base Editor&#39;s Processivity Through Site-Directed Mutagenesis of rAPOBEC1 
     It was reasoned that the processivity of the base editor could be modulated by making point mutations in the deaminase enzyme. The incorporation of mutations that slightly reduce the catalytic activity of deaminase in which the base editor could still catalyze on average one round of cytidine deamination but was unlikely to access and catalyze another deamination within the relevant timcscale were pursued. In effect, the resulting base editor would have a narrower deamination window. 
     rAPOBEC1 mutations probed in this work are listed in Table 5. Some of the mutations resulted in slight apparent impairment of rAPOBEC1 catalysis, which manifested as preferential editing of one cytidine over another when multiple cytidines are found within the deamination window. Combining some of these mutations had an additive effect, allowing the base editor to discriminate substrate cytidines with higher stringency. Some of the double mutants and the triple mutant allowed selective editing of one cytidine among multiple cytidines that are right next to one another ( FIG. 57 ). 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 rAPOBEC1 Point Mutations Investigated 
               
            
           
           
               
               
               
            
               
                 rAPOBEC1 mutation  
                 Corresponding mutation  
                   
               
               
                 studied in this work 
                 in APOBEC3G 
                 Reference 
               
               
                   
               
               
                 H121R/H122R 
                 D315R/D316R 
                 Holden, L. G. et al. 
               
               
                 R126A 
                 R320A 
                 Chen, K-M. et al. 
               
               
                 R126E 
                 R320E 
                 Chen, K-M. et al. 
               
               
                 R118A 
                 R313A 
                 Chen, K-M. et al. 
               
               
                 W90A 
                 W285A 
                 Chen, K-M. et al. 
               
               
                 W90Y 
                 W285Y 
                   
               
               
                 R312E 
                 R326E 
               
               
                   
               
            
           
         
       
     
     Base Editor PAM Expansion and Processivity Modulation 
     The next generation of base editors were designed to expand editable cytidines in the genome by using other RNA-guided DNA binders ( FIG. 58 ). Using a NGG PAM only allows for a single target within the “window” whereas the use of multiple different PAMs allows for Cas9 to be positioned anywhere to effect selective deamination. A variety of new base editors have been created from Cas9 variants ( FIG. 59  and Table 4). Different PAM sites (NGA,  FIG. 60 ; NGCG,  FIG. 61 ; NNGRRT,  FIG. 62 ; and NNHRRT, FIG. 63 ) were explored. Selective deamination was successfully achieved through kinetic modulation of cytidine deaminase point mutagenesis ( FIG. 65  and Table 5). 
     The effect of various mutations on the deamination window was then investigated in cell culture using spacers with multiple cytidines ( FIGS. 66 and 67 ). 
     Further, the effect of various mutations on different genomic sites with limited numbers of cytidines was examined ( FIGS. 68 to 71 ). It was found that approximately one cytidine will be edited within the deamination windown in the spacer, while the rest of the cytidines will be left intact. Overall, the preference for editing is as follows: C 6 &gt;C 5 &lt;&lt;C 7 ≈C 4 . 
     Base Editing Using Cpf1 
     Cpf1, a Cas9 homolog, can be obtained as AsCpf1, LbCpf1, or from any other species. Schematics of fusion constructs, including BE2 and BE3 equivalents, are shown in  FIG. 73 . The BE2 equivalent uses catalytically inactive Cpf2 enzyme (dCpf1) instead of Cas9, while the BE3 equivalent includes the Cpf1 mutant, which nicks the target strand. The bottom schematic depicts different fusion architectures to combine the two innovations illustrated above it ( FIG. 73 ). The base editing results of HEK293T cell TTTN PAM sites using Cpf1 BE2 were examined with different spacers ( FIGS. 64A to 64C ). In some embodiments, Cpf1 may be used in place of a Cas9 domain in any of the base editors provided herein. In some embodiments, the Cpf1 is a protein that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% identical to SEQ ID NO 313. 
     
       
         
           
               
            
               
                 Full Protein Sequence of Cpf1 (SEQ ID NO: 313): 
               
               
                 MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKA 
               
               
                   
               
               
                 KQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKS 
               
               
                   
               
               
                 AKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGI 
               
               
                   
               
               
                 ELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSSNDIPTSII 
               
               
                   
               
               
                 YRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKT 
               
               
                   
               
               
                 SEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGI 
               
               
                   
               
               
                 NEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVT 
               
               
                   
               
               
                 TMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLT 
               
               
                   
               
               
                 DLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKY 
               
               
                   
               
               
                 LSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLA 
               
               
                   
               
               
                 QISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSED 
               
               
                   
               
               
                 KANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNF 
               
               
                   
               
               
                 ENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENK 
               
               
                   
               
               
                 GEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKN 
               
               
                   
               
               
                 GSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSI 
               
               
                   
               
               
                 DEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGR 
               
               
                   
               
               
                 PNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIA 
               
               
                   
               
               
                 NKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEI 
               
               
                   
               
               
                 NLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMK 
               
               
                   
               
               
                 TNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYN 
               
               
                   
               
               
                 AIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGG 
               
               
                   
               
               
                 VLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYE 
               
               
                   
               
               
                 SVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSR 
               
               
                   
               
               
                 LINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESD 
               
               
                   
               
               
                 KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNM 
               
               
                   
               
               
                 PQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN 
               
            
           
         
       
     
     EXAMPLE 10: Increased Fidelity of Base Editing 
     Examining the difference between plasmid delivery of BE3 and HF-BE3, it was found that the two edit on-target loci with comparable efficiency ( FIGS. 74 and 75 ). However, HF-BE3 edited off-target loci much less than BE3, meaning that HF-BE3 has a much higher DNA specificity than BE3 ( FIG. 76 ). Deaminase protein lipofection to HEK cells demonstrated that protein delivery of BE3 results in comparable on-target activity, but much better specificity, than plasmid DNA delivery of BE3. Using improved transfection procedures and better plasmids (n=2), the experiment used the following conditions: protein delivery was 125 nM Cas9:sgRNA complex, plasmid delivery was 750 ng BE3/HF-BE3 plasmid+250 ng sgRNA plasmid, and lipofection was with 1.5 μL of Lipofectamine 2000 per well. EMX-1 off target site 2 and FANCF off-target site 1 showed the most off-target editing with BE3, compared to all of the off-targets assayed ( FIGS. 77 and 78 ), while HEK-3 showed no significant editing at off-targets for any of the delivery methods ( FIG. 79 ). HEK-4 shows some C-to-G editing on at the on-target site, while its off-target sites 1, 3, and 4 showed the most off-target editing of all the assayed sites ( FIG. 80 ). 
     Delivery of BE3 Protein via Micro-Injection to Zebrafish 
     TYR guide RNAs were tested in an in vitro assay for sgRNA activity ( FIGS. 81 and 82 ). The % HTS reads show shows many C residues were converted to T residues during a 2 h incubation with purified BE3 protein and PCR of the resulting product. Experiments used an 80-mer synthetic DNA substate with the target deamination site in 60 bp of its genomic context. This is not the same as % edited DNA strands because only one strand was nicked, so the product is not amplified by PCR. The proportion of HTS reads edited is equal to x/(2-x), where x is the actual proportion of THS reads edited. For 60% editing, the actual proportion of bases edited is 75%. “Off target” is represents BE3 incubated with the same DNA substrate, while bound to an off-target sgRNA. It was found sgRNAs sgRH_13, sgHR_17, and possibly sgHR_ 16 appeared to be promising targets for in vivo injection experiments. 
     The delivery of BE3 protein in was tested in vivo in zebrafish. Zebrafish embryos (n=16-24) were injected with either scramled sgRNA, sgHR_13, sgHR_16, or sgHR_17 and purified BE3. Three embryos from each condition were analyzed independently (single embryo) and for each condition, all of the injected embryos were pooled and sequenced as a pool. The results are shown in  FIGS. 83 to 85 . 
     EXAMPLE 11: Uses of Base Editors to Treat Disease 
     Base editors or complexes provided herein (e.g., BE3) may be used to modify nucleic acids. For example, base editors may be used to change a cytosine to a thymine in a nucleic acid (e.g., DNA). Such changes may be made to, inter alia, alter the amino acid sequence of a protein, to destroy or create a start codon, to create a stop codon, to distupt splicing donors, to disrupt splicing acceptors or edit regulatory sequences. Examples of possible nucleotide changes are shown in  FIG. 86 . 
     Base editors or complexes provided herein (e.g., BE3) may be used to edit an isoform of Apolipoprotein E in a subject. For example, an Apolipoprotein E isoform may be edited to yield an isoform associated with a lower risk of developing Alzheimer&#39;s disease. Apolipoprotein E has four isoforms that differ at amino acids 112 and 158. APOE4 is the largest and most common genetic risk factor for late-onset Alzheimer&#39;s disease. Arginine residue 158 of APOE4, encoded by the nucleic acid sequence CGC, may be changed to a cysteine by using a base editor (e.g., BE3) to change the CGC nucleic acid sequence to TGC, which encodes cysteine at residue 158. This change yields an APOE3r isoform, which is associated with lower Alzheimer&#39;s disease risk. See  FIG. 87 . 
     It was tested whether base editor BE3 could be used to edit APOE4 to APOE3r in mouse astrocytes ( FIG. 88 ). APOE 4 mouse astrocytes were nucleofected with Cas9+ template or BE3, targeting the nucleic acid encoding Arginine 158 of APOE4. The Cas9+ template yielded only 0.3% editing with 26% indels, while BE3 yielded 75% editing with 5% indels. Two additional base-edited cytosines are silent and do not yield changes to the amino acid sequence ( FIG. 88 ). 
     Base editors or complexes provided herein may be used to treat prion protein diseases such as Creutzfeldt-Jakob disease and fatal familial insomnia, for example, by introducing mutations into a PRNP gene. Reverting PRNP mutations may not yield therapeutic results, and intels in PRNP may be pathogenic. Accordingly, it was tested whether PRNP could be mutated using base editors (e.g., BE3) to introduce a premature stop codon in the PRNP gene. BE3, associated with its guide RNA, was introduced into HEK cells or glioblastoma cells and was capable of editing the PRNP gene to change the encoded arginine at residue 37 to a stop codon. BE3 yielded 41% editing ( FIG. 89 ). 
     Additional genes that may be edited include the following: APOE editing of Arg 112 and Arg 158 to treat increased Alzheimer&#39;s risk; APP editing of Ala 673 to decrease Alzheimer&#39;s risk; PRNP editing of Arg 37 to treat fatal familial insomnia and other prion protein diseases; DMD editing of the exons 23 and 51 splice sites to treat Duchenne muscular dystrophy; FTO editing of intron 1 to treat obesity risk; PDS editing of exon 8 to treat Pendred syndrome (genetic deafness); TMC1 editing of exon 8 to treat congenital hearing loss; CYBB editing of various patient-relevant mutations to treat chronic granulomatous disease. Additional diseases that may be treated using the base editors provided herein are shown in Table 6. below. 
     UGI also plays a key role. Knocking out UDG (which UGI inhibits) was shown to dramatically improve the cleanliness and efficiency of C to T base editing ( FIG. 90 ). Furthermore, base editors with nickase and without UGI were shown to produce a mixture of outcomes, with very high indel rates ( FIG. 91 ). 
     EXAMPLE 12: Expanding the Targeting Scope of Base Editing 
     Base editing is a new approach to genome editing that uses a fusion protein containing a catalylically defective  Streptococcus pyogenes  Cas9, a cytidine deaminase, and an inhibitor of base excision repair to induce programmable, single-nucleotide C→T (or G→A) 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 1 . The development of five new C→T (or G→A) base editors that use natural and engineered Cas9 variants with different protospacer-adjacent motif (PAM) specificities to expand the number of sites that can be targeted by base editing by 2.5-fold are described herein. Additionally, new base editors containing mutated cytidine deaminase domains that narrow the width of the apparent editing window from approximately 5 nucleotides to 1 or 2 nucleotides were engineered, enabling the discrimination of neighboring C nucleotides that would previously be edited with comparable efficiency. Together, these developments substantially increase the targeting scope of base editing. 
     CRISPR-Cas9 nucleases have been widely used to mediate targeted genome editing 2 . In most genome editing applications, Cas9 forms a complex with a 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-homologuous 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). 3,4  Unfortunately, under most non-perturbalive conditions HDR is inefficient, dependent on cell state and cell type, and dominated by a larger frequency of indels. 3,4  As most of the known genetic variations associated with human disease are point mutations 5 , methods that can more efficiently and cleanly make precise point mutations are needed. 
     Base editing, which enables targeted replacement of a C:G base pair with a T:A base pair in a programmable manner without inducing DSBs 1 . has been recently described. Base editing uses a fusion protein between a catalytically inactivated (dCas9) or nickase form of  Streptococcus pyogenes  Cas9 (SpCas9), a cytidine deaminase such as APOBEC1, and an inhibitor of base excision repair such as uracil glycosylase inhibitor (UGI) to convert cytidines into uridines within a five-nucleotide window specified by the sgRNA. 1  The third-generation base editor, BE3, converts C:G base pairs to T:A base pairs, including disease-relevant point mutations, in a variety of cell lines with higher efficiency and lower indel frequency than what can be achieved using other genome editing methods 1 . Subsequent studies have validated the deaminase-dCas9 fusion approach in a variety of settings 6-7 . 
     Efficient editing by BE3 requires the presence of an NGG PAM that places the target C within a five-nucleotide window near the PAM-distal end of the protospacer (positions 4-8, counting the PAM as positions 21-23) 1 . This PAM requirement substantially limits the number of sites in the human genome that can be efficiently targeted by BE3, as many sites of interest lack an NGG 13- to 17-nucleotides downstream of the target C. Moreover, the high activity and processivity of BE3 results in conversion of all Cs within the editing window to Ts, which can potentially introduce undesired changes to the target locus. Herein, new C:G to T:A base editors that address both of these limitations are described. 
     It was thought that any Cas9 homolog that binds DNA and forms an “R-loop” complex 8  containing a single-stranded DNA bubble could in principle be converted into a base editor. These new base editors would expand the number of targetable loci by allowing non-NGG PAM sites to be edited. The Cas9 homolog from  Staphylococcus aureus  (SaCas9) is considerably smaller than SpCas9 (1053 vs. 1368 residues), can mediate efficient genome editing in mammalian cells, and requires an NNGRRT PAM 9 . SpCas9 was replaced with SaCas9 in BE3 to generate SaBE3 and transfected HEK293T cells with plasmids encoding SaBE3 and sgRNAs targeting six human genomic loci ( FIGS. 92A and 92B ). After 3 d, the genomic loci were subjected to high-throughput DNA sequencing (HTS) to quantify base editing efficiency. SaBE3 enabled C to T base editing of target Cs at a variety of genomic sites in human cells, with very high conversion efficiencies (approximately 50-75% of total DNA sequences converted from C to T, without enrichment for transfected cells) arising from targeting Cs at positions 6-11. The efficiency of SaBE3 on NNGRRT-containing target sites in general exceeded that of BE3 on NGG-containing target sites 1 . Perhaps due to its higher average efficiency, SaBE3 can also result in detectable base editing at target Cs at positions outside of the canonical BE3 activity window ( FIG. 92C ). In comparison, BE3 showed significantly reduced editing under the same conditions (0-11%), in accordance with the known SpCas9 PAM preference ( FIG. 106A ) 10 . These data show that SaBE3 can facilitate very efficient base editing at sites not accessible to BE3. 
     The targeting range of base editors was further expanded by applying recently engineered Cas9 variants that expand or alter PAM specificities. Joung and coworkers recently reported three SpCas9 mutants that accept NGA (VQR-Cas9), NGAG (EQR-Cas9), or NGCG(VRER-Cas9) PAM sequences 11 . In addition, Joung and coworkers engineered a SaCas9 variant containing three mutations (SaKKH-Cas9) that relax its PAM requirement to NNNRRT 12 . The SpCas9 portion of BE3 was replaced with these four Cas9 variants to produce VQR-BE3, EQR-BE3, VRER-BE3, and SaKKH-BE3, which target NNNRRT, NGA, NGAG, and NGCG PAMs respectively. HEK293T cells were transfected with plasmids encoding these constructs and sgRNAs targeting six genomic loci for each new base editor, and measured C to T base conversions using HTS. 
     SaKKH-BE3 edited sites with NNNRRT PAMs with efficiencies up to 62% of treated, non-enriched cells ( FIG. 92D ). As expected, SaBE3 was unable to efficiently edit targets containing PAMs that were NNNHRRT (where H=A, C, or T) ( FIG. 92D ). VQR-BE3, EQR-BE3, and VRER-BE3 exhibited more modest, but still substantial base editing efficiencies of up to 50% of treated, non-enriched cells at genomic loci with the expected PAM requirements with an editing window similar to that of BE3 ( FIGS. 92E and 92F ). Base editing efficiencies of VQR-BE3, EQR-BE3, and VRER-BE3 in general closely paralleled the reported PAM requirements of the corresponding Cas9 nucleases; for example. EQR-BE3 was unable to efficiently edit targets containing NGAH PAM sequences (FIG.  92 F). In contrast, BE3 was unable to edit sites with NGA or NGCG PAMs efficiently (0-3%), likely due to its PAM restrictions ( FIG. 106B ). 
     Collectively, the properties of SaBE3, SaKKH-BE3, VQR-BE3, EQR-BE3, and VRER-BE3 establish that base editors exhibit a modularity that facilitates their ability to exploit Cas9 homologs and engineered variants. 
     Next, base editors with altered activity window widths were developed. All Cs within the activity window of BE3 can be efficiently converted to Ts 1 . The ability to modulate the width of this window would be useful in cases in which it is important to edit only a subset of Cs present in the BE3 activity window. 
     The length of the linker between APOBEC1 and dCas9 was previously observed to modulate the number of bases that are accessible by APOBEC1 in vitro 1 . In HEK293T cells, however, varying the linker length did not significantly modulate the width of the editing window, suggesting that in the complex cellular milieu, the relative orientation and flexibility of dCas9 and the cytidine deaminase are not strongly determined by linker length ( FIG. 96 ). Next, it was thought that truncating the 5 40   end of the sgRNA might narrow the base editing window by reducing the length of single-stranded DNA accessible to the deaminase upon formation of the RNA-DNA heteroduplex. HEK293T cells were co-transfected with plasmids encoding BE3 and sgRNAs of different spacer lengths targeting a locus with multiple Cs in the editing window. No consistent changes in the width of base editing when using truncated sgRNAs with 17- to 19-base spacers were observed ( FIGS. 95A to 95B ). Truncating the sgRNA spacer to fewer than 17 bases resulted in large losses in activity ( FIG. 95A ). 
     As an alternative approach, it was thought that mutations to the deaminase domain might narrow the width of the editing window through multiple possible mechanisms. First, some mutations may alter substrate binding, the conformation of bound DNA, or substrate accessibility to the active site in ways that reduce tolerance for non-optimal presentation of a C to the deaminase active site. Second, because the high activity of APOBEC1 likely contributes to the deamination of multiple Cs per DNA binding event, 1,13,14  mutations that reduce the catalytic efficiency of the deaminase domain of a base editor might prevent it from catalyzing successive rounds of deamination before dissociating from the DNA. Once any C:G to T:A editing event has taken place, the sgRNA no longer perfectly matches the target DNA sequence and re-binding of the base editor to the target locus should be less favorable. Both strategies were tested in an effort to discover new base editors that distinguish among multiple cytidines within the original editing window. 
     Given the absence of an available APOBEC1 structure, several mutations previously reported to modulate the catalytic activity of APOBEC3G, a cytidine deaminase from the same family that shares 42% sequence similarity of its active site-containing domain to that of APOBEC1, were identified 15 . Corresponding APOBEC1 mutations were incorporated into BE3 and evaluated their effect on base editing efficiency and editing window width in HEK293T cells at two C-rich genomic sites containing Cs at positions 3,4, 5, 6, 8, 9, 10, 12, 13, and 14 (site A); or containing Cs at positions 5, 6, 7, 8, 9, 10, 11, and 13 (site B). 
     The APOBEC1 mutations R118A and W90A each led to dramatic loss of base editing efficiency ( FIG. 97C ). R132E led to a general decrease in editing efficiency but did not change the substantially narrow the shape of the editing window ( FIG. 97C ). In contrast, several mutations that narrowed the width of the editing window while maintaining substantial editing efficiency were found ( FIGS. 93A and 97C ). The “editing window width” was defined to represent the artificially calculated window width within which editing efficiency exceeds the half-maximal value for that target. The editing window width of BE3 for the two C-rich genomic sites tested was 5.0 (site A) and 6.1 (site B) nucleotides. 
     R126 in APOBEC1 is predicted to interact with the phosphate backbone of ssDNA 13 . Previous studies have shown that introducing the corresponding mutation into APOBEC3G decreased catalysis by at least 5-fold 14 . Interestingly, when introduced into APOBEC1 in BE3, R126A and R126E increased or maintained activity relative to BE3 at the most strongly edited positions (C5, C6, and C7), while decreasing editing activity at other positions ( FIGS. 93A and 97C ). Each of these two mutations therefore narrowed the width of the editing window at site A and site B to 4.4 and 3.4 nucleotides (R126A), or to 4.2 and 3.1 nucleotides (R126E). respectively ( FIGS. 93A and 97C ). 
     W90 in APOBEC1 (corresponding to W285 in APOBEC3G) is predicted to form a hydrophobic pocket in the APOBEC3G active site and assist in substrate binding 13 . Mutating this residue to Ala abrogated APOBEC3G&#39;s catalytic activity 13 . In BE3, W90A almost completely abrogated base editing efficiency ( FIG. 97C ). In contrast, it was found that W90Y only modestly decreased base editing activity while narrowing the editing window width at site A and site B to 3.8 and 4.9 nucleotides, respectively ( FIG. 93A ). These results demonstrate that mutations to the cytidine deaminase domain can narrow the activity window width of the corresponding base editors. 
     W90Y, R126E, and R132E, the three mutations that narrowed the editing window without drastically reducing base editing activity, were combined into doubly and triply mulated base editors. The double mutant W90Y+R126E resulted in a base editor (YE1-BE3) with BE3-like maximal editing efficiencies, but substantially narrowed editing window width (width at site A and site B=2.9 and 3.0 nucleotides, respectively ( FIG. 93A ). The W90Y+R132E base editor (YE2-BE3) exhibited modestly lower editing efficiencies (averaging 1.4-fold lower maximal editing yields across the five sites tested compared with BE3), and also substantially narrowed editing window width (width at site A and site B=2.7 and 2.8 nucleotides, respectively) ( FIG. 97C ). The R126E+R132E double mutant (EE-BE3) showed similar maximal editing efficiencies and editing window width as YE2-BE3 ( FIG. 97C ). The triple mutant W90Y+R126E+R132E (YEE-BE3) exhibited 2.0-fold lower average maximal editing yields but very little editing beyond the C6 position and an editing window width of 2.1 and 1.4 nucleotides for site A and site B, respectively ( FIG. 97C ). These data taken together indicate that mutations in the cytidine deaminase domain can strongly affect editing window widths, in some cases with minimal or only modest effects on editing efficiency. 
     The base editing outcomes of BE3, YE 1-BE3, YE2-BE3, EE-BE3, and YEE-BE3 were further compared in HEK293T cells targeting four well-studied human genomic sites that contain multiple Cs within the BE3 activity window 1 . These target loci contained target Cs at positions 4 and 5 (HEK site 3), positions 4 and 6 (HEK site 2), positions 5 and 6 (EMX1), or positions 6, 7, 8, and 11 (FANCF). BE3 exhibited little (&lt;1.2-fold) preference for editing any Cs within the position 4-8 activity window. In contrast, YE1-BE3, exhibited a 1.3-fold preference for editing C5 over C4 (HEK site 3), 2.6-fold preference for C6 over C4 (HEK site 2), 2.0-fold preference for C5 over C6 (EMX1), and 1.5-fold preference for C6 over C7 (FANCF) ( FIG. 93B ). YE2-BE3 and EE-BE3 exhibited somewhat greater positional specificity (narrower activity window) than YE1-BE3, averaging 2.4-fold preference for editing C5 over C4 (HEK site 3), 9.5-fold preference for C6 over C4 (HEK site 2), 2.9-fold preference for C5 over C6 (EMX1), and 2.6-fold preference for C7 over C6 (FANCF) ( FIG. 93B ). YEE-BE3 showed the greatest positional selectivity, with a 2.9-fold preference for editing C5 over C4 (HEK site 3), 29.7-fold preference for C6 over C4 (HEK site 2), 7.9-fold preference for C5 over C6 (EMX1), and 7.9-fold preference for C7 over C6 (FANCF) ( FIG. 93B ). The findings establish that mutant base editors can discriminate between adjacent Cs, even when both nucleotides are within the BE3 editing window. 
     The product distributions of these four mutants and BE3 were further analyzed by HTS to evaluate their apparent proccssivity. BE3 generated predominantly T4-T5 (HEK site 3), T4-T6 (HEK site 2), and T5-T6 (EMX1) products in treated HEK293T cells, resulting in, on average, 7.4-fold more products containing two Ts, than products containing a single T. In contrast, YE1-BE3, YE2-BE3, EE-BE3, and YEE-BE3 showed substantially higher preferences for singly edited C4-T5, C4-T6, and T5-C6 products ( FIG. 93C ). YE1-BE3 yielded products with an average single-T to double-T product ratio of 1.4. YE2-BE3 and EE-BE3 yielded products with an average single-T to double-T product ratio of 4.3 and 5.1, respectively ( FIG. 93C ). Consistent with the above results, the YEE-BE3 triple mutant favored single-T products by an average of 14.3-fold across the three genomic loci. ( FIG. 93C ). For the target site in which only one C is within the target window (HEK site 4, at position C5), all four mutants exhibited comparable editing efficiencies as BE3 ( FIG. 98 ). These findings indicate that these BE3 mutants have decreased apparent processivity and can favor the conversion of only a single C at target sites containing multiple Cs within the BE3 editing window. These data also suggest a positional preference of C5&gt;C6&gt;C7=C4 for these mutant base editors, although this preference could differ depending on the target sequence. 
     The window-modulating mutations in APOBEC1 were applied to VQR-BE3, allowing selective base editing of substrates at sites targeted by NGA PAM ( FIG. 107A ). However, when these mutations were applied to SaKKH-BE3, a linear decrease in base editing efficiency was observed without the improvement in substrate selectivity, suggesting a different kinetic equilibrium and substrate accessibility of this base editor than those of BE3 and its variants ( FIG. 107B ). 
     The five base editors with altered PAM specificities described in this study together increase the number of disease-associated mutations in the ClinVar database that can in principle be corrected by base editing by 2.5-fold ( FIGS. 94A and 94B ). Similarly, the development of base editors with narrowed editing windows approximately doubles the fraction of ClinVar entries with a properly positioned NGG PAM that can be corrected by base editing without comparable modification of a non-target C (from 31% for BE3 to 59% for YEE-BE3) ( FIGS. 94A and 94B ). 
     In summary, the targeting scope of base editing was substantially expanded by developing base editors that use Cas9 variants with different PAM specificities, and by developing a collection of deaminase mutants with varying editing window widths. In theory, base editing should be possible using other programmable DNA-binding proteins (such as Cpf1 16 ) that create a bubble of single-stranded DNA that can serve as a substrate for a single-strand-specific nucleotide deaminase enzyme. 
     Materials and Methods 
     Cloning. PCR was performed using Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs). Plasmids for BE and sgRNA were constructed using USER cloning (New England Biolabs), obtained from previously reported plasmids 1 . DNA vector amplification was carried out using NEB 10beta competent cells (New England Biolabs). 
     Cell culture. HEK293T (ATCC CRL-3216) were cultured 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% CO 2 . Immortalized rat astrocytes containing the ApoE4 isoform of the APOE gene (Taconic Biosciences) were maintained 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 Geneticin (ThermoFisher Scientific). 
     Transfections. HEK293T cells were seeded on 48-well collagen-coated BioCoat plates (Corning) and transfected at approximately 85% confluency. 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. 
     High-throughput DNA sequencing of genomic DNA samples. Transfected cells were harvested after 3 d and the genomic DNA was isolated using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter) according to the manufacturer&#39;s instructions. Genomic regions of interest were amplified by PCR with flanking HTS primer pairs listed in the Supplementary Sequences. PCR amplification was carried out with Phusion hot-start II DNA polymerase (ThermoFisher) according to the manufacturer&#39;s instructions. PCR products were purified using RapidTips (Diffinity Genomics). Secondary PCR was performed to attach sequencing adaptors. The products were gel-purified and quantified using the KAPA Library Quantification Kit-Illumina (KAPA Biosystems). Samples were sequenced on an Illumina MiSeq as previously described 1 . 
     Data analysis. Nucleotide frequencies were assessed using a previously described MATLAB script 1 . Briefly, the reads were aligned to the reference sequence via the Smith-Waterman algorithm. Base calls with Q-scores below 30 were replaced with a placeholder nucleotide (N). This quality threshold results in nucleotide frequencies with an expected theoretical error rate of 1 in 1000. 
     Analyses of base editing processivity were performed using a custom python script. This program trims sequencing reads to the 20 nucleotide protospacer sequence as determined by a perfect match for the 7 nucleotide sequences that should flank the target site. These targets were then consolidated and sorted by abundance to assess the frequency of base editing products. 
     Bioinformatic analysis of the ClinVar database of human disease-associated mutations was performed in a manner similar to that previously described but with small adjustments 1 . These adjustments enable the identification of targets with PAMs of customizable length and sequence. In addition, this improved script includes a priority ranking of target C positions (C5&gt;C6&gt;C7&gt;C8≈C4), thus enabling the identification of target sites in which the on-target C is cither the only cytosine within the window or is placed at a position with higher predicted editing efficiency than any off-target C within the editing window. 
     References for Example 12 
     
         
         1 Komor, A. C. et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.  Nature  533, 420-424 (2016). 
         2 Sander, J. D. &amp; Joung. J. K. CRISPR-Cas systems for editing, regulating and targeting genomes.  Nature biotechnology  32, 347-355 (2014). 
         3 Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems.  Science  339, 819-823(2013). 
         4 Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system.  Nat. Protocols  8, 2281-2308 (2013). 
         5 Landrum, M. J. et al. ClinVar: public archive of interpretations of clinically relevant variants.  Nucleic Acids Res.  44, D862-D868 (2015). 
         6 Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.  Science  353, aaf8729-1-8 (2016). 
         7 Ma, Y. et al. Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells.  Nat. Methods  doi:10.1038/nmeth.4027 (2016). 
         8 Jiang, F. et al. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage.  Science  351, 867-71 (2016). 
         9 Ran, F. A. et al. In vivo genome editing using  Staphylococcus aureus  Cas9.  Nature  520, 186-191 (2015). 
         10 Zhang, Y. et al. Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells.  Sci. Rep.  4, (2014). 
         11 Kleinsliver, B. P. et. al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities.  Nature  523, 481-485 (2015). 
         12 Kleinsliver, B. P. et. al. Broadening the targeting range of  Staphylococcus aureus  CRISPR-Cas9 by modifying PAM recognition.  Nat. Biotechnol.  33, 1293-1298 (2015). 
         13 Holden, L. G. et al. Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implications.  Nature  452, 121-124 (2008). 
         14 Chen, K.-M. et al. Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G.  Nature  452, 116-119 (2008). 
         15 Harris, R. S., Petersen-Mahrt, S. K. &amp; Neuberger, M. S. RNA Editing Enzyme APOBEC1 and Some of Its Homologs Can Act as DNA Mutators.  Molecular Cell  10, 1247-1253(2002). 
         16 Zetsche, B. et al. Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System.  Cell  163, 759-771 (2015). 
       
    
     EXAMPLE  13   
     Using improved transfection procedures and better plasmids, biological replicates (n=3) were used to install the four HF mutations into the Cas9 portion of BE3. The muations do not significantly effect on-targeting editing with plasmid delivery ( FIG. 99 ). At the tested concentration, BE3 protein delivery works; however, the on-target editing is lower than for plasmid delivery ( FIG. 100 ). Protein delivery of BE3 with the HF mutations installed reduces on-targeting editing efficiency but still yields some edited cells ( FIG. 101 ). 
     Both lipofection and installing HF mutations were shown to decrease off-target deamination events. For the four sites shown in  FIG. 102 , the off-target sitest (OT) with the highest GUIDE-Seq reads and deamination events were assayed (Komor el al.,  Nature,  2016). The specificity ratio was calculated by dividing the off-target editing by the on-target editing at the closest corresponding C. In cases where off-target editing was not detectable, the ratio was set to 100. Thus, a higher specificity ratio indicates a more specific construct. BE3 plasmid delivery showed much higher off-target/on-target editing than protein delivery of BE3, plasmid delivery of HF-BE3, or protein delivery of HF-BE3 ( FIGS. 102 and 105 ). 
     Purified proteins HF-BE3 and BE3 were analyzed in vitro for their capabilities to convert C to T residues at different positions in the spacer with the most permissive motif. Both BE3 and HF-BE3 proteins were found to have the same “window” for base editing ( FIGS. 103 and 104 ). 
     A list of the disease targets is given in Table 9. The base to be edited in Table 9 is indicated in bold and underlined. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Base Editor Disease Targets 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 GENE 
                 DISEASE 
                 SPACER 
                 PAM 
                 EDITOR 
                 DEFECT 
                 CELL 
               
               
                   
               
               
                 RB1 
                 RETINOBLASTOMA 
                 AAT   C   TAGTAAA 
                 AAAAGT 
                 SAKKH-BE3 
                 SPLICING 
                 J82 
               
               
                   
                   
                 TAAATTGATGT 
                   
                   
                 IMPAIRMENT 
                   
               
               
                   
               
               
                 PTEN 
                 CANCER 
                 GACCAA   C   GGCT 
                 TGA 
                 VQR-BE3 
                 W111R 
                 MC116 
               
               
                   
                   
                 AAGTGAAGA 
                   
                   
                   
                   
               
               
                   
               
               
                 PIK3CA 
                 CANCER 
                 TC   C   TTTCTTCA 
                 ACTGGT 
                 SAKKH-BE3 
                 K111R 
                 CRL-5853 
               
               
                   
                   
                 CGGTTGCCT 
                   
                   
                   
                   
               
               
                   
               
               
                 PIK3CA 
                 CANCER 
                 CTC   C   TGCTCAG 
                 AGA 
                 VQR-BE3 
                 Q546R 
                 CRL-2505 
               
               
                   
                   
                 TGATTTCAG 
                   
                   
                   
                   
               
               
                   
               
               
                 TP53 
                 CANCER 
                 TGT   C   ACACATG 
                 TGG 
                 YEE-BE3 
                 N239D 
                 SNU475 
               
               
                   
                   
                 TAGTTGTAG 
                   
                   
                   
                   
               
               
                   
               
               
                 HRAS 
                 CANCER 
                 CCTCC   C   GGCCG 
                 AGG 
                 YEE-BE3 
                 Q61R 
                 MC/CAR 
               
               
                   
                   
                 GCGGTATCC 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Exemplary diseases that may be treated using base editors.  
               
            
           
           
               
               
               
               
               
            
               
                   
                 gene 
                 Base 
                   
                   
               
               
                 Disease target 
                 symbol 
                 changed 
                 sgRNA (PAM) 
                 Base editor 
               
               
                   
               
               
                 Prion disease 
                 PRNP 
                 R37* 
                 GGCAGC C GATACCCGGGGCA(GGG) 
                 BE3 
               
               
                   
                   
                   
                 GGGCAGC C GATACCCGGGGC(AGG) 
                   
               
               
                   
               
               
                 Pendred syndrome 
                 Slc26a4 
                 c.919-2A&gt;G 
                 TTATTGTC C GAAATAAAAGA(AGA) 
                 BE3 
               
               
                   
                   
                   
                 ATTGTC C GAAATAAAAGAAG(AGG) 
                 (VQR SaCas9) 
               
               
                   
                   
                   
                 TTGTC C GAAATAAAAGAAGA(GGA) 
                   
               
               
                   
                   
                   
                 GTC C GAAATAAAAGAAGAGGAAAA(AAT) 
                   
               
               
                   
                   
                   
                 GTC C GAAATAAAAGAAGAGGAAAAA(ATT) 
                   
               
               
                   
               
               
                 Congenital  
                 Tmc1 
                 c.545A&gt;G 
                 CAGGAAG C ACGAGGCCACTG(AGG) 
                 BE3 
               
               
                 deafness 
                   
                   
                 AACAGGAAG C ACGAGGCCAC(TGA) 
                 YE-BE3 
               
               
                   
                   
                   
                 AGGAAG C ACGAGGCCACTGA(GGA) 
                 YEE-BE3 
               
               
                   
               
               
                 Acquired  
                 SNHL 
                 S33F 
                 TTGGATT C TGGAATCCATTC(TGG) 
                 BE3 
               
               
                 deafness 
                   
                   
                   
                   
               
               
                   
               
               
                 Alzheimer&#39;s  
                 APP 
                 A673T 
                 TCTGCATCCATCTTCACTTC(AGA) 
                 BE3 VQR 
               
               
                 Disease 
                   
                   
                   
                   
               
               
                   
               
               
                 Niemann-Pick  
                 NPC1 
                 I1061T 
                 CTTACAGCCAGTAATGTCAC(CGA) 
                 BE3 VQR 
               
               
                 Disease Type C 
               
               
                   
               
               
                 The protospacer and PAM sequences are shown in the sgRNA (PAM) column. 
               
               
                 The PAM sequence is shown in parentheses and with the base to be edited indicated by underlining. 
               
            
           
         
       
     
       
     
       
         
           
               
             
               
                 Lengthy table referenced here 
               
             
            
               
                 US20220220462A1-20220714-T00002 
               
               
                 Please refer to the end of the specification for access instructions. 
               
            
           
         
       
     
       
     
       
         
           
               
             
               
                 Lengthy table referenced here 
               
             
            
               
                 US20220220462A1-20220714-T00003 
               
               
                 Please refer to the end of the specification for access instructions. 
               
            
           
         
       
     
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     EQUIVALENTS AND SCOPE 
     Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims. 
     Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context. The disclosure of a group that includes “or” between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed. 
     It is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitation, element, clause, or descriptive term, from one or more of the claims or from one or more relevant portion of the description, is introduced into another claim. For example, a claim that is dependent on another claim can be modified to include one or more of the limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of making or using the composition according to any of the methods of making or using disclosed herein or according to methods known in the art, if any, are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. 
     Where elements are presented as lists, e.g., in Markush group format, it is to be understood that every possible subgroup of the elements is also disclosed, and that any element or subgroup of elements can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where an embodiment, product, or method is referred to as comprising particular elements, features, or steps, embodiments, products, or methods that consist, or consist essentially of, such elements, features, or steps, are provided as well. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed. 
     Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in some embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For purposes of brevity, the values in each range have not been individually spelled out herein, but it will be understood that each of these values is provided herein and may be specifically claimed or disclaimed. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range. 
     In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein. 
       
     
       
         
           
               
             
               
                 LENGTHY TABLES 
               
             
            
               
                 The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (https://seqdata.uspto.gov/?pageRequest=docDetail&amp;DocID=US20220220462A1). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).