ADENINE BASE EDITORS WITH REDUCED OFF-TARGET EFFECTS

RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application, U.S.S.N. 62/835,490, filed April 17, 2019, which is incorporated by reference herein.

FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant numbers AI142756, HG009490, EB022376, and GM118062 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE DISCLOSURE

[0003] Base editors enable the precise installation of targeted point mutations in genomic DNA without creating double- stranded DNA breaks (DSBs) (1-3). Adenine base editors (ABEs) convert a target A·T base pair to a G*C base pair (1). Because the mutation of G*C base pairs to A·T base pairs is the primary form of de novo mutation (4), ABEs have the potential to correct almost half of known human pathogenic point mutations (5). The adenine base editor, ABE7.10, can perform remarkably clean and efficient A*T-to-G*C conversion in DNA with very low levels of undesirable byproducts such as small insertions or deletions (indels) in cultured cells, adult mice, plants, and other organisms (1, 6-10).

[0004] Off-target base editing can arise from guide RNA-dependent or guide RNA- independent editing events (1, 3). The former results from RNA-guided binding of the Cas9 domain to DNA sites that are similar, but not identical, to the target DNA locus (7, 20-23).

[0005] Separately, adenine base editors may induce off-target editing of cellular RNA.

Unintended editing of cellular RNA could introduce deleterious effects on the function of translated proteins, and potential associated cytotoxicity. There is an unrecognized need in the art for adenine base editors that have reduced RNA editing activity while retaining high DNA editing efficiency.

SUMMARY OF THE DISCLOSURE

[0006] The discovery and widespread implementation of the CRISPR/Cas system has dramatically expanded the toolbox for genome engineering and has revolutionized the future prospects of basic biological research, data storage in living systems, agricultural science, and medicine. The recent development of adenine base editors by fusion of a deaminase to Cas9 enables guide RNA (gRNA)-targeted single nucleotide deamination for A:T base pair conversion to G:C using adenine base editors within a specific target window. Base editing has been broadly demonstrated with high efficiency in a range of species, including human zygotes.

[0007] Various engineered base editors with improved DNA editing efficiencies have been developed. Reference is made to Komor, A.C. et al, Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity, Sci Adv 3 (2017); Rees, H.A. et al., Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery, Nat. Commun. 8, 15790 (2017); U.S. Patent Publication No. 2018/0073012, published March 15, 2018; U.S. Patent Publication No. 2017/0121693, published May 4, 2017; International Publication No. WO 2017/070633, published April 27, 2017; and U.S. Patent Publication No. 2015/0166980, published June 18, 2015, U.S. Patent No. 9,840,699, issued December 12, 2017; and U.S. Patent No. 10,077,453, issued September 18, 2018, each of which are incorporated herein in their entireties. Base editors (BEs) may be fusions of a Cas

(“CRIS PR-associated”) domain and a nucleobase (or“base”) modification domain (e.g., a natural or evolved deaminase, such as an adenosine deaminase domain). In some cases, base editors may also include proteins or domains that affect cellular DNA repair processes to increase the efficiency and/or stability of the resulting single-nucleotide change.

[0008] Base editors reported to date contain a catalytically impaired Cas9 domain fused to a nucleobase modification domain. The Cas9 domain directs the nucleobase modification domain to directly convert one base to another at a guide RNA-programmed target site. Two classes of base editors have been developed to date: Cytosine base editors (CBEs), which convert C*G to T·A, and adenine base editors (ABEs), which convert A·T to G*C .

Collectively, CBEs and ABEs enable the correction of all four types of transition mutations (C to T, G to A, A to G, and T to C). As half of known disease-associated gene variants are point mutations, and transition mutations account for -60% of known pathogenic point mutations, base editors are being widely used to study and treat genetic diseases in a variety of cell types and organisms, including animal models of human genetic diseases.

[0009] ABEs are especially useful for the study and correction of pathogenic alleles, as nearly half of pathogenic point mutations in principle can be corrected by converting an A·T base pair to a G*C base pair. Many of the ABEs reported to date include a single polypeptide chain containing a heterodimer of a wild-type E. coli TadA monomer (ecTadA, or TadA) that plays a structural role during base editing and a laboratory-evolved E. coli TadA monomer TadA7.10 (also referred to herein as“TadA*”) that catalyzes deoxyadenosine deamination, and a Cas9 (D10A) nickase. Wild type E. coli TadA acts as a homodimer to deaminate an adenosine located in a tRNA anticodon loop, generating inosine (I). Although early ABE variants required a heterodimeric TadA containing an N-terminal wild-type TadA monomer for maximal activity, Joung el al. showed that later ABE variants have comparable activity with and without the wild-type TadA monomer.

[0010] The present disclosure is based, at least in part, on the mutagenesis of existing adenine base editors to provide variant ABEs that have reduced off-target effects while retaining high DNA editing efficiency. The adenosine deaminase domain of the ABE7.10 base editor comprises a heterodimer of two adenosine deaminases, one of which is TadA7.10, a deoxy adenosine deaminase that was previously evolved from an E. coli tRNA adenosine deaminase to act on single- stranded DNA. (The other deaminase of the heterodimer is a wild-type ecTadA.) TadA7.10 is also comprised within the deaminase domain of ABEmax, which is a variant of ABE7.10 that has been codon-optimized for expression in human cells. TadA7.10 comprises the following substitutions in ecTadA: W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, R152P, E155V, I156F, and K157N.

Reference is made to International Publication No. WO 2018/027078, published August 2, 2018; International Publication No. WO 2019/079347 published April 25, 2019; International Publication No. WO 2019/226593, published November 28, 2019; U.S. Patent Publication No. 2018/0073012, published March 15, 2018, which issued as U.S. Patent No. 10,113,163, on October 30, 2018; and U.S. Patent Publication No. 2017/0121693, published May 4, 2017, which issued as U.S. Patent No. 10,167,457 on January 1, 2019.

[0011] Many ABEs reported to date comprise single polypeptide chains containing three fused protein components: a wild-type E. coli TadA monomer that plays a structural role during base editing, a laboratory-evolved E. coli TadA monomer TadA7.10 that catalyzes deoxy adenosine deamination, and a Cas9 (D10A) nickase (1, 3) (see FIGs. 1A, 13A). E. coli TadA natively acts as a homodimer to deaminate an adenine located in a tRNA anticodon loop (25), generating inosine (I) (the adenosine is deaminated to a hypoxanthine).

[0012] It was hypothesized that the wild-type TadA monomer, which natively acts on RNA but has strict sequence requirements (25, 26), and/or the evolved TadA7.10 monomer, which was evolved to accept ssDNA as a substrate and to have broad sequence compatibility, may be able to catalyze the deamination of cellular RNA (1, 3) (see FIG. IB). While no substantial ABE7.10-mediated adenosine-to-inosine (A-to-I) editing was previously observed in HEK293T cells among a handful of abundant transcripts sequenced at modest depth (1), the association of elevated endogenous A-to-I editing in the transcriptome with disease (27) warranted a more comprehensive examination of possible ABE-mediated RNA editing. In particular, recent studies have identified aberrant A-to-I editing as a mechanism by which tumors can develop a resistance to immune checkpoint blockade (28).

[0013] Guide RNA-dependent off-target base editing has been reduced through strategies including installation of mutations that increase DNA specificity into the Cas9 component of base editors, adding 5' guanosine nucleotides to the sgRNA, or delivery of the base editor as a ribonucleoprotein complex (RNP) (19, 22, 24). Guide RNA-independent off-target editing can arise from binding of the deaminase domain of a base editor to C or A bases in a Cas9- independent manner (3). Recent studies characterized guide RNA-independent off-target

DNA editing activity of BE3, the original CBE, in mouse embryos (18) and in rice (17). In contrast with BE3, ABE7.10 in these studies did not result in detectable guide RNA- independent off-target DNA mutations (17, 18). The efficiency of base editors was recently improved through codon- and nuclear localization sequence-optimization to generate

ABEmax (15). ABEmax was shown to generate low but detectable levels of widespread adenosine-to-inosine editing in cellular RNAs. The present disclosure is aimed to satisfy a heretofore unrecognized need in the art for the reduction of off-target editing of RNA induced by the deaminase domains of ABEmax and other current adenine base editors.

[0014] To address the above-described deficiency in the art, targeted mutagenesis guided by an analysis of deaminase structure was applied to wild-type ecTadA and TadA7.10 deaminases to minimize the ribonucleotide deaminating activity of the domain comprising both deaminases, i.e., the adenosine deaminating activity in RNA. TadA(V106W), an exemplary variant emerging from these mutagenesis experiments, contains a single substitution relative to TadA7.10 at residue 106. TadA(E59A), another exemplary variant emerging from these mutagenesis experiments, contains a single substitution of ecTadA at residue 59. Accordingly, the present disclosure provides TadA(V106W), TadA(E59A), and other TadA7.10 deaminase variants. Adenosine deaminase domains comprising one or more of these variants exhibit reduced off-target effects, such as reduced RNA deamination activity. The present disclosure also provides improved adenine base editors that comprise an adenosine deaminase domain comprising a TadA variant, such as a TadA(E59A), and/or a

TadA7.10 variant, such as TadA(V106W). Accordingly, the disclosure provides adenine base editors that are variants of ABE7.10, or ABEmax. The disclosure also provides editing methods, kits and compositions that make use of these ABEmax variants, which minimize the induction of RNA editing in cells.

[0015] Accordingly, in some aspects, the present disclosure provides adenine base editors that comprise fusion proteins comprising a nucleic acid DNA binding protein (or napDNAbp) domain and an adenosine deaminase domain. The napDNAbp domain may comprise a Cas9 protein, or a variant thereof, e.g., a Cas9 nickase. The adenosine deaminase domain may comprise one or more adenosine deaminases. In certain embodiments, the adenosine deaminase domain comprises a dimer of a first and second adenosine deaminase. The dimer may be a heterodimer, comprising a first adenosine deaminase that is different from a second adenosine deaminase. The first adenosine deaminase may be positioned N-terminal to the second adenosine deaminase. In various embodiments, the one or more adenosine deaminases are connected by a linker (e.g., a peptide linker).

[0016] In various embodiments, the first adenosine deaminase is an E. coli TadA (ecTadA) or a variant thereof. In some embodiments, the first adenosine deaminase is an ecTadA having an amino acid substitution at E59 of ecTadA. For instance, this substitution may be an E59A or an E59Q substitution. In some embodiments, the amino acid substitution at residue 59 inactivates the catalytic region of the adenosine deaminase.

[0017] In various embodiments, the second adenosine deaminase is an ecTadA or variant thereof. In some embodiments, the second adenosine deaminase is an ecTadA having some or all of the amino acid substitutions comprised within the deaminase TadA7.10 of the adenine base editor ABEmax. The second adenosine deaminase may comprise a variant of TadA7.10 that comprises one or more amino acid substitutions relative to the amino acid sequence of TadA7.10. In certain embodiments, the deaminase comprises a TadA7.10 variant comprising an amino acid substitution at V 106 of TadA7.10. For instance, this substitution may comprise a V106W, V106F, V106Q, or a V106M substitution in the amino acid sequence of TadA7.10. In other embodiments, the deaminase comprises an TadA7.10 variant comprising an amino acid substitution at N 108 of TadA7.10. For instance, this substitution may comprise an N108W of TadA7.10. In certain embodiments, the deaminase comprises a TadA7.10 variant comprising an amino acid substitution at R47 of TadA7.10.

For instance, this substitution may comprise an R46W, R46F, R46Q, or an R46M of

TadA7.10. In certain embodiments, the second adenosine deaminase comprises two or more amino acid substitutions selected from V106W, V106F, V106Q, or V106M, N108, and R46W, R46F, R46Q, or R46M of TadA7.10.

[0018] The adenosine deaminase domains provided herein (e.g., a heterodimer of adenosine deaminases connected by a linker) comprises a first adenosine deaminase comprising an ecTadA having an amino acid substitution at E59 of ecTadA, and a second adenosine deaminase comprises an TadA7.10 variant comprising an amino acid substitution at V106 of TadA7.10. In certain embodiments, the adenosine deaminase domain comprises a first adenosine deaminase comprising an E59A substitution, and a second deaminase comprising a V106W substitution. In certain embodiments, the adenosine deaminase domain comprises a first adenosine deaminase comprising an E59A substitution, and a second deaminase comprising an N108W substitution. In certain embodiments, the adenosine deaminase domain comprises a first adenosine deaminase comprising an E59A substitution, and a second deaminase comprising a V106W substitution and/or a N108W substitution and/or an

R47Q substitution.

[0019] In some embodiments, the adenine base editors provided herein may be capable of preserving DNA editing efficiency, and in some embodiments demonstrate improved DNA editing efficiencies, relative to existing adenine base editors, such as ABE7.10. In some embodiments, the ABEs described herein exhibit reduced off-target editing effects while retaining high on-target editing efficiencies. In certain embodiments, the disclosed ABEs exhibit reduced Cas9-independent off-target editing effects while retaining high on-target editing efficiencies. In certain embodiments, the disclosed ABEs exhibit reduced off-target editing effects in cellular mRNA.

[0020] In some embodiments, the adenine base editors provided herein are capable of limiting formation of indels in a DNA substrate. In some embodiments, the ABEs provided herein have an expanded target window for editing a DNA substrate than canonical ABEs (e.g., a target window that corresponds to protospacer positions 4-11, 8-14, or 9-14 of the target sequence, wherein protospacer position 0 corresponds to the position of the transcription start site of the target gene). In some embodiments, the adenosine deaminases disclosed herein may be compatible with a variety of Cas homologs, including small-sized, circularly permuted, and evolved Cas homologs.

[0021] The present specification further provides methods of DNA editing that make use of the improved adenine base editors. The methods may induce (or yield, provide or cause) an average adenosine (A) to inosine (I) (A-to-I) editing frequency in cellular mRNA transcripts of 0.3% or less, as measured by high throughput screening. In some embodiments, the methods induce (or provide or cause) an average adenosine (A) to inosine (I) (A-to-I) editing frequency across the mRNA transcriptome of a human cell (e.g. an HEK293 cell) of about 0.2% or less.

[0022] In some aspects, the present disclosure provides compositions comprising the adenine base editors with reduced off-target effects, such as reduced RNA editing effects, as described herein, e.g., fusion proteins comprising an nCas9 domain and an adenosine deaminase domain (e.g., a heterodimer of a first and second adenosine deaminase), and one or more guide RNAs, e.g., a single-guide RNA (“sgRNA”).

[0023] In some aspects, the present disclosure provides for nucleic acid molecules encoding and/or expressing the adenine base editors as described herein, and the adenosine deaminase domains thereof, as well as expression vectors or constructs for expressing the adenine base editors described herein and a gRNA, host cells comprising said nucleic acid molecules and expression vectors, and one or more gRNAs, and compositions for delivering and/or administering nucleic acid-based embodiments described herein. The nucleic acid sequences may be codon-optimized for expression in the cells of any organism of interest. In certain embodiments, the nucleic acid sequence is codon-optimized for expression in human cells.

In other embodiments, cells containing such nucleic acid molecules and expression vectors are provided.

[0024] The present specification further provides complexes comprising the adenine base editors described herein and a gRNA bound to the Cas9 domain of the fusion protein, such as a single guide RNA. The guide RNA may be 15-100 nucleotides in length and comprise a sequence of at least 10, at least 15, or at least 20 contiguous nucleotides that is

complementary to a target nucleotide sequence.

[0025] In other aspects, the disclosure provides kits for expressing and/or transducing host cells with an expression construct encoding the fusion protein and gRNA. It further provides kits for administration of expressed fusion protein and expressed gRNA molecules to a host cell. The disclosure further provides host cells stably or transiently expressing the fusion protein and gRNA, or a complex thereof.

[0026] Methods are also provided for editing a target nucleic acid molecule, e.g., a single nucleobase within a genome, with an adenine base editor described herein, that generate (or cause) reduced off-target effects, e.g. editing of cellular mRNA. Such methods involve transducing (e.g., via transfection) cells with a plurality of complexes each comprising a fusion protein (e.g., a fusion protein comprising a Cas9 nickase (nCas9) domain and an adenosine deaminase domain) and a gRNA molecule. In certain embodiments, the methods involve the transfection of nucleic acid constructs (e.g., plasmids) that each (or together) encode the components of a complex of fusion protein and gRNA molecule. In other embodiments, the methods disclosed herein involve the introduction into cells of a complex comprising a fusion protein and gRNA molecule that has been expressed and cloned outside of these cells.

[0027] In some embodiments, the disclosed editing methods result in an actual or average off-target DNA editing frequency of about 2.0% or less. In some embodiments, the editing method results in less than 5% indel formation in the nucleic acid substrate (e.g. a DNA substrate).

[0028] In some embodiments, methods of treatment using the disclosed base editors are provided. The methods described herein may comprise treating a subject having or at risk of developing a disease, disorder, or condition, comprising administering to the subject a fusion protein as described herein, a polynucleotide as described herein, a vector as described herein, or a pharmaceutical composition as described herein.

[0029] By decoupling DNA and RNA editing activities, the novel adenosine deaminase variants and ABE7.10 variants provided herein increase the precision of adenine base editing by minimizing both RNA and DNA off-target editing activity. These variants may be especially useful for applications that demand minimal RNA editing and high DNA specificity.

[0030] The details of one or more embodiments of the disclosure are set forth herein. Other features, objects, and advantages of the disclosure will be apparent from the Detailed

Description, Examples, Figures, and Claims. References cited in this application are incorporated herein by reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0032] FIGs. 1A to II show RNA and DNA editing activity of each TadA monomer in ABEmax. FIG. 1A illustrates that ABEmax (shown as a schematic model) comprises three proteins fused in a single chain: TadA-TadA*-Cas9(D10A). FIG. IB illustrates the two TadA monomers (shown as a schematic model) in ABEmax. The schematic models in FIG. 1A and FIG. IB are generated from independently solved Cas9 (pdb id: 4un3) and E. coli TadA (pdb id: lz3a) structures, as the structure of ABE has not yet been solved. FIG. 1C shows the average A-to-I conversion frequency in three mRNA transcripts from each treatment analyzed by HTS. FIG. ID shows the number of adenosines within a 220- to 240- nucleotide region of the indicated mRNA that are converted to inosine (read as a G after cDNA synthesis and DNA sequencing) at a detectable level (>0.1%). Cas9 (D10A) controls show the number of adenosines that are edited by endogenous cellular adenosine deaminases. The amplified regions of RSF1D1, CTNNB1 and IP90 mRNA have 46, 59, and 77 sequenced adenosines, respectively. FIG. IE shows DNA base editing at seven genomic loci from ABEmax or by ABEmax with mutations at catalytic Glu 59 in TadA or TadA* (TadA7.10). The protospacer position of the target A and the sequence context of the A are shown. FIG.

IF shows RNA editing frequencies at various adenosines within the RSF1D1 amplicon after treatment with the indicated base editors. The adenosine homologous to TadA’s native substrate is at position 152 within the amplicon. FIG. 1G shows that on-target DNA base editing with the LDLR sgRNA leads to a U-to-C edit in the LDLR mRNA in the

transcriptome-wide RNA-seq data. Alignments were visualized in the Integrated Genomics

Viewer (IGV), and aligned to hg38. FIG. 1H illustrates transcriptome-wide RNA-Seq analysis showing the number of high confidence (Phred quality score > 20, see Methods) A- to-I variant calls after treatment with the indicated base editors. The dotted line represents the number of A-to-I conversions in the transcriptome from endogenous deaminase activity as measured in the Cas9 (D10A) control samples. FIG. II shows the average frequency (%) of

A-to-I RNA editing across all transcripts. For FIGs. 1A-1F, data are shown as individual data points and mean ± s.d. for n=3 independent biological replicates performed on different days.

For FIGs. 1H and II, data are shown as mean ± s.e.m. The alignment was generated by combining reads from three independent biological replicates, performed on different days.

[0033] FIGs. 2A to 21 show the design and testing of ABE7.10 variants (or ABEmax variants) with reduced RNA editing activity. Views of the structure of S. aureus TadA bound to a minimized version of its native substrate (tRNA ^Ar82 ) (PDB: 2B3J) (25), showing the residues homologous to Arg 47 (FIG. 2A), Asp 108 (FIG. 2B), and Ala 106 (FIG. 2C) in E. coli TadA. Asp 108 is mutated to Asn 108 in the evolved TadA*, while Ala 106 is mutated to

Val 106 in TadA* (1). FIG. 2D shows DNA base editing at seven genomic loci from

ABEmax or ABEmax mutants. FIG. 2E shows the number of adenosines converted to inosine at a detectable level (>0.1%) within a 220- to 240-nt region of the indicated mRNA by

ABEmax or ABEmax mutants. The amplified regions of RSL1D1, CTNNB1 and IP90 mRNA have 46, 59, and 77 sequenced adenosines, respectively. The Cas9(D10A) controls show the number of adenosines that are edited due to endogenous A-to-I editing activity.

FIG. 2F shows average A-to-I RNA editing frequencies by ABEmax or ABEmax mutants among 46 adenosines in RSL1D1, 59 in CTNNB1, and 77 in IP90 mRNA transcripts. FIG.

2G shows that on-target DNA base editing with the LDLR sgRNA leads to a U-to-C edit in the LDLR mRNA in the transcriptome-wide RNA-seq data. Alignments were visualized in the Integrated Genomics Viewer (IGV), and aligned to hg38. FIG. 2H illustrates

transcriptome-wide RNA-Seq analysis showing the number of high confidence (Phred quality score > 20, see Methods) A-to-I variant calls after treatment with the indicated base editors.

The dotted line represents the number of A-to-I conversions in the transcriptome from endogenous deaminase activity as measured in the Cas9(D10A) control samples. FIG. 21 shows the average frequency (%) of A-to-I RNA editing across all transcripts. For FIGs. 2C-

2F, data are shown as individual data points and mean ± s.d. for n=3 independent biological replicates performed on different days. For FIGs. 2H and 21, data are shown as mean ± s.e.m. The alignment was generated by combining reads from three independent biological replicates, performed on different days.

[0034] FIGs. 3A to 3C show analysis of A-to-I RNA edits found in transcriptome-wide RNA sequencing. FIG. 3A shows classification of the position in which an A-to-I RNA edit was found.“5 kb downstream” refers to mutations that occur within 5 kb downstream of a coding gene and“5 kb upstream” refers to mutations that occur within the region 5 kb upstream of a coding gene. FIG. 3B illustrates that for edits in protein coding regions of mRNAs, edits were classified into synonymous or non-synonymous mutations. FIG. 3C shows that for non- synonymous A-to-I edits in protein-coding regions of RNA, SIFT was used to predict the effect on protein function for these edits. High- or low-confidence calls (indicated in parentheses in the figure) were made according to the standard parameters of the prediction software (see Methods).

[0035] FIGs. 4A to 4D show indel frequencies associated with ABEmax and engineered ABEmax mutants. FIG. 4A shows catalytically disabled ABE7.10 variants. FIG. 4B shows ABEmax(TadA E59A) variants with mutations at Arg 47 in TadA*. FIG. 4C shows

ABEmax(TadA E59A) variants with mutations at Asn 108 in TadA*. FIG. 4D shows ABEmax(TadA E59A) variants with mutations at Val 106 in TadA*. Individual data points and mean+s.d. for n=3 independent biological replicates, performed on different days.

[0036] FIGs. 5A to 5B illustrate DNA base editing and indel formation in HeLa cells from ABEmax and ABEmax mutants. To measure DNA base editing (FIG. 5A) and indel formation (FIG. 5B), HeLa cells were lipofected with the indicated base editor plasmid combined with the indicated sgRNA plasmid. After 48 hours, genomic DNA was harvested, amplified by PCR, and subjected to HTS. Data are shown as individual data points and mean+s.d. for n=3 independent biological replicates performed at different times.

[0037] FIGs. 6A to 6F illustrate DNA base editing, indel formation, and RNA editing in U20S and K562 cells harvested 48 hours after nucleofection with ABEmax, ABEmax mutants, or Cas9(D10A). DNA base editing efficiencies (FIG. 6A) and indel frequencies (FIG. 6B) were measured in indicated cells 48 hours days after nucleofection by HTS. RNA from nucleofected U20S or K562 cells was harvested simultaneously with genomic DNA, and reverse transcription and HTS were used to assess the frequency of sequenced adenosines in three mRNA transcripts with measurable A-to-I conversion in U20S cells (FIG. 6C), the average frequency of A-to-I conversion in three mRNA transcripts in U20S cells (FIG. 6D), the frequency of sequenced adenosines in three mRNA transcripts with measurable A-to-I conversion in K562 cells (FIG. 6E), and the average frequency of A-to-I conversion in three mRNA transcripts in K562 cells (FIG. 6F). Data are shown as individual data points and mean+s.d. for n=3 independent biological replicates.

[0038] FIGs. 7A to 7D illustrate DNA base editing, indel formation, and RNA editing in HEK293T cells harvested 5 days after transfection with ABEmax or ABEmax mutants. DNA base editing efficiencies (FIG. 7A) and indel frequencies (FIG. 7B) were measured in HEK293T cells 5 days after transfection. RNA from transfected HEK293T cells was harvested simultaneously with genomic DNA, and reverse transcription and HTS was used to assess the frequency of sequenced adenosines with measurable A-to-I conversion (FIG. 7C) and the average frequency of A-to-I conversion in three mRNA transcripts (FIG. 7D). Data are shown as individual data points and mean+s.d. for n=3 independent biological replicates performed at different times.

[0039] FIG. 8 shows off-target DNA base editing associated with the HEK site 2 locus by ABEmax and ABEmax mutants. Off-target genomic DNA loci for the HEK site 2 sgRNA previously identified by GUIDE-Seq (31) were analyzed by HTS following treatment with the indicated ABE7.10 variants. Data are shown as individual data points and mean+s.d. for n=3 independent biological replicates, performed on different days.

[0040] FIG. 9 shows off-target DNA base editing associated with the HEK site 3 locus by ABEmax and ABEmax mutants. Off-target genomic DNA loci for the HEK site 3 sgRNA previously identified by GUIDE-Seq (31) were analyzed by HTS following treatment with the indicated ABE7.10 variants. Data are shown as individual data points and mean+s.d. for n=3 independent biological replicates, performed on different days.

[0041] FIG. 10 shows off-target DNA base editing associated with the HEK site 4 locus by ABEmax and ABE7.10 mutants. Off-target genomic DNA loci for the HEK site 4 sgRNA previously identified by GUIDE-Seq (31) were analyzed by HTS following treatment with the indicated ABE7.10 variants. Data are shown as individual data points and mean+s.d. for n=3 independent biological replicates, performed on different days.

[0042] FIGs. 11A to 11D demonstrate results of DNA base editing, indel formation, and RNA editing in HEK293T cells harvested 48 hours after transfection with ABEmax, ABEmaxAW, ABEmaxQW, or ABEmax(TadA* A106V). DNA base editing efficiencies (FIG. 11 A) and indel frequencies (FIG. 11B) were measured in HEK293T cells harvested 48 hours after transfection. RNA from transfected HEK293T cells was harvested simultaneously with genomic DNA, and reverse transcription and HTS was used to assess the frequency of sequenced adenosines with measurable A-to-I conversion (FIG. 11C) and the average frequency of A-to-I conversion in three mRNA transcripts (FIG. 1 ID). Data are shown as individual data points and mean+s.d. for n=3 independent biological replicates performed at different times.

[0043] FIG. 12 depicts A-to-I RNA editing across the transcriptome for ABEmax,

ABEmaxAW, ABEmax(TadA E59A) and Cas9(D10A). A-to-I variant calls were plotted by transcript location. Bins 1,000,000 nucleotides wide are represented by each colored band. The number of high confidence A-to-I edits per bin are plotted to show the density of A-to-I edits per bin.

[0044] FIGs. 13A to 13B show plasmid maps including the architecture of ABEmax (FIG. 13A) and ABEmaxAW (FIG. 13B).

[0045] FIG. 14 depicts an alignment of the amino acid sequences of TadA deaminases derived from various species and the consensus E. coli TadA amino acid sequence.

DEFINITIONS

[0046] 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.

[0047] As used herein, the term“adenosine deaminase domain” refers to a domain within a fusion protein comprising two or more adenosine deaminases. For instance, an adenosine deaminase domain may comprise a heterodimer of a first adenosine deaminase and a second deaminase domain, connected by a linker.

[0048]“Base editing” refers to genome editing technology that involves the conversion of a specific nucleic acid base into another at a targeted genomic locus. In certain embodiments, this can be achieved without requiring double- stranded DNA breaks (DSB), or single stranded breaks ( i.e ., nicking). To date, other genome editing techniques, including CRISPR- based systems, begin with the introduction of a DSB at a locus of interest. Subsequently, cellular DNA repair enzymes mend the break, commonly resulting in random insertions or deletions (indels) of bases at the site of the DSB. However, when the introduction or correction of a point mutation at a target locus is desired rather than stochastic disruption of the entire gene, these genome editing techniques are unsuitable, as correction rates are low ( e.g . typically 0.1% to 5%), with the major genome editing products being indels. In order to increase the efficiency of gene correction without simultaneously introducing random indels, the present inventors previously modified the CRISPR/Cas9 system to directly convert one DNA base into another without DSB formation. 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 is incorporated by reference herein. [0049] The following base editor, which effects transitions (pyrimidine to pyrimidine, or purine to purine) mutations are relevant to the methods disclosed herein.

• Adenine base editor (or“ABE”). This type of editor converts an A:T Watson- Crick nucleobase pair to a G:C Watson-Crick nucleobase pair. Because the corresponding Watson-Crick paired bases are also interchanged as a result of the conversion, this category of base editor may also be referred to as a thymine base editor (or“TBE”).

[0050] The term“base editor (BE)” as used herein, refers to an agent comprising a polypeptide that is capable of making a modification to a base ( e.g ., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the base editor is capable of deaminating a base within a nucleic acid such as a base within a DNA molecule.

In the case of an adenine base editor, the base editor is capable of deaminating an adenine (A) in DNA. Such base editors may include a nucleic acid programmable DNA binding protein (napDNAbp) fused to an adenosine deaminase. Some base editors include CRISPR-mediated fusion proteins that are utilized in the base editing methods described herein. In some embodiments, the base editor comprises a nuclease-inactive Cas9 (dCas9) fused to a deaminase which binds a nucleic acid in a guide RNA-programmed manner via the formation of an R-loop, but does not cleave the nucleic acid. For example, the dCas9 domain of the fusion protein may include a D10A and a H840A mutation (which renders Cas9 capable of cleaving only one strand of a nucleic acid duplex), as described in PCT/US2016/058344, which published as WO 2017/070632 on April 27, 2017 and is incorporated herein by reference in its entirety. The DNA cleavage domain of S. pyogenes Cas9 includes two subdomains, the HNH nuclease subdomain and the RuvCl subdomain. The HNH subdomain cleaves the strand complementary to the gRNA (the“targeted strand”, or the strand in which editing or deamination occurs), whereas the RuvCl subdomain cleaves the non

complementary strand containing the PAM sequence (the“non-edited strand”). The RuvCl mutant D10A generates a nick in the targeted strand, while the HNH mutant H840A generates a nick on the non-edited strand (see Jinek et al, Science , 337:816-821(2012); Qi el al, Cell. 28; 152(5): 1173-83 (2013)).

[0051] The term“base editor” encompasses the CRISPR-mediated fusion proteins utilized in the multiplexed base editing methods described herein as well as any base editor known or described in the art at the time of this filing or developed in the future. Reference is made to Rees & Liu, Base editing: precision chemistry on the genome and transcriptome of living cells, Nat. Rev. Genet. 2018;19(12):770-788; as well as U.S. Patent Publication No.

2018/0073012, published March 15, 2018, which issued as U.S. Patent No. 10,113,163, on October 30, 2018; U.S. Patent Publication No. 2017/0121693, published May 4, 2017, which issued as U.S. Patent No. 10,167,457 on January 1, 2019; International Publication No. WO

2017/070633, published April 27, 2017; U.S. Patent Publication No. 2015/0166980, published June 18, 2015; International Publication No. WO 2017/070633, published April 27,

2017; International Publication No. WO 2018/027078, published August 2, 2018;

International Application No PCT/US2018/056146, filed October 16, 2018, which published as Publication No. WO 2019/079347 on April 25, 2019; International Application No

PCT/US2019/033848, filed May 23, 2019, which published as Publication No. WO

2019/226593 on November 28, 2019; U.S. Patent Publication No. 2015/0166980, published

June 18, 2015; U.S. Patent No. 9,840,699, issued December 12, 2017; U.S. Patent No.

10,077,453, issued September 18, 2018; International Publication No. WO 2019/023680, published January 31, 2019; International Publication No. WO 2018/0176009, published

September 27, 2018; International Application No. PCT/US2019/47996, filed August 23,

2019; International Application No. PCT/US2019/049793, filed September 5, 2019;

International Application No. PCT/US2019/61685, filed November 15, 2019; International

Application No. PCT/US2019/57956, filed October 24, 2019, the contents of each of which are incorporated herein by reference in their entireties.

[0052] The term“Cas9” or“Cas9 nuclease” or“Cas9 domain” refers to a CRISPR-associated protein 9, or variant thereof, and embraces any naturally occurring Cas9 from any organism, any naturally-occurring Cas9 equivalent or fragment thereof, any Cas9 homolog, ortholog, or paralog from any organism, and any variant of a Cas9, naturally-occurring or engineered.

The term Cas9 is not meant to be particularly limiting and may be referred to as a“Cas9 or variant thereof.” Exemplary Cas9 proteins are described herein and also described in the art. The present disclosure is unlimited with regard to the particular Cas9 that is employed in the CRISPR-mediated fusion proteins utilized in the disclosure.

[0053] 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. Cas9 variants include functional fragments of Cas9. 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 a 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 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.

[0054] As used herein, the term“dCas9” refers to a nuclease-inactive Cas9 or nuclease-dead Cas9, or a variant thereof, and embraces any naturally occurring dCas9 from any organism, any naturally-occurring dCas9 equivalent or functional fragment thereof, any dCas9 homolog, ortholog, or paralog from any organism, and any variant of a dCas9, naturally- occurring or engineered. The term dCas9 is not meant to be particularly limiting and may be referred to as a“dCas9 or variant thereof.” Exemplary dCas9 proteins and method for making dCas9 proteins are further described herein and/or are described in the art and are incorporated herein by reference. Any suitable mutation which inactivates both Cas9 endonucleases, such as D10A and H840A mutations in the wild-type S. pyogenes Cas9 amino acid sequence, or D10A and N580A mutations in the wild-type S. aureus Cas9 amino acid sequence, may be used to form the dCas9.

[0055] As used herein, the term“nCas9” or“Cas9 nickase” refers to a Cas9 or a variant thereof, which cleaves or nicks only one of the strands of a target cut site thereby introducing a nick in a double strand DNA molecule rather than creating a double strand break. This can be achieved by introducing appropriate mutations in a wild-type Cas9 which inactivates one of the two endonuclease activities of the Cas9. Any suitable mutation which inactivates one Cas9 endonuclease activity but leaves the other intact is contemplated, such as one of D10A or H840A mutations in the wild-type S. pyogenes Cas9 amino acid sequence, or a D10A mutation in the wild-type S. aureus Cas9 amino acid sequence, may be used to form the nCas9.

[0056]“CRISPR” is a family of DNA sequences (i.e., CRISPR clusters) in bacteria and archaea that represent snippets of prior infections by a virus that have invaded the prokaryote. The snippets of DNA are used by the prokaryotic cell to detect and destroy DNA from subsequent attacks by similar viruses and effectively constitute, along with an array of CRISPR-associated proteins (including Cas9 and homologs thereof) and CRISPR-associated RNA, a prokaryotic immune defense system. In nature, CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In certain types of CRISPR systems (e.g., type II

CRISPR systems), correct processing of pre-crRNA requires a trans-encoded small RNA

(tracrRNA), endogenous ribonuclease 3 (me) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently,

Cas9/crRNA/tracrRNA endonucleolytic ally cleaves linear or circular nucleic acid target complementary to the RNA. Specifically, 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“gRNA”) may be engineered so as to incorporate embodiments of both the crRNA and tracrRNA into a single RNA species— the guide RNA. See, e.g., Jinek M., el al., Science 337:816-821(2012), the entire contents of which is herein 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. CRISPR biology, as well as Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g.,“Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti J.J., el al, Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001);“CRISPR RNA maturation by trans- encoded small RNA and host factor RNase III.” Deltcheva E., el al, Nature 471:602-

607(2011); and“A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., et al, Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes, S. thermophiles, C. ulcerans, S. diphtheria, S.

syrphidicola, P. intermedia, S. taiwanense, S. iniae, B. baltica, P. torquis, S. thermophilus , L. innocua, C. jejuni and N. meningitidis. 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.

[0057] The term“deaminase” or“deaminase domain” refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenine or adenosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenosine in deoxyribonucleic acid (DNA) to inosine (and thus the conversion of adenine base to hypoxanthine base). [0058] The deaminases provided herein may be from any organism, such as a bacterium. In some embodiments, the deaminase or deaminase domain is a variant of a naturally-occurring deaminase from an organism. In some embodiments, the deaminase or deaminase domain does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring deaminase.

[0059] Adenosine deaminases ( e.g . engineered adenosine deaminases, evolved adenosine deaminases) provided herein may be may be enzymes that convert adenosine (A) to inosine

(I) in DNA or RNA. Such adenosine deaminase can lead to an A:T to G:C base pair conversion. In some embodiments, the deaminase is a variant of a naturally-occurring deaminase from an organism. In some embodiments, the deaminasedoes not occur in nature.

For example, in some embodiments, the deaminase 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.

[0060] In some embodiments, the adenosine deaminase is derived from a bacterium, such as, E.coli, S. aureus, S. typhi, S. putrefaciens, H. influenzae, or C. crescentus. In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is an E. coli TadA deaminase (ecTadA). In some embodiments, the TadA deaminase is a truncated E. coli TadA deaminase. For example, the truncated ecTadA may be missing one or more N-terminal amino acids relative to a full-length ecTadA. In some embodiments, the truncated ecTadA may be missing 1, 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to the full length ecTadA. In some embodiments, the truncated ecTadA may be missing 1, 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to the full length ecTadA. In some embodiments, the ecTadA deaminase does not comprise an N-terminal methionine. Reference is made to U.S. Patent Publication No. 2018/0073012, published March 15, 2018, which is incorporated herein by reference.

[0061] As used herein, the term“DNA binding protein” or“DNA binding protein domain” refers to any protein that localizes to and binds a specific target DNA nucleotide sequence (e.g. a gene locus of a genome). This term embraces RNA-programmable proteins, which associate (e.g. form a complex) with one or more nucleic acid molecules (i.e., which includes, for example, guide RNA in the case of Cas systems) that direct or otherwise program the protein to localize to a specific target nucleotide sequence (e.g., DNA sequence) that is complementary to the one or more nucleic acid molecules (or a portion or region thereof) associated with the protein. Exemplary RNA-programmable proteins are CRISPR-Cas9 proteins, as well as Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g. engineered or modified), and may include a Cas9 equivalent from any type of CRISPR system (e.g. type II, V, VI), including Casl2a (a type-V

CRISPR-Cas system) (formerly known as Cpfl), C2cl (a type V CRISPR-Cas system), C2c2

(a type VI CRISPR-Cas system), C2c3 (a type V CRISPR-Cas system), a GeoCas9, a

CjCas9, a Casl2b, a Casl2g, a Casl2h, a Casl2i, a Casl3b, a Casl3c, a Casl3d, a Casl4, a

Csn2, an xCas9, an SpCas9-NG, a Cas9-KKH, a circularly permuted Cas9, an Argonaute

(Ago), a SmacCas9, or a Spy-macCas9. Further Cas-equivalents are described in Makarova et al,“C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector,” Science 2016; 353(6299), the contents of which are incorporated herein by reference.

[0062] The term“DNA editing efficiency,” as used herein, refers to the number or proportion of intended base pairs that are edited. For example, if a base editor edits 10% of the base pairs that it is intended to target (e.g., within a cell or within a population of cells), then the base editor can be described as being 10% efficient. Some aspects of editing efficiency embrace the modification (e.g. deamination) of a specific nucleotide within DNA, without generating a large number or percentage of insertions or deletions (i.e., indels). It is generally accepted that editing while generating less than 5% indels (as measured over total target nucleotide substrates) is high editing efficiency. The generation of more than 20% indels is generally accepted as poor or low editing efficiency. Indel formation may be measured by techniques known in the art, including high-throughput screening of sequencing reads.

[0063] The term“off-target editing frequency,” as used herein, refers to the number or proportion of unintended base pairs, e.g. DNA base pairs, that are edited. On-target and off- target editing frequencies may be measured by the methods and assays described herein, further in view of techniques known in the art, including high-throughput sequencing reads. As used herein, high-throughput sequencing involves the hybridization of nucleic acid primers (e.g., DNA primers) with complementarity to nucleic acid (e.g., DNA) regions just upstream or downstream of the target sequence or off-target sequence of interest. Because the DNA target sequence and the Cas9-independent off-target sequences are known a priori in the methods disclosed herein, nucleic acid primers with sufficient complementarity to regions upstream or downstream of the target sequence and Cas9-independent off-target sequences of interest may be designed using techniques known in the art, such as the

PhusionU PCR kit (Fife Technologies), Phusion HS II kit (Fife Technologies), and Illumina MiSeq kit. Since many of the Cas9-dependent off-target sites have high sequence identity to the target site of interest, nucleic acid primers with sufficient complementarity to regions upstream or downstream of the Cas9-dependent off-target site may likewise be designed using techniques and kits known in the art. These kits make use of polymerase chain reaction

(PCR) amplification, which produces amplicons as intermediate products. The target and off- target sequences may comprise genomic loci that further comprise protospacers and PAMs.

Accordingly, the term“amplicons,” as used herein, may refer to nucleic acid molecules that constitute the aggregates of genomic loci, protospacers and PAMs. High-throughput sequencing techniques used herein may further include Sanger sequencing and/or whole genome sequencing (WGS).

[0064] The terms“RNA editing activity,”“RNA editing effects” and“RNA off-target editing,” as used herein, refer to the introduction of modifications (e.g. deaminations) to nucleotides within cellular RNA, e.g. messenger RNA (mRNA). An important goal of DNA base editing efficiency is the modification (e.g. deamination) of a specific nucleotide within DNA, without introducing modifications of similar nucleotides within RNA. RNA editing effects are“low” or“reduced” when a detected mutation is introduced into RNA molecules at a frequency of 0.3% or less. For reference, the ABEmax base editor introduces edits into RNA at a frequency of about 0.50%. RNA editing effects are“low” or“reduced” when a mutation is detected at a magnitude that is less than about 70,000 edits within an analyzed mRNA transcriptome. The number of RNA edits may be measured by techniques known in the art, including high-throughput screening of sequencing reads and RNA-seq. The effects of RNA editing on the function of a protein translated from the edited mRNA transcript may be predicted by use of the SIFT (“Sorting Intolerant from Tolerant”) algorithm, which bases predictions on sequence homology and the physical properties of amino acids.

[0065] The term“on-target editing,” as used herein, refers to the introduction of intended modifications (e.g., deaminations) to nucleotides (e.g., adenine) in a target sequence, such as using the base editors described herein. The term“off-target DNA editing,” as used herein, refers to the introduction of unintended modifications (e.g. deaminations) to nucleotides (e.g. adenine) in a sequence outside the canonical base editor binding window (i.e., from one protospacer position to another, typically 2 to 8 nucleotides long). Off-target DNA editing can result from weak or non-specific binding of the gRNA sequence to the target sequence.

[0066] 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 composition may refer to the amount of the composition that is sufficient to edit a target site of a nucleotide sequence, e.g. a genome. In some embodiments, an effective amount of a composition provided herein, e.g. of a composition comprising a nuclease-inactive Cas9 domain, a deaminase domain, a gRNA and optionally a growth factor and anti-apoptotic factor, may refer to the amount of the composition that is sufficient to induce editing of a target site specifically bound and edited by the fusion protein. In some embodiments, an effective amount of a composition provided herein may refer to the amount of the composition sufficient to induce editing having the following characteristics: > 50% product purity, < 5% indels, and an editing window of 2-8 nucleotides. As will be appreciated by the skilled artisan, the effective amount of an agent, e.g. a composition or a fusion protein-gRNA complex, 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.

[0067] The term“evolved base editor” or“evolved base editor variant” refers to a base editor formed as a result of mutagenizing a reference or starting-point base editor. The term refers to embodiments in which the nucleobase modification domain is evolved or a separate domain is evolved. Mutagenizing a reference or starting-point base editor may comprise mutagenizing an adenosine deaminase. Amino acid sequence variations may include one or more mutated residues within the amino acid sequence of a reference base editor, e.g., as a result of a change in the nucleotide sequence encoding the base editor that results in a change in the codon at any particular position in the coding sequence, the deletion of one or more amino acids (e.g., a truncated protein), the insertion of one or more amino acids, or any combination of the foregoing. The evolved base editor may include variants in one or more components or domains of the base editor (e.g., variants introduced into one or more adenosine deaminases).

[0068] The term“fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two 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. 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.

[0069] The term“host cell,” as used herein, refers to a cell that can host, replicate, and transfer a phage vector useful for a continuous evolution process as provided herein. In embodiments where the vector is a viral vector, a suitable host cell is a cell that may be infected by the viral vector, can replicate it, and can package it into viral particles that can infect fresh host cells. A cell can host a viral vector if it supports expression of genes of viral vector, replication of the viral genome, and/or the generation of viral particles. One criterion to determine whether a cell is a suitable host cell for a given viral vector is to determine whether the cell can support the viral life cycle of a wild-type viral genome that the viral vector is derived from. For example, if the viral vector is a modified M13 phage genome, as provided in some embodiments described herein, then a suitable host cell would be any cell that can support the wild-type M13 phage life cycle. Suitable host cells for viral vectors useful in continuous evolution processes are well known to those of skill in the art, and the disclosure is not limited in this respect. In some embodiments, the viral vector is a phage and the host cell is a bacterial cell. In some embodiments, the host cell is an E. coli cell. Suitable

E. coli host strains will be apparent to those of skill in the art, and include, but are not limited to, New England Biolabs (NEB) Turbo, ToplOF’, DH12S, ER2738, ER2267, and XLl-Blue

MRF’ . These strain names are art recognized and the genotype of these strains has been well characterized. The term“fresh,” as used herein interchangeably with the terms“non- infected” or“uninfected” in the context of host cells, refers to a host cell that has not been infected by a viral vector comprising a gene of interest as used in a continuous evolution process provided herein. A fresh host cell can, however, have been infected by a viral vector unrelated to the vector to be evolved or by a vector of the same or a similar type but not carrying the gene of interest.

[0070] In some embodiments, the host cell is a prokaryotic cell, for example, a bacterial cell. In some embodiments, the host cell is an E. coli cell. In some embodiments, the host cell is a eukaryotic cell, for example, a yeast cell, an insect cell, or a mammalian cell. The type of host cell, will, of course, depend on the viral vector employed, and suitable host cell/viral vector combinations will be readily apparent to those of skill in the art.

[0071] The term“linker,” as used herein, refers to a chemical group or a molecule linking two molecules or domains, e.g. dCas9 and a deaminase. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other domains 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 domain. Chemical groups include, but are not limited to, disulfide, hydrazone, and azide domains. 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. In some embodiments, the linker is an XTEN linker. In some embodiments, the linker is a 32-amino acid linker. In other embodiments, the linker is a 30-, 31-, 33- or 34- amino acid linker.

[0072] As used herein, the term“low toxicity” refers to the maintenance of a viability above 60% in a population of cells following application of a base editing method or administration of a composition disclosed herein. The term may also refer to prevention of apoptosis (cell death) in a population of cells of more than 40%. For instance, a genome editing method that leads to less than 30% (e.g. 25%, 20%, 15%, 10%, or 5%) cell death exhibits low toxicity. Cell toxicity may be assessed by an appropriate staining assay, e.g. Annexin V and propidium iodide staining assays, and subsequent flow cytometry (e.g. FACS).

[0073] 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; a deletion or insertion of one or more residues within a sequence; or a substitution of a residue within a sequence of a genome in a subject to be corrected. 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)). Mutations can include a variety of categories, such as single base polymorphisms, microduplication regions, indel, and inversions, and is not meant to be limiting in any way. Mutations can include“loss-of- function” mutations which is a result of a mutation that reduces or abolishes a protein activity. Most loss-of-function mutations are recessive, because in a heterozygote the second chromosome copy carries an unmutated version of the gene coding for a fully functional protein whose presence compensates for the effect of the mutation. There are some exceptions where a loss-of-function mutation is dominant, one example being

haploinsufficiency, where the organism is unable to tolerate the approximately 50% reduction in protein activity suffered by the heterozygote. This is the explanation for a few genetic diseases in humans, including Marfan syndrome, which results from a mutation in the gene for the connective tissue protein called fibrillin. Mutations also embrace“gain-of-function” mutations, which is one which confers an abnormal activity on a protein or cell that is otherwise not present in a normal condition. Many gain-of-function mutations are in regulatory sequences rather than in coding regions, and can therefore have a number of consequences. For example, a mutation might lead to one or more genes being expressed in the wrong tissues, these tissues gaining functions that they normally lack. Alternatively the mutation could lead to overexpression of one or more genes involved in control of the cell cycle, thus leading to uncontrolled cell division and hence to cancer. Because of their nature, gain-of-function mutations are usually dominant.

[0074] The terms“non-naturally occurring” or“engineered” are used interchangeably and indicate the involvement of the hand of man. These terms, when referring to nucleic acid molecules or polypeptides (e.g. deaminases) mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and/or as found in nature (e.g. an amino acid sequence not found in nature).

[0075] The term“nucleic acid,” as used herein, refers to 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 may 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 may 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,

inosinedenosine, 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).

[0076] As used herein to modify guide RNA molecules, the term“backbone” refers to the component of the guide RNA that comprises the core region, also known as the

crRNA/tracrRNA. The backbone is separate from the guide sequence, or spacer, region of the guide RNA, which has complementarity to a protospacer of a nucleic acid molecule.

[0077] The term“nucleic acid programmable DNA binding protein (napDNAbp)” refers to any protein that may associate (e.g., form a complex) with one or more nucleic acid molecules (i.e., which may broadly be referred to as a“napDNAbp-programming nucleic acid molecule” and includes, for example, guide RNA in the case of Cas systems) which direct or otherwise program the protein to localize to a specific target nucleotide sequence

(e.g., a gene locus of a genome) that is complementary to the one or more nucleic acid molecules (or a portion or region thereof) associated with the protein, thereby causing the protein to bind to the nucleotide sequence at the specific target site. This term napDNAbp embraces CRISPR-Cas9 proteins, as well as Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or modified), and may include a Cas9 equivalent from any type of CRISPR system (e.g., type II,

V, VI), including Casl2a (a type-V CRISPR-Cas system) (formerly known as Cpfl), C2cl (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system), C2c3 (a type V

CRISPR-Cas system), a GeoCas9, a CjCas9, a Cas 12b, a Casl2g, a Casl2h, a Casl2i, a

Casl3b, a Casl3c, a Casl3d, a Casl4, a Csn2, an xCas9, an SpCas9-NG, a Cas9-KKH, a circularly permuted Cas9, an Argonaute (Ago), a SmacCas9, or a Spy-macCas9. The napDNAbp may be a Cas9 domain that comprises a nuclease active Cas9 domain, a nuclease inactive Cas9 (dCas9) domain, or a Cas9 nickase (nCas9) domain. Further Cas equivalents are described in Makarova et al.,“C2c2 is a single-component programmable RNA-guided

RNA-targeting CRISPR effector,” Science 2016; 353 (6299), the contents of which are incorporated herein by reference. However, the nucleic acid programmable DNA binding protein (napDNAbp) that may be used in connection with this disclosure are not limited to

CRISPR-Cas systems. The claimed invention embraces any such programmable protein, such as the Argonaute protein from Natronobacterium gregoryi (NgAgo) which may also be used for DNA-guided genome editing. NgAgo-guide DNA system does not require a PAM sequence or guide RNA molecules, which means genome editing can be performed simply by the expression of generic NgAgo protein and introduction of synthetic oligonucleotides on any genomic sequence. See Gao et al, DNA-guided genome editing using the Natronobacterium gregoryi Argonaute. Nature Biotechnology 2016; 34(7):768-73, which is incorporated herein by reference.

[0078] In some embodiments, the napDNAbp is a 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 interchangeabley 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 (or equivalent) 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 homologous to a tracrRNA as depicted in Figure IE of 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. Patent No. 9,340,799, entitled“mRNA-Sensing Switchable gRNAs,” and International Patent Application No. PCT/US2014/054247, filed September 6, 2013, published as WO 2015/035136 and entitled“Delivery System For Functional Nucleases,” the entire contents of each are herein incorporated by reference. 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 (Csnl) from Streptococcus pyogenes (see, e.g.,“Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti J.J. et al.., Proc. Natl. Acad. Sci. U.S.A. 98:4658- 4663(2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E. et al, Nature 471:602-607(2011); and“A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M. et al, Science 337:816- 821(2012), the entire contents of each of which are incorporated herein by reference.

[0079] The napDNAbp 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 napDNAbp nucleases, such as Cas9, for site-specific cleavage (e.g., to modify a genome) are known in the art (see e.g., Cong, L. el 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 Acid Res. (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).

[0080] The term“napDNAbp -programming nucleic acid molecule” or equivalently“guide sequence” refers the one or more nucleic acid molecules which associate with and direct or otherwise program a napDNAbp protein to localize to a specific target nucleotide sequence (e.g., a gene locus of a genome) that is complementary to the one or more nucleic acid molecules (or a portion or region thereof) associated with the protein, thereby causing the napDNAbp protein to bind to the nucleotide sequence at the specific target site. A non limiting example is a guide RNA of a Cas protein of a CRISPR-Cas genome editing system.

[0081] A nuclear localization signal or sequence (NLS) is an amino acid sequence that tags, designates, or otherwise marks a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface. Different nuclear localized proteins may share the same NLS. An NLS has the opposite function of a nuclear export signal (NES), which targets proteins out of the nucleus. Thus, a single nuclear localization signal can direct the entity with which it is associated to the nucleus of a cell. Such sequences may be of any size and composition, for example more than 25, 25, 15, 12, 10, 8, 7, 6, 5, or 4 amino acids, but will preferably comprise at least a four to eight amino acid sequence known to function as a nuclear localization signal (NLS). In some embodiments, the disclosed NLSs are bipartite NLSs (“bpNLS”).

[0082] The term“promoter” is art-recognized and refers to a nucleic acid molecule with a sequence recognized by the cellular transcription machinery and able to initiate transcription of a downstream gene. A promoter may be constitutively active, meaning that the promoter is always active in a given cellular context, or conditionally active, meaning that the promoter is only active in the presence of a specific condition. For example, a conditional promoter may only be active in the presence of a specific protein that connects a protein associated with a regulatory element in the promoter to the basic transcriptional machinery, or only in the absence of an inhibitory molecule. A subclass of conditionally active promoters are inducible promoters that require the presence of a small molecule“inducer” for activity.

Examples of inducible promoters include, but are not limited to, arabinose-inducible promoters, Tet-on promoters, and tamoxifen-inducible promoters. A variety of constitutive, conditional, and inducible promoters are well known to the skilled artisan, and the skilled artisan will be able to ascertain a variety of such promoters useful in carrying out the present disclosure, which is not limited in this respect. In various embodiments, the disclosure provides vectors with appropriate promoters for driving expression of the nucleic acid sequences encoding the fusion proteins (or one or more individual components thereof).

[0083] 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.

[0084] 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.

[0085] The term“target site” refers to a sequence within a nucleic acid molecule that is edited by a fusion protein (e.g. a dCas9-deaminase fusion protein provided herein). The target site further refers to the sequence within a nucleic acid molecule to which a complex of the fusion protein and gRNA binds.

[0086] 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, disorder, or condition, 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, disorder, or condition, 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 prevention or recurrence.

[0087] As used herein, e.g. for the purposes of reporting a specific number of loci, the terms “unique loci” and“unique genomic loci” refer to distinct genomic sequences (e.g. distinct coding sequences) wherein all copies of a distinct sequence in the genome are collectively counted (or reported) only once; in contrast, each copy of a“non-unique locus” or“repetitive element” is counted for purposes of reporting a specific number of loci.

[0088] As used herein, the term“variant” refers to a protein having characteristics that deviate from what occurs in nature that retains at least one functional i.e. binding, interaction, or enzymatic ability and/or therapeutic property thereof. A“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 the wild type protein. For instance, a variant of Cas9 may comprise a Cas9 that has one or more changes in amino acid residues as compared to a wild type Cas9 amino acid sequence. As another example, a variant of a deaminase may comprise a deaminase that has one or more changes in amino acid residues as compared to a wild type deaminase amino acid sequence, e.g. following ancestral sequence reconstruction of the deaminase. These changes include chemical modifications, including substitutions of different amino acid residues truncations, covalent additions (e.g. of a tag), and any other mutations. This term also embraces fragments of a wild type protein.

[0089] The level or degree of which the property is retained may be reduced relative to the wild type protein but is typically the same or similar in kind. Generally, variants are overall very similar, and in many regions, identical to the amino acid sequence of the protein described herein. A skilled artisan will appreciate how to make and use variants that maintain all, or at least some, of a functional ability or property.

[0090] The variant proteins may comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, identical to, for example, the amino acid sequence of a wild-type protein, or any protein provided herein (e.g. Cas9 protein, fusion protein, and fusion protein protein). Further polypeptides provided in the disclosure are encoded by polynucleotides which hybridize to the complement of a nucleic acid molecule encoding a protein such as a Cas9 protein under stringent hybridization conditions (e.g. hybridization to filter bound DNA in 6x Sodium chloride/S odium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.2.times.SSC, 0.1% SDS at about 50-65 degrees Celsius), under highly stringent conditions (e.g. hybridization to filter bound DNA in 6x sodium chloride/S odium citrate

(SSC) at about 45 degrees Celsius, followed by one or more washes in O.lxSSC, 0.2% SDS at about 68 degrees Celsius), or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F. M. et al, eds., 1989 Current

Protocol in Molecular Biology , Green publishing associates, Inc., and John Wiley & Sons

Inc., New York, at pp. 6.3.1-6.3.6 and 2.10.3).

[0091] By a polypeptide having an amino acid sequence at least, for example, 95%

“identical” to a query amino acid sequence, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

[0092] As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, for instance, the amino acid sequence of a protein such as a Cas9 protein, can be determined conventionally using known computer programs.

A preferred method for determining the best overall match between a query sequence (a sequence of the present disclosure) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. {Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k- tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter. [0093] If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results.

This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C- terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present disclosure. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.

[0094] As used herein, the term“wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.

[0095] These and other exemplary substituents are described in more detail in the Detailed Description, Examples, and claims. The invention is not intended to be limited in any manner by the above exemplary listing of substituents.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0096] The present disclosure provides adenine base editors that are variants of ABEmax that feature a significantly lower RNA editing footprint while retaining DNA editing fidelity. The disclosed adenine base editors that comprise an adenosine deaminase domain (e.g., a variant of an adenosine deaminase that deaminates deoxyadenosine in DNA as described herein) and a napDNAbp domain (e.g., a Cas9 protein) capable of binding to a specific nucleotide sequence. The deamination of an adenosine by an adenosine deaminase may lead to a point mutation from adenine (A) to guanine (G), a process referred to herein as base editing. For example, the adenosine may be converted to an inosine residue. Within the constraints of a DNA polymerase active site, inosine pairs most stably with C and therefore is read or replicated by the cell’s replication machinery as a guanine (G). Such base editors are useful inter alia for targeted editing of nucleic acid sequences. Such base editors may be used for targeted editing of DNA in vitro , e.g., for the generation of mutant cells or animals. Such base editors may be used for 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, or for multiplexed editing of a genome. And these base editors may be used for the introduction of targeted mutations in vivo, e.g., the correction of genetic defects or the introduction of deactivating mutations in disease- associated genes in a subject, or for multiplexed editing of a genome. The adenine base editors described herein may be utilized for the targeted editing of G to A mutations (e.g., targeted genome editing). The disclosure provides deaminases, base editors, nucleic acids, vectors, cells, compositions, methods, kits, and uses that utilize the deaminases and base editors provided herein. In particular aspects, base editing methods comprising contacting a nucleic acid molecule with an adenine base editor and a guide RNA that has complementarity to a target sequence are disclosed; as well as kits and pharmaceutical compositions for the administration of ABE7.10 variants to a host cell.

[0097] ABE7.10 (ABEmax) was shown to generate detectable levels of widespread adenosine-to-inosine editing in cellular RNAs. Using structure-guided principles to design mutations in both deaminase domains, new ABE variants were developed that retain their ability to edit DNA efficiently but show greatly reduced off-target effects, such as reduced RNA editing activity, off-target DNA editing activity, and indel byproduct formation, in three mammalian cell lines.

[0098] Given the lack of an elucidated structure of ABE or of the E. coli TadA homodimer bound to RNA, the crystal structure of was used for targeted mutagenesis of wild-type E. coli TadA deaminases to design that reduce undesired RNA editing activity. S. aureus TadA has high sequence homology to E. coli TadA (25). Three TadA* residues were identified, predicted to interact with the RNA substrate as targets for substitutions that might impair TadA* -mediated RNA deamination. It was hypothesized that impeding the ability of TadA* to accommodate 2'-hydroxyl groups that are present in RNA, but absent in DNA, by replacing these three amino acids with larger or more hydrophobic residues (Gin, Phe, Trp, or Met) could further improve the DNA versus RNA editing specificity of ABEmax comprising an adenosine deaminase domain comprising a mutated TadA(E59A) (or

ABEmax(TadA(E59W)). Arginine 47 is predicted to form a hydrogen bond with the 2'- hydroxyl group of the substrate adenosine (FIG. 2A). Arg 47 was replaced in TadA* with Gin, Phe, Trp, or Met in an effort to abrogate this interaction. A series of ABEmax mutants was generated with TadA* substitutions at either Aspartine 108 (FIG. 2B) or Valine 106 (FIG. 2C), two residues that are located close to the catalytic site of TadA, and that mutated from Asp 108 and Ala 106 during the evolution of TadA*(l). Aspartine 108 is predicted to directly hydrogen bond with the 2 '-hydroxyl group of the uridine immediately 5' of the substrate adenosine (FIG. 2B), and replacement of Alanine 106 might fill some of the space that accommodates this uridine, including its 2' hydroxyl group, with larger and more hydrophobic side chains (FIG. 2C). Asn 108 was replaced in ABEmax TadA* with Gin, Phe,

Trp, Lys, or Met, and Val 106 in ABEmax TadA* with Gin, Phe, Trp, or Met, in an effort to disrupt the ability of TadA* to accommodate ribonucleotides by eliminating the possibility of forming hydrogen bonds with 2' hydroxyl groups in RNA or by steric occlusion. An additional mutation of Aspartine 108 to lysine was also designed.

[0099] Using structure-guided mutagenesis approaches, ABE7.10 variants were designed with mutations in both TadA domains demonstrated greatly reduced RNA editing while maintaining efficient target DNA editing, improving DNA specificity, and reducing indel byproduct formation. An ABE7.10 variant comprising an adenosine deaminase domain comprising TadA(E59W) and TadA7.10(V106W) generated particularly low levels of off- target effects. TadA7.10(V106W) comprises the following substitutions in ecTadA: W23R, H36L, P48A, R51L, L84F, A106W, D108N, H123Y, S146C, D147Y, R152P, E155V, I156F, and K157N. Another ABE7.10 variant comprising an adenosine deaminase domain comprising TadA(E59W) and TadA7.10(N108W) generated particularly low levels of off- target effects. TadA7.10(N108W) comprises the following substitutions in ecTadA: W23R, H36L, P48A, R51L, L84F, A106V, D108W, H123Y, S146C, D147Y, R152P, E155V, I156F, and K157N.

[0100] Off-target activity may arise because of imperfect hybridization of the napDNAbp- guide RNA complex to sequences that share identity with the target sequence. Otherwise, off-target activity may occur independently of the napDNAbp-guide RNA complex arise as a result of stochastic binding of the adenine base editor to DNA sequences (often sequences that do not share high sequence identity with the target sequence) due to an intrinsic affinity of the base editor of the nucleotide modification domain (e.g., the deaminase domain) of the base editor with DNA. NapDNAbp -independent (e.g., Cas9-independent) editing events arise in particular when the base editor is overexpressed in the system under evaluation, such as a cell or a subject.

[0101] In the experiments described herein, A-to-I editing attributable to the overexpression of ABEmax, the most efficient ABE variant reported to date (15), was measured with high sensitivity. Targeted deep sequencing of individual abundant mRNA transcripts and transcriptome-wide RNA-seq techniques were utilized to demonstrate that ABEmax induced low levels of widespread adenosine-to-inosine (A-to-I) editing across the transcriptome. Comparison of RNA editing rates between ABEmax mutants with catalytically disabled deaminase domains revealed that both the wild-type E. coli TadA monomer that plays a structural role during base editing and laboratory-evolved E. coli TadA7.10 (TadA*) that catalyzes deoxy adenosine deamination contribute to RNA editing. This may represent the first recognition of off-target RNA editing in ABEmax and thus the first recognition of this deficiency in the art.

[0102] Specifically, the novel ABEmax variants disclosed herein provide average RNA editing frequencies as low as 0.068% (among 182 total adenosines in three analyzed mRNA transcripts), which are levels that approach those observed from a Cas9 nickase-alone control and represent a 7.2-fold reduction relative to the 0.49% average RNA editing frequency of ABEmax (see FIG. 2F). The novel ABEmax variants disclosed herein provide average overall magnitudes of detectable RNA edits among the 182 total adenosines analyzed of as low as 26+10, which is similar to the background of 12+6 for Cas9 nickase alone and significantly reduced from an average of 94+8 with ABEmax (see FIG. 2E). These editing frequencies were analyzed using high-throughput screening (HTS).

[0103] On a human cell transcriptome-wide basis, as analyzed by RNA-seq, the novel ABEmax variants disclosed herein provide average RNA editing frequencies as low as 0.14%, levels nearly equivalent to those observed from Cas9 nickase alone and represent a significant reduction compared with the 0.22% average RNA editing frequency of ABEmax (see FIGs. 2G, 2H). These novel ABEmax variants provide average overall detectable transcriptome edits of about 57,700 edits, levels similar to the background of 53,300 for Cas9 nickase alone and significantly lower (by 10,608 edits) than those ABEmax (see FIG. 2E).

[0104] Notably, the disclosed ABEmax variants retain, and in some cases show improved, the high DNA editing fidelity of ABEmax. These variants were shown to generate reduced indel formation (3.7-fold fewer indels) relative to ABEmax at seven target DNA loci, as analyzed by HTS (see FIGs. 4A-4D). These variants generated an average off-target DNA editing frequency as low as 0.79+0.18%, a 2.7-fold improvement relative to ABEmax. These results may indicate an important correlation: Mutations that reduce the tolerance of ABEmax for RNA editing may also increase the DNA specificity of base editing, likely by reducing DNA binding interactions that support productive editing of off-target loci.

[0105] Accordingly, in some aspects, the disclosure provides fusion proteins (adenine base editors) that comprise an adenosine deaminase domain ( e.g ., an adenosine deaminase that deaminates deoxyadenosine in DNA as described herein) and a napDNAbp domain (e.g., a Cas9 protein) capable of binding to a specific nucleotide sequence. Exemplary fusion proteins comprise a Cas9 domain and an adenosine deaminase domain. The Cas9 domain may be any of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) provided herein. In some embodiments, any of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) provided herein may be fused with any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase domain comprises a single adenosine deaminase enzyme. In other embodiments, the adenosine deaminase domain comprises two adenosine deaminases, e.g., a heterodimer of adenosine deaminases.

[0106] The deamination of an adenosine by an adenosine deaminase can lead to a point mutation, this process is referred to herein as base editing. For example, the adenosine may be converted to an inosine residue, which typically base pairs with a cytosine residue. Such fusion proteins are useful inter alia for targeted editing of nucleic acid sequences. Such fusion proteins may be used for targeted editing of DNA in vitro, e.g., for the generation of mutant cells or animals; for the introduction of targeted mutations, e.g., for the correction of genetic defects in cells ex vivo, e.g., in cells obtained from a subject that are subsequently re introduced into the same or another subject; and for the introduction of targeted mutations in vivo, e.g., the correction of genetic defects or the introduction of deactivating mutations in disease-associated genes in a subject. As an example, diseases that may be treated by making an A to G, or a T to C mutation, may be treated using the base editors provided herein.

Without wishing to be bound by any particular theory certain anemias, such as sickle cell anemia, may be treated by inducing expression of hemoglobin, such as fetal hemoglobin, which is typically silenced in adults. As one example, mutating the thymine to a cytosine at position -198 in the promoter controlling HBG1 and/or HBG2 gene expression results in increased expression of the HBG1 and HBG2 proteins, respectively. Another example, a class of disorders that results from a G to A mutation in a gene is iron storage disorders, where the HFE gene comprises a G to A mutation that results in expression of a C282Y mutant HFE protein. See International Publication No. WO 2019/079347, published April 25, 2019, herein incorporated by reference. Thus, the adenine base editors described herein may be utilized for the targeted editing of such G to A mutations (e.g., targeted genome editing). The disclosure provides deaminases, cells, compositions, methods, kits, systems, etc. that utilize the disclosed deaminases and adenine base editors.

[0107] In some embodiments, the adenine base editors provided herein may be made by fusing together one or more protein domains, thereby generating a fusion protein. In certain embodiments, the fusion proteins provided herein comprise one or more features that improve the base editing activity (e.g., efficiency, selectivity, and specificity) of the fusion proteins. For example, the fusion proteins provided herein may comprise a Cas9 domain that has reduced nuclease activity. In some embodiments, the fusion proteins provided herein may have a Cas9 domain that does not have nuclease activity (dCas9), or a Cas9 domain that cuts one strand of a duplexed DNA molecule, referred to as a Cas9 nickase (nCas9). Without wishing to be bound by any particular theory, the presence of the catalytic residue ( e.g .,

H840) maintains the activity of the Cas9 to cleave the non-edited (e.g., non-deaminated) strand containing a T opposite the targeted A. Mutation of the catalytic residue (e.g., DIO to

A10) of Cas9 prevents cleavage of the edited strand containing the targeted A residue. Such

Cas9 variants are able to generate a single-strand DNA break (nick) at a specific location based on the gRNA-defined target sequence, leading to repair of the non-edited strand, ultimately resulting in a T to C change on the non-edited strand.

[0108] The adenosine deaminase domains of the disclosed fusion proteins comprise variants of wild-type deaminase enzymes. These variants comprise an amino acid sequence that 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 wild type enzyme. In some embodiments, the adenosine deaminase domains may comprise an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more than 30 amino acids that differ relative to the amino acid sequence of the wild type enzyme. These differences may comprise nucleotides that have been inserted, deleted, or substituted relative to the amino acid sequence of the wild type enzyme. In some embodiments, the adenosine deaminase domains contain stretches of about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 300, about 400, about 500, or more than 500 consecutive amino acids in common with the wild type enzyme. In some embodiments, the adenosine deaminase domains comprise truncations at the N-terminus or C-terminus relative to the wild-type enzyme. In some embodiments, the adenosine deaminase domains comprise truncations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more than 30 amino acids at the N-terminus or C- terminus relative to the wild-type or base sequence.

[0109] Still further, the present disclosure provides for methods of making the adenine base editors, as well as methods of using the base editors or nucleic acid molecules encoding the base editors in applications including editing a nucleic acid molecule, e.g., a genome. The disclosure accordingly provides methods for editing a target nucleic acid molecule, e.g., a single nucleobase within a genome, with a base editing system described herein (e.g., in the form of an evolved base editor as described herein, or a vector or construct encoding same). Such methods involve transducing (e.g., via transfection) cells with a plurality of complexes each comprising a fusion protein (e.g., a fusion protein comprising a napDNAbp (nCas9) domain and an adenosine deaminase domain) and a gRNA molecule. In some embodiments, the gRNA is bound to the napDNAbp domain of the fusion protein. In some embodiments, each gRNA comprises a guide sequence of at least 10 contiguous nucleotides (e.g., 10, 11,

12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides) that is complementary to a target sequence. In certain embodiments, the methods involve the transfection of nucleic acid constructs (e.g., plasmids) that each (or together) encode the components of a complex of fusion protein and gRNA molecule.

[0110] In certain embodiments of the disclosed methods, a nucleic acid construct that encodes the fusion protein is transfected into the cell separately from the plasmid that encodes the gRNA molecule. In certain embodiments, these components are encoded on a single construct and transfected together. In other embodiments, the methods disclosed herein involve the introduction into cells of a complex comprising a fusion protein and gRNA molecule that has been expressed and cloned outside of these cells.

[0111] It should be appreciated that any fusion protein, e.g., any of the fusion proteins provided herein, may be introduced into the cell in any suitable way, either stably or transiently. In some embodiments, a fusion protein may be transfected into the cell. In some embodiments, the cell may be transduced or transfected with a nucleic acid construct that encodes a fusion protein. For example, e a cell may be transduced (e.g., with a virus encoding a fusion protein), or transfected (e.g., with a plasmid encoding a fusion protein) with a nucleic acid that encodes a fusion protein, or the translated fusion protein. Such transduction may be a stable or transient transduction. In some embodiments, cells expressing a fusion protein or containing a fusion protein may be transduced or transfected with one or more gRNA molecules, for example when the fusion protein comprises a Cas9 (e.g., nCas9) domain. In some embodiments, a plasmid expressing a fusion protein may be introduced into cells through electroporation, transient (e.g., lipofection) and stable genome integration (e.g., piggybac) and viral transduction or other methods known to those of skill in the art.

[0112] In certain embodiments, the methods described above result in a cutting (or nicking) one strand of the double- stranded DNA, for example, the strand that includes the thymine (T) of the target A:T nucleobase pair opposite the strand containing the target adenine (A) that is being deaminated. This nicking result serves to direct mismatch repair machinery to the non- edited strand, ensuring that the chemically modified nucleobase is not interpreted as a lesion by the machinery. This nick may be created by the use of an nCas9.

[0113] The specification also provides methods for efficiently editing a target nucleic acid molecule, e.g., a single nucleobase of a genome, with a base editing system described herein

(e.g., in the form of an base editor as described herein or a vector or construct encoding same), thereby installing an edit. Still further, the disclosure provides therapeutic methods for treating a genetic disease and/or for altering or changing a genetic trait or condition by contacting a target nucleic acid molecule, e.g., a target nucleic acid molecule in the genome of an organism, with a base editing system (e.g., in the form of an base editor protein or a vector encoding same) and conducting base editing to treat the genetic disease and/or change the genetic trait (e.g., eye color).

[0114] The target nucleotide sequence may comprise a target sequence (e.g., a point mutation) associated with a disease, disorder, or condition, such as sickle cell anemia. The target sequence may comprise a G to A point mutation associated with a disease, disorder, or condition, and wherein the deamination of the mutant A base results in mismatch repair- mediated correction to a sequence that is not associated with a disease, or disorder, or condition. The target sequence may instead comprise an C to T point mutation associated with a disease, disorder, or condition, and wherein the deamination of the A base that is paired with the mutant T base results in mismatch repair-mediated correction to a sequence that is not associated with a disease, or disorder, or condition. The target sequence may encode a protein, and where the point mutation is in a codon and results in a change in the amino acid encoded by the mutant codon as compared to a wild-type codon. The target sequence may also be at a splice site, and the point mutation results in a change in the splicing of an mRNA transcript as compared to a wild-type transcript. In addition, the target may be at a non-coding sequence of a gene, such as a promoter, and the point mutation results in increased or decreased expression of the gene.

[0115] Exemplary target genes include HBG1, HBG2, and HFE, for each of which a sickle cell anemia phenotype is frequently caused by an A:T to G:C point mutation.

[0116] In various embodiments, application of the disclosed adenine base editors results in the deamination of a target site. In some cases, the deamination of a mutant A results in a change of the amino acid encoded by the mutant codon, which in some cases can result in the expression of a wild-type amino acid. The application of the base editors can also result in a change of the mRNA transcript, and even restoring the mRNA transcript to a wild-type state.

[0117] The methods described herein involving contacting a base editor with a target nucleotide sequence can occur in vitro, ex vivo, or in vivo in a subject. In certain

embodiments, the subject has been diagnosed with a disease, disorder, or condition, such as, but not limited to, a disease, disorder, or condition associated with a point mutation in the HBG1 gene, the HBG2 gene, or the HFE gene. The methods described herein involving contacting a base editor with a target nucleotide sequence in the genome of an organism, e.g. a human. [0118] In another aspect, the specification discloses pharmaceutical compositions comprising any of the presently disclosed base editor fusion proteins. In one aspect, the specification discloses a pharmaceutical composition comprising any one of the presently disclosed complexes of fusion proteins and gRNA. In one aspect, the specification discloses a pharmaceutical composition comprising polynucleotides encoding the fusion proteins disclosed herein and polynucleotides encoding a gRNA, or polynucleotides encoding both.

[0119] In another aspect, the specification discloses a pharmaceutical composition comprising any one of the presently disclosed vectors. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition further comprises a lipid and/or polymer. In certain embodiments, the lipid and/or polymer is cationic. The preparation of such lipid particles is well known. See, e.g. U.S. Patent Nos. 4,880,635; 4,906,477;

4,911,928; 4,917,951; 4,920,016; 4,921,757; and 9,737,604, each of which is incorporated herein by reference.

[0120] In the examples provided herein, exemplary adenine base editors having the general structure of an evolved fusion protein, such as ecTadA (D108X; X=W, Q, F, K, or M)- XTEN-nCas9, catalyze A to G transition mutations in cells such as eukaryotic cells (e.g., HEK293T mammalian cells). In other examples exemplary adenine base editors contain two ecTadA domains and a nucleic acid programmable DNA binding protein (napDNAbp). The two ecTadA domains may be the same (e.g., a homodimer), or two different ecTadA domains (e.g., a heterodimer of a first adenosine deaminase and a second deaminase (e.g., wild-type ecTadA and ecTadA (A106V/D108W))). For example base editors may have the general structure ecTadA-ecTadA*-nCas9, where ecTadA* represents an evolved ecTadA

comprising one or more mutations of SEQ ID NO: 86. Additional examples of base editors containing ecTadA variants provided herein demonstrate an improvement in performance of the base editors in mammalian cells.

[0121] Without wishing to be bound by any particular theory, the adenine base editors described herein work by using ecTadA variants to deaminate A bases in DNA, causing adenosine to guanine mutations via inosine formation. Inosine preferentially hydrogen bonds with C, resulting in an A to G mutation during DNA replication. When covalently tethered to Cas9 (or another nucleic acid programmable DNA binding protein), the adenosine deaminase (e.g., ecTadA) is localized to a gene of interest and catalyzes A to G mutations in the ssDNA substrate. This editor may be used to target and revert single nucleotide polymorphisms (SNPs) in disease-relevant genes, which require A to G reversion. This editor can also be used to target and revert single nucleotide polymorphisms (SNPs) in disease-relevant genes, which require T to C reversion by mutating the A, opposite of the T, to a G. The T may then be replaced with a C, for example by base excision repair mechanisms, or may be changed in subsequent rounds of DNA replication. Thus, the adenine base editors described herein may deaminate the A nucleobase to give a nucleotide sequence that is not associated with a disease or disorder. In some aspects, the adenine base editors described herein may be useful for deaminating an adenosine (A) nucleobase in a gene promoter. In some embodiments, deamination leads to induce transcription of the gene. The induction of transcription of a gene leads to an increase in expression of the protein encoded by the gene (e.g., the gene product). A guide RNA (gRNA) bound to the base editor comprises a guide sequence that is complementary to a target nucleic acid sequence in the promoter.

Adenosine deaminases

[0122] The disclosure provides fusion proteins that comprise one or more adenosine deaminases having one or more substitutions in ecTadA, and fusion proteins that comprise one ore more adenosine deaminases having one or more substitutions in TadA7.10. In some aspects, such fusion proteins are capable of deaminating adenosine in a nucleic acid sequence (e.g., DNA or RNA). As one example, any of the fusion proteins provided herein may be base editors (e.g., adenine base editors). In various embodiments, the adenosine deaminases of the disclosed base editors hydrolytically deaminate a targeted adenosine in a nucleic acid of interest to an inosine, which is read as a guanosine (G) by DNA polymerase enzymes. Without wishing to be bound by any particular theory, dimerization of adenosine deaminases (e.g., in cis or in trans) may improve the ability (e.g., efficiency) of the fusion protein to modify a nucleic acid base, for example, to deaminate adenine.

[0123] Exemplary, non-limiting, embodiments of adenosine deaminases are provided herein. In some embodiments, the adenosine deaminase domain of any of the disclosed base editors comprises a single adenosine deaminase, or a monomer. In some embodiments, the adenosine deaminase domain comprises 2, 3, 4 or 5 adenosine deaminases. In some embodiments, the adenosine deaminase domain comprises two adenosine deaminases, or a dimer. In some embodiments, the deaminase domain comprises a dimer of an engineered (or evolved) deaminase and a wild-type deaminase, such as a wild-type E. coli deaminase. In some embodiments, any of the fusion proteins may comprise 2, 3, 4 or 5 adenosine deaminases. In some embodiments, any of the fusion proteins provided herein comprise two adenosine deaminases. Exemplary, non-limiting, embodiments of adenosine deaminases are provided herein. It should be appreciated that the mutations provided herein (e.g., mutations in ecTadA) may be applied to adenosine deaminases in other adenine base editors, for example those provided in International Publication No. WO 2018/027078, published August

2, 2018; International Publication No. WO 2019/079347, published April 25, 2019;

International Application No PCT/US2019/033848, filed May 23, 2019, which published as

International Publication No. WO 2019/226593 on November 28, 2019; U.S. Patent

Publication No. 2018/0073012, published March 15, 2018, which issued as U.S. Patent No.

10,113,163, on October 30, 2018; U.S. Patent Publication No. 2017/0121693, published May

4, 2017, which issued as U.S. Patent No. 10,167,457 on January 1, 2019; International

Publication No. WO 2017/070633, published April 27, 2017; U.S. Patent Publication No.

2015/0166980, published June 18, 2015; U.S. Patent No. 9,840,699, issued December 12,

2017; and U.S. Patent No. 10,077,453, issued September 18, 2018, all of which are incorporated herein by reference in their entireties.

[0124] In some embodiments, any of the adenosine deaminases provided herein are capable of deaminating adenine. In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenine in a deoxy adenosine residue of DNA. The adenosine deaminase may be derived from any suitable organism ( e.g ., E. coli). In some embodiments, the adenosine deaminase is a naturally-occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA). An amino acid sequence alignment of exemplary TadA deaminases derived from Bacillus subtilis (set forth in full as SEQ ID NO: 89), S. aureus (SEQ ID NO: 88), and S. pyogenes (SEQ ID NO: 110) as compared to the consensus sequence of E. coli TadA is provided as FIG. 14. Exemplary amino acid substitutions in the amino acid sequence of E. co/z)TadA, such as substitutions in amino acid residues 46, 59, 106, or 108, and the homologous mutations in the B. subtilis , S. aureus , and S. pyogenes TadA deaminases, are shown. Accordingly, one of skill in the art would be able to generate mutations in any naturally-occurring adenosine deaminase (e.g., having homology to ecTadA) that corresponds to any of the mutations described herein, e.g., any of the mutations identified in ecTadA. In some embodiments, the adenosine deaminase is from a prokaryote. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenosine deaminase is from E. coli.

[0125] In some embodiments, the adenosine deaminase is a naturally-occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA). ecTadA natively operates as a homodimer, with one monomer catalyzing deamination, and the other monomer acting as a docking station for the tRNA substrate. In other embodiments, the adenosine deaminase may be modified.

Modified adenosine deaminases may be obtained by, e.g., evolving a reference version using targeted mutagenesis, targeted mutagenesis informed by crystallographic structure, or a continuous evolution process (e.g., PACE) described herein so that the deaminase is effective at editing a DNA target. In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenine. In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenine in a deoxyadenosine residue of DNA.

Reference is made to International Publication No. WO 2018/027078, published August 2,

2018; International Publication No. WO 2019/079347 published April 25, 2019; International

Publication No. WO 2019/226593, published November 28, 2019; U.S. Patent Publication

No. 2018/0073012, published March 15, 2018, which issued as U.S. Patent No. 10,113,163, on October 30, 2018; and U.S. Patent Publication No. 2017/0121693, published May 4, 2017, which issued as U.S. Patent No. 10,167,457, on January 1, 2019; and Rees & Liu, Base editing: precision chemistry on the genome and transcriptome of living cells, Nat Rev Genet.

2018 Dec; 19(12):770-788, the disclosures of which are herein incorporated by reference in their entireties. In various embodiments, the deaminase provided herein is a dimer of two adenosine deaminases. In various embodiments, the deaminase provided herein is a homodimer of two TadA deaminases. In various embodiments, the deaminase provided herein is a heterodimer of a wild-type TadA deaminase and an evolved variant of a TadA deaminase. In various embodiments, the deaminase provided herein is a dimer of two adenosine deaminases that is linked covalently or non-covalently to a napDNAbp.

[0126] In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least

99.5% identical to any one of the amino acid sequences set forth in any one of SEQ ID NOs:

86-107 and 110, or to any of the adenosine deaminases provided herein. It should be appreciated that adenosine deaminases provided herein may include one or more mutations

(e.g., any of the mutations provided herein). The disclosure provides adenosine deaminases with a certain percent identiy plus any of the mutations or combinations thereof described herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26,

27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to any one of the amino acid sequences set forth in SEQ ID NOs:

86-107 and 110, or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth in SEQ ID NOs: 86-107 and

110, or any of the adenosine deaminases provided herein.

[0127] In some embodiments, the adenosine deaminase comprises a E59X mutation in ecTadA SEQ ID NO: 86, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In particular embodiments, the adenosine deaminase comprises a E59A mutation in SEQ ID NO: 86, or a corresponding mutation in another adenosine deaminase.

[0128] In some embodiments, the adenosine deaminase comprises a D108X mutation in ecTadA SEQ ID NO: 86, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a D108W, D108Q, D108F, D108K, or D108M mutation in SEQ ID NO: 86, or a corresponding mutation in another adenosine deaminase. In particular embodiments, the adenosine deaminase comprises a D108W mutation in SEQ ID NO: 86, or a corresponding mutation in another adenosine deaminase. It should be appreciated, however, that additional deaminases may similarly be aligned to identify homologous amino acid residues that may be mutated as provided herein (see FIG. 14).

[0129] In some embodiments, the adenosine deaminase comprises an N108W mutation in SEQ ID NO: 96 (TadA7.10), an embodiment also referred to as TadA 7.10 (N108W). Its sequence is provided as SEQ ID NO: 98.

[0130] In some embodiments, the adenosine deaminase comprises an A106X mutation in ecTadA SEQ ID NO: 86, or a corresponding mutation in another adenosine deaminase, where X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises an A106V mutation in SEQ ID NO: 86, or a corresponding mutation in another adenosine deaminase.

In some embodiments, the adenosine deaminase comprises an A106Q, A106F, A106W, or A106M mutation in SEQ ID NO: 86, or a corresponding mutation in another adenosine deaminase.

[0131] In particular embodiments, the adenosine deaminase comprises a V106W mutation in SEQ ID NO: 96, an embodiment also referred to as TadA 7.10 (V106W). Its sequence is provided as SEQ ID NO: 97. [0132] In some embodiments, the adenosine deaminase comprises a R47X mutation in SEQ

ID NO: 86, or a corresponding mutation in another adenosine deaminase, where the presence of X indicates any amino acid other than the corresponding amino acid in the wild-type adenosine deaminase. In some embodiments, the adenosine deaminase comprises a R47Q,

R47F, R47W, or R47M mutation in SEQ ID NO: 86, or a corresponding mutation in another adenosine deaminase.

[0133] In particular embodiments, the adenosine deaminase comprises a R47Q, R47F, R47W, or R47M mutation in SEQ ID NO: 96.

[0134] In particular embodiments, the adenosine deaminase comprises a V106Q mutation and an N108W mutation in SEQ ID NO: 96. In particular embodiments, the adenosine deaminase comprises a V106W mutation, an N108W mutation, and an R47Z mutation, wherein Z is selected from the residues consisting of Q, F, W and M, in SEQ ID NO: 86.

[0135] It should be appreciated that any of the mutations provided herein ( e.g ., based on the ecTadA amino acid sequence of SEQ ID NO: 86) may be introduced into other adenosine deaminases, such as S. aureus TadA (saTadA), or other adenosine deaminases (e.g., bacterial adenosine deaminases), such as those sequences provided below. See FIG. 14. It would be apparent to the skilled artisan how to identify amino acid residues from other adenosine deaminases that are homologous to the mutated residues in ecTadA. Thus, any of the mutations identified in ecTadA may be made in other adenosine deaminases that have homologous amino acid residues. It should also be appreciated that any of the mutations provided herein may be made individually or in any combination in ecTadA or another adenosine deaminase. For example, an adenosine deaminase may contain a D108N, a A106V, and/or a R47Q mutation in ecTadA SEQ ID NO: 86, or a corresponding mutation in another adenosine deaminase.

[0136] In some embodiments, the adenosine deaminase comprises one, two, or three mutations selected from the group consisting of D108, A106, and R47 in SEQ ID NO: 86, or a corresponding mutation or mutations in another adenosine deaminase. In some

embodiments, the adenosine deaminase comprises one, two, or three substitutions selected from the group consisting of D108W, A106W, and R47Q in SEQ ID NO: 86, or a corresponding mutation or mutations in another adenosine deaminase. An adenosine deaminase domain comprising TadA(E59W) and TadA7.10(V106W) generated particularly low levels of off-target effects. Another adenosine deaminase domain comprising

TadA(E59W) and TadA7.10(N108W) generated particularly low levels of off-target effects.

[0137] In other aspects, the disclosure provides adenine base editors with broadened target sequence compatibility. In general, native ecTadA deaminates the adenine in the sequence UAC (e.g., the target sequence) of the anticodon loop of tRNA ^Arg . Without wishing to be bound by any particular theory, in order to expand the utility of ABEs comprising one or more ecTadA deaminases, such as any of the adenosine deaminases provided herein, the adenosine deaminase proteins were optimized to recognize a wide variey of target sequences within the protospacer sequence without compromising the editing efficiency of the adenosine nucleobase editor complex. In some embodiments, the target sequence is an A in the middle of a 5'-NAN-3' sequence, wherein N is T, C, G, or A. In some embodiments, the target sequence comprises 5'-TAC-3'. In some embodiments, the target sequence comprises

5'-GAA-3'.

[0138] In some embodiments, the adenosine deaminase is an N-terminal truncated E. coli TadA. In certain embodiments, the adenosine deaminase comprises the amino acid sequence: MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPT AHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKT GAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD

(SEQ ID NO: 86).

[0139] In some embodiments, the TadA deaminase is a full-length E. coli TadA deaminase (ecTadA). For example, in certain embodiments, the adenosine deaminase comprises the amino acid sequence:

MRRAFIT G VFFLS E VEF S HE YWMRH ALTLAKRA WDERE VP V G A VLVHNNRVIGEG WNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIG RVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEI KAQKKAQS STD (SEQ ID NO: 87)

[0140] It should be appreciated, however, that additional adenosine deaminases useful in the present application would be apparent to the skilled artisan and are within the scope of this disclosure. For example, the adenosine deaminase may be a homolog of an AD AT.

Exemplary AD AT homologs include, without limitation:

[0141] Staphylococcus aureus TadA:

MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAH AEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCS GS LMNLLQQS NFNHRAIVDKG VLKE AC S TLLTTFFKNLRANKKS TN (SEQ ID NO:

88)

[0142] Bacillus subtilis TadA:

MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEQRSIAHAEML

VID E AC KALGT WRLEG ATLY VTLEPCPMC AG A V VLS R VEK V VF G AFDPKGGC S GTLMN

LLQEERFNHQ AE V V S G VLEEEC GGMLS AFFRELRKKKK A ARKNLS E (SEQ ID NO:

89)

[0143] Salmonella typhimurium (S. typhimurium ) TadA:

MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEG WNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIG RVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIK ALKKADRAEGAGPAV (SEQ ID NO: 90)

[0144] Shewanella putrefaciens (S. putrefaciens ) TadA:

MDE YWMQVAMQM AEKAEAAGE VPV GA VLVKDGQQIATGYNLS IS QHDPT AHAEI LCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGT VVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE

(SEQ ID NO: 91)

[0145] Haemophilus influenzae F3031 (H. influenzae ) TadA:

MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQS DPT AH AEIIALRN G AKNIQN YRLLN S TLY VTLEPCTMC AG AILHS RIKRLVFG AS D YK TGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSD

K (SEQ ID NO: 92)

[0146] Caulobacter crescentus ( C . crescentus) TadA:

MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAH DPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADD PKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI (SEQ ID NO: 93)

[0147] Geobacter sulfurreducens (G. sulfurreducens) TadA:

MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSN DPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDP KGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALF IDERKVPPEP (SEQ ID NO: 94)

[0148] Streptococcus pyogenes ( S . pyogenes ) TadA

MPYSLEEQTYFMQEALKEAEKSLQKAEIPIGCVIVKDGEIIGRGHNAREESNQAIMHA EIMAINEANAHEGNWRLLDTTLFVTIEPCVMCSGAIGLARIPHVIYGASNQKFGGADS LYQILTDERLNHRVQVERGLLAADCANIMQTFFRQGRERKKIAKHLIKEQSDPFD

(SEQ ID NO: 110)

[0149] Exemplary adenosine deaminase variants of the disclosure are described below. In certain embodiments, the adenosine deaminase has a sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% sequence identity to one of the following:

[0150] (Ec)TadA, catalytically inactive (E59A)

MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPT AHAAIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAK TG A AGS LMD VLHHPGMNHRVEITEGILADEC A ALLS DFFRMRRQEIKAQKK AQS S T

D (SEQ ID NO: 95)

[0151] TadA 7.10

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPT AHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKT GAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

(SEQ ID NO: 96)

[0152] TadA 7.10 (V106W)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPT AHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNAK TGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSST

D (SEQ ID NO: 97)

[0153] TadA 7.10 (N108W)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPT AHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRWAK TGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSST

D (SEQ ID NO: 98)

[0154] TadA 7.10 (N108Q)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPT AHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRQAKT GAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

(SEQ ID NO: 99)

[0155] TadA 7.10 (V106F)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPT AHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGFRNAKT GAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

(SEQ ID NO: 100)

[0156] TadA 7.10 (V106Q)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPT

AHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGQRNAKT GAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

(SEQ ID NO: 101)

[0157] TadA 7.10 (V106M)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPT AHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGMRNAK TGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSST

D (SEQ ID NO: 102)

[0158] TadA 7.10 (R47F)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNFAIGLHDPT AHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKT GAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

(SEQ ID NO: 103)

[0159] TadA 7.10 (R47W)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNWAIGLHDPT AHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKT GAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

(SEQ ID NO: 104)

[0160] TadA 7.10 (R47Q)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNQAIGLHDPT AHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKT GAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

(SEQ ID NO: 105)

[0161] TadA 7.10 (R47M)

MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNMAIGLHDPT AHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKT GAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

(SEQ ID NO: 106)

[0162] TadA (E59Q)

MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPT AHAQIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAK TG A AGS LMD VLHHPGMNHRVEITEGILADEC A ALLS DFFRMRRQEIKAQKK AQS S T

D (SEQ ID NO: 107)

[0163] Any two or more of the adenosine deaminases described herein may be connected to one another (e.g. by a linker) within an adenosine deaminase domain of the fusion proteins provided herein. For instance, the fusion proteins provided herein may contain only two adenosine deaminases. In some embodiments, the adenosine deaminases are the same. In some embodiments, the adenosine deaminases are any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminases are different. In some

embodiments, the first adenosine deaminase is any of the adenosine deaminases provided herein, and the second adenosine is any of the adenosine deaminases provided herein, but is not identical to the first adenosine deaminase. In some embodiments, the fusion protein comprises two adenosine deaminases ( e.g a first adenosine deaminase and a second adenosine deaminase). In some embodiments, the fusion protein comprises a first adenosine deaminase and a second adenosine deaminase. In some embodiments, the first adenosine deaminase is N-terminal to the second adenosine deaminase in the fusion protein. In some embodiments, the first adenosine deaminase is C-terminal to the second adenosine deaminase in the fusion protein. In some embodiments, the first adenosine deaminase and the second deaminase are fused directly or via a linker.

[0164] In particular embodiments, the base editors disclosed herein comprise a heterodimer of a first adenosine deaminase that is N-terminal to a second adenosine deaminase, wherein the first adenosine deaminase comprises a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 95; and the second adenosine deaminase comprises a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 97.

[0165] In other embodiments, the second adenosine deaminase of the base editors provided herein comprises a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 96 (TadA 7.10), wherein any sequence variation may only occur in amino acid positions other than R47, V106 or N108 of SEQ ID NO: 96. In other words, these embodiments must contain amino acid substitutions at R47, V106 or N108 of SEQ ID NO: 96.

[0166] In other embodiments, the second adenosine deaminase of the heterodimer comprises a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 107. In other embodiments, second adenosine deaminase comprises a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NOs: 98 or 99. In other embodiments, second adenosine deaminase comprises a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% sequence identity to a sequence selected from SEQ ID NOs: 100-102. In other embodiments, second adenosine deaminase comprises a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% sequence identity to a sequence selected from SEQ ID NOs: 103-106. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,

39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations compared to any one of the amino acid sequences set forth in SEQ ID NOs: 96-107, and 110 (e.g., TadA7.10), or any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase comprises an amino acid sequence that has at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, or at least 170 identical contiguous amino acid residues as compared to any one of the amino acid sequences set forth in SEQ ID NOs: 96-107 and 110 (e.g., TadA7.10), or any of the adenosine deaminases provided herein. napDNAbp domains

[0167] The adenine base editors described herein comprise a nucleic acid programmable DNA binding (napDNAbp) domain. The napDNAbp is associated with at least one guide nucleic acid (e.g., guide RNA), which localizes the napDNAbp to a DNA sequence that comprises a DNA strand (i.e., a target strand) that is complementary to the guide nucleic acid, or a portion thereof (e.g., the protospacer of a guide RNA). In other words, the guide nucleic- acid“programs” the napDNAbp domain to localize and bind to a complementary sequence of the target strand. Binding of the napDNAbp domain to a complementary sequence enables the nucleobase modification domains (e.g., adenosine deaminase domain) of the base editor to access and enzymatically deaminate a target adenine base in the target strand.

[0168] The napDNAbp domain can be a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. As outlined above, CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences

complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer.

The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3 '-5' exonucleolytically. In nature, DNA-binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply“gNRA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek et al., Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference.

[0169] Without wishing to be bound by any particular theory, the binding mechanism of a napDNAbp - guide RNA complex, in general, includes the step of forming an R-loop whereby the napDNAbp induces the unwinding of a double-strand DNA target, thereby separating the strands in the region bound by the napDNAbp. The guideRNA protospacer then hybridizes to the“target strand.” This displaces a“non-target strand” that is

complementary to the target strand, which forms the single strand region of the R-loop. In some embodiments, the napDNAbp includes one or more nuclease activities, which cuts the DNA leaving various types of lesions (e.g., a nick in one strand of the DNA). For example, the napDNAbp may comprises a nuclease activity that cuts the non-target strand at a first location, and / or cuts the target strand at a second location. Depending on the nuclease activity, the target DNA can be cut to form a“double- stranded break” whereby both strands are cut. In other embodiments, the target DNA can be cut at only a single site, i.e., the DNA is“nicked” on one strand.

[0170] The below description of various napDNAbps which can be used in connection with the disclosed nucleobase modification domains (adenosine deaminase domains) is not meant to be limiting in any way. The base editors may comprise the canonical SpCas9, or any ortholog Cas9 protein, or any variant Cas9 protein— including any naturally occurring variant, mutant, or otherwise engineered version of Cas9— that is known or which can be made or evolved through a directed evolution or otherwise mutagenic process. In various embodiments, the napDNAbp has a nickase activity, i.e., only cleave one strand of the target DNA sequence. In other embodiments, the napDNAbp has an inactive nuclease, e.g., are “dead” proteins. Other variant Cas9 proteins that may be used are those having a smaller molecular weight than the canonical SpCas9 (e.g., for easier delivery) or having modified or rearranged primary amino acid sequence (e.g., the circular permutant forms). The base editors described herein may also comprise Cas9 equivalents, including Casl2a/Cpfl and Casl2b proteins. The napDNAbps used herein (e.g., an SpCas9 or SpCas9 variant) may also may also contain various modifications that alter/enhance their PAM specifities. The disclosure contemplates any Cas9, Cas9 variant, or Cas9 equivalent which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% sequence identity to a reference Cas9 sequence, such as a reference SpCas9 canonical sequence (set forth in SEQ ID NO: 141), a reference SaCas9 canonical sequence (set forth in SEQ ID NO: 127) or a reference Cas9 equivalent (e.g., Casl2a/Cpfl). [0171] In some embodiments, the napDNAbp directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the

complement of the target sequence. In some embodiments, the napDNAbp directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A in reference to the canonical SpCas9 sequence, or to equivalent amino acid positions in other Cas9 variants or Cas9 equivalents.

[0172] As used herein, the term“Cas protein” refers to a full-length Cas protein obtained from nature, a recombinant Cas protein having a sequences that differs from a naturally occurring Cas protein, or any fragment of a Cas protein that nevertheless retains all or a significant amount of the requisite basic functions needed for the disclosed methods, i.e., (i) possession of nucleic-acid programmable binding of the Cas protein to a target DNA, and (ii) ability to nick the target DNA sequence on one strand. The Cas proteins contemplated herein embrace CRISPR Cas9 proteins, as well as Cas9 equivalents, variants (e.g., Cas9 nickase

(nCas9) or nuclease inactive Cas9 (dCas9)) homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and may include a Cas9 equivalent from any type of CRISPR system (e.g., type II, V, VI), including

Cpfl (a type-V CRISPR-Cas systems), C2cl (a type V CRISPR-Cas system), C2c2 (a type

VI CRISPR-Cas system) and C2c3 (a type V CRISPR-Cas system). Further Cas-equivalents are described in Makarova et ah,“C2c2 is a single-component programmable RNA-guided

RNA-targeting CRISPR effector,” Science 2016; 353(6299), the contents of which are incorporated herein by reference.

[0173] The term“Cas9” or“Cas9 domain” embraces any naturally occurring Cas9 from any organism, any naturally-occurring Cas9 equivalent or functional fragment thereof, any Cas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a Cas9, naturally-occurring or engineered. The term Cas9 is not meant to be particularly limiting and may be referred to as a“Cas9 or equivalent.” Exemplary Cas9 proteins are further described herein and/or are described in the art and are incorporated herein by reference. The present disclosure is unlimited with regard to the particular napDNAbp that is employed in the base editors of the disclosure.

[0174] Additional Cas9 sequences and structures are well known to those of skill in the art (see, e.g.,“Complete genome sequence of an Ml 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 L, Hauer M., Doudna J.A., Charpentier E. Science 337:816-

821(2012), the entire contents of each of which are incorporated herein by reference), and also provided below.

[0175] Examples of Cas9 and Cas9 equivalents are provided as follows; however, these specific examples are not meant to be limiting. The base editors of the present disclosure may use any suitable napDNAbp, including any suitable Cas9 or Cas9 equivalent.

Wild type canonical SpCas9

[0176] In one embodiment, the base editor constructs described herein may comprise the “canonical SpCas9” nuclease from S. pyogenes, which has been widely used as a tool for genome engineering. This Cas9 protein is a large, multi-domain protein containing two distinct nuclease domains. Point mutations can be introduced into Cas9 to abolish one or both nuclease activities, resulting in a nickase Cas9 (nCas9) or dead Cas9 (dCas9), respectively, that still retains its ability to bind DNA in a sgRNA-programmed manner. In principle, when fused to another protein or domain, Cas9 or variant thereof (e.g., nCas9) can target that protein to virtually any DNA sequence simply by co-expression with an appropriate sgRNA. As used herein, the canonical SpCas9 protein refers to the wild type protein from

Streptococcus pyogenes having the following amino acid sequence:

_ _

[0177] The base editors described herein may include canonical SpCas9, or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with a wild type Cas9 sequence provided above. These variants may include SpCas9 variants containing one or more mutations, including any known mutation reported with the

SwissProt Accession No. Q99ZW2 entry, which include:

[0178] Other wild type SpCas9 sequences that may be used in the present disclosure, include:

[0179] The base editors described herein may include any of the above SpCas9 sequences, or any variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

Wild type Cas9 orthologs

[0180] In other embodiments, the Cas9 protein can be a wild type Cas9 ortholog from another bacterial species. For example, the following Cas9 orthologs can be used in connection with the base editor constructs described in this disclosure. In addition, any variant Cas9 orthologs having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any of the below orthologs may also be used with the disclosed base editors.

[0181] The base editors described herein may include any of the above Cas9 ortholog sequences, or any variants thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0182] The napDNAbp may include any suitable homologs and/or orthologs or naturally occurring enzymes, such as Cas9. Cas9 homologs and/or orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus . Preferably, the Cas moiety is configured (e.g, mutagenized, recombinantly engineered, or otherwise obtained from nature) as a nickase, i.e., capable of cleaving only a single strand of the target doubpdditional 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. In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 3. In some embodiments, the Cas9 protein comprises an amino acid sequence that is 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 protein as provided by any one of the Cas9 orthologs in the above tables. Dead napDNAbp variants

[0183] In some embodiments, the disclosed base editors may comprise a catalytically inactive, or“dead,” napDNAbp domain. Exemplary catalytically inactive domains in the disclosed base editors are dead S. pyogenes Cas9 (dSpCas9) and S. pyogenes Cas9 nickase (SpCas9n).

[0184] In certain embodiments, the base editors described herein may include a dead Cas9, e.g., dead SpCas9, which has no nuclease activity due to one or more mutations that inactivate both nuclease domains of SpCas9, namely the RuvC domain (which cleaves the non-protospacer DNA strand) and HNH domain (which cleaves the protospacer DNA strand). The nuclease inactivation may be due to one or mutations that result in one or more substitutions and/or deletions in the amino acid sequence of the encoded protein, or any variants thereof having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0185] In certain embodiments, the base editors described herein may include a dead Cas9, e.g., dead SpCas9, which has no nuclease activity due to one or more mutations that inactivate both nuclease domains of SpCas9, namely the RuvC domain (which cleaves the non-protospacer DNA strand) and HNH domain (which cleaves the protospacer DNA strand). The D10A and N580A mutations in the wild-type S. aureus Cas9 amino acid sequence may be used to form a dSaCas9. Accordingly, in some embodiments, the napDNAbp domain of the base editors provided herein comprises a dSaCas9 that has D10A and N580A mutations relative to the wild-type SaCas9 sequence (SEQ ID NO: 127).

[0186] As used herein, the term“dCas9” refers to a nuclease-inactive Cas9 or nuclease-dead Cas9, or a functional fragment thereof, and embraces any naturally occurring dCas9 from any organism, any naturally-occurring dCas9 equivalent or functional fragment thereof, any dCas9 homolog, ortholog, or paralog from any organism, and any mutant or variant of a dCas9, naturally-occurring or engineered. The term dCas9 is not meant to be particularly limiting and may be referred to as a“dCas9 or equivalent.” Exemplary dCas9 proteins and method for making dCas9 proteins are further described herein and/or are described in the art and are incorporated herein by reference.

[0187] In other 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. In other embodiments, Cas9 variants having mutations other than D10A and H840A are provided which may result in the full or partial inactivate of the endogenous Cas9 nuclease activity (e.g., nCas9 or dCas9, respectively). Such mutations, by way of example, include other amino acid substitutions at DIO and H820, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and/or the RuvCl subdomain) with reference to a wild type sequence such as Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1). In some embodiments, variants or homologues of Cas9 (e.g., variants of Cas9 from Streptococcus pyogenes (NCBI Reference

Sequence: NC_017053.1)) 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 NCBI Reference Sequence: NC_017053.1. In some embodiments, variants of dCas9 (e.g., variants of NCBI Reference Sequence: NC_017053.1) are provided having amino acid sequences which are shorter, or longer than NC_017053.1 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.

[0188] In some embodiments, the napDNAbp domain of any of the disclosed base editors comprises a dead S. pyogenes Cas9 (dSpCas9). In some embodiments, the napDNAbp domain of any of the disclosed based editors is comprises at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 108. In some embodiments, the napDNAbp domain of any of the disclosed base editors comprises the amino acid sequence of SEQ ID NO: 108.

[0189] In one embodiment, the dead Cas9 may be based on the canonical SpCas9 sequence of Q99ZW2 and may have the following sequence, which comprises a D10A and an H810A substitutions (underlined and bolded), or a variant of SEQ ID NO: 108 having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto:

napDNAbp nickase variants

[0190] In some embodiments, the disclosed base editors may comprise a napDNAbp domain that comprises a nickase. In some embodments, the base editors described herein comprise a Cas9 nickase. The term“Cas9 nickase” of“nCas9” refers to a variant of Cas9 which is capable of introducing a single-strand break in a double strand DNA molecule target. In some embodiments, the Cas9 nickase comprises only a single functioning nuclease domain. The wild type Cas9 (e.g., the canonical SpCas9) comprises two separate nuclease domains, namely, the RuvC domain (which cleaves the non-protospacer DNA strand) and HNH domain (which cleaves the protospacer DNA strand). In one embodiment, the Cas9 nickase comprises a mutation in the RuvC domain which inactivates the RuvC nuclease activity. For example, mutations in aspartate (D) 10, histidine (H) 983, aspartate (D) 986, or glutamate (E)

762, have been reported as loss-of-function mutations of the RuvC nuclease domain and the creation of a functional Cas9 nickase (e.g., Nishimasu et ak,“Crystal structure of Cas9 in complex with guide RNA and target DNA,” Cell 156(5), 935-949, which is incorporated herein by reference). Thus, nickase mutations in the RuvC domain could include D10X,

H983X, D986X, or E762X, wherein X is any amino acid other than the wild type amino acid.

In certain embodiments, the nickase could be D10A, of H983A, or D986A, or E762A, or a combination thereof.

[0191] In some embodiments, the napDNAbp domain of any of the disclosed base editors comprises an S. pyogenes Cas9 nickase (SpCas9n). In some embodiments, the napDNAbp domain of any of the disclosed based editors is comprises at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 109 or 153. In some embodiments, the napDNAbp domain of any of the disclosed base editors comprises the amino acid sequence of SEQ ID NO: 109. In some embodiments, the napDNAbp domain of any of the disclosed base editors comprises the amino acid sequence of SEQ ID NO: 153.

[0192] In some embodiments, the napDNAbp domain of any of the disclosed base editors comprises an S. aureus Cas9 nickase (SaCas9n). In some embodiments, the napDNAbp domain of any of the disclosed based editors is comprises at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 151. In some embodiments, the napDNAbp domain of any of the disclosed base editors comprises the amino acid sequence of SEQ ID NO: 151.

[0193] In various embodiments, the Cas9 nickase can having a mutation in the RuvC nuclease domain and have one of the following amino acid sequences, or a variant thereof having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

[0194] In another embodiment, the Cas9 nickase comprises a mutation in the HNH domain which inactivates the HNH nuclease activity. For example, mutations in histidine (H) 840 or asparagine (R) 863 have been reported as loss-of-function mutations of the HNH nuclease domain and the creation of a functional Cas9 nickase (e.g., Nishimasu et ah,“Crystal structure of Cas9 in complex with guide RNA and target DNA,” Cell 156(5), 935-949, which is incorporated herein by reference). Thus, nickase mutations in the HNH domain could include H840X and R863X, wherein X is any amino acid other than the wild type amino acid.

In certain embodiments, the nickase could be H840A or R863A or a combination thereof.

[0195] In various embodiments, the Cas9 nickase can have a mutation in the HNH nuclease domain and have one of the following amino acid sequences, or a variant thereof having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least

99% sequence identity thereto.

[0196] In some embodiments, the N-terminal methionine is removed from a Cas9 nickase, or from any Cas9 variant, ortholog, or equivalent disclosed or contemplated herein. For example, methionine-minus Cas9 nickases include the following sequences, or a variant thereof having an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.

Other Cas9 variants

[0197] The napDNAbp domains used in the base editors described herein may also include other Cas9 variants that area 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 any reference Cas9 protein, including any wild type Cas9, or mutant Cas9 (e.g., a dead Cas9 or Cas9 nickase), or circular permutant Cas9, or other variant of Cas9 disclosed herein or known in the art. In some embodiments, a 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 a reference Cas9. In some embodiments, the Cas9 variant comprises a fragment of a reference 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 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 (e.g., SEQ ID NO: 141).

[0198] In some embodiments, the disclosure also may utilize Cas9 fragments which retain their functions and which are fragments of any herein disclosed Cas9 protein. In some embodiments, the Cas9 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.

[0199] In various embodiments, the base editors disclosed herein may comprise one of the Cas9 variants described as follows, or a Cas9 variant thereof having 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 any reference Cas9 variants.

Other Cas9 equivalents

[0200] In some embodiments, the base editors described herein can include any Cas9 equivalent. As used herein, the term“Cas9 equivalent” is a broad term that encompasses any napDNAbp protein that serves the same function as Cas9 in the present base editors despite that its amino acid primary sequence and/or its three-dimensional structure may be different and/or unrelated from an evolutionary standpoint. Thus, while Cas9 equivalents include any Cas9 ortholog, homolog, mutant, or variant described or embraced herein that are

evolutionarily related, the Cas9 equivalents also embrace proteins that may have evolved through convergent evolution processes to have the same or similar function as Cas9, but which do not necessarily have any similarity with regard to amino acid sequence and/or three dimensional structure. The base editors described here embrace any Cas9 equivalent that would provide the same or similar function as Cas9 despite that the Cas9 equivalent may be based on a protein that arose through convergent evolution.

[0201] For example, CasX is a Cas9 equivalent that reportedly has the same function as Cas9 but which evolved through convergent evolution. Thus, the CasX protein described in Liu et al.,“CasX enzymes comprises a distinct family of RNA-guided genome editors,” Nature, 2019, Vol.566: 218-223, is contemplated to be used with the base editors described herein.

In addition, any variant or modification of CasX is conceivable and within the scope of the present disclosure.

[0202] Cas9 is a bacterial enzyme that evolved in a wide variety of species. However, the Cas9 equivalents contemplated herein may also be obtained from archaea, which constitute a domain and kingdom of single-celled prokaryotic microbes different from bacteria.

[0203] In some embodiments, Cas9 equivalents may refer to CasX or CasY, which have been described in, for example, Burstein et ah,“New CRISPR-Cas systems from

uncultivated microbes.” Cell Res. 2017 Feb 21. doi: 10.1038/cr.2017.21, the entire contents of which is hereby incorporated by reference. Using genome-resolved metagenomics, a number of CRISPR-Cas systems were identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in little- studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems were discovered, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. In some embodiments, Cas9 refers to CasX, or a variant of CasX. In some embodiments, Cas9 refers to a CasY, or a variant of CasY. It should be appreciated that other RNA-guided DNA binding proteins may be used as a nucleic acid programmable DNA binding protein (napDNAbp), and are within the scope of this disclosure. Also see Liu et ah, “CasX enzymes comprises a distinct family of RNA-guided genome editors,” Nature, 2019, Vol.566: 218-223. Any of these Cas9 equivalents are contemplated.

[0204] In some embodiments, the Cas9 equivalent comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring CasX or CasY protein. In some embodiments, the napDNAbp is a naturally-occurring CasX or CasY protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a wild-type Cas moiety or any Cas moiety provided herein.

[0205] In various embodiments, the nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpfl, C2cl, C2c2, C2C3, Argonaute, Casl2a, and Casl2b. One example of a nucleic acid programmable DNA- binding protein that has different PAM specificity than Cas9 is Clustered Regularly

Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (Cpfl). Similar to Cas9, Cpfl is also a class 2 CRISPR effector. It has been shown that Cpfl mediates robust DNA interference with features distinct from Cas9. Cpfl is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif (TTN,

TTTN, or YTN). Moreover, Cpfl cleaves DNA via a staggered DNA double-stranded break.

Out of 16 Cpfl-family proteins, two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells. Cpfl proteins are known in the art and have been described previously, for example Yamano et ak,“Crystal structure of Cpfl in complex with guide RNA and target DNA.” Cell (165) 2016, p. 949-962; the entire contents of which is hereby incorporated by reference. The state of the art may also now refer to Cpfl enzymes as Casl2a.

[0206] In still other embodiments, the Cas protein may include any CRISPR associated protein, including but not limited to Casl2a, Casl2b, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (sometimes referred to as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2. Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof, and preferably comprising a nickase mutation (e.g., a mutation corresponding to the D10A mutation of the wild type SpCas9 polypeptide of SEQ ID NO: 141).

[0207] In various other embodiments, the napDNAbp can be any of the following proteins: a Cas9, a Cpfl, a CasX, a CasY, a C2cl, a C2c2, a C2c3, a GeoCas9, a CjCas9, a Casl2a, a Casl2b, a Casl2g, a Casl2h, a Casl2i, a Casl3b, a Casl3c, a Casl3d, a Casl4, a Csn2, an xCas9, an SpCas9-NG, a circularly permuted Cas9, or an Argonaute (Ago), a Cas9-KKH, a SmacCas9, a Spy-macCas9, an SpCas9-VRQR, an SpCas9-NRRH, an SpaCas9-NRTH, an SpCas9-NRCH, or a variant thereof.

[0208] In certain embodiments, the base editors contemplated herein can include a Cas9 protein that is of smaller molecular weight than the canonical SpCas9 sequence. In some embodiments, the smaller-sized Cas9 variants may facilitate delivery to cells, e.g., by an expression vector, nanoparticle, or other means of delivery. The canonical SpCas9 protein is 1368 amino acids in length and has a predicted molecular weight of 158 kilodaltons. The term“small-sized Cas9 variant”, as used herein, refers to any Cas9 variant— naturally occurring, engineered, or otherwise— that is less than at least 1300 amino acids, or at least less than 1290 amino acids, or than less than 1280 amino acids, or less than 1270 amino acid, or less than 1260 amino acid, or less than 1250 amino acids, or less than 1240 amino acids, or less than 1230 amino acids, or less than 1220 amino acids, or less than 1210 amino acids, or less than 1200 amino acids, or less than 1190 amino acids, or less than 1180 amino acids, or less than 1170 amino acids, or less than 1160 amino acids, or less than 1150 amino acids, or less than 1140 amino acids, or less than 1130 amino acids, or less than 1120 amino acids, or less than 1110 amino acids, or less than 1100 amino acids, or less than 1050 amino acids, or less than 1000 amino acids, or less than 950 amino acids, or less than 900 amino acids, or less than 850 amino acids, or less than 800 amino acids, or less than 750 amino acids, or less than

700 amino acids, or less than 650 amino acids, or less than 600 amino acids, or less than 550 amino acids, or less than 500 amino acids, but at least larger than about 400 amino acids and retaining the required functions of the Cas9 protein.

[0209] In various embodiments, the base editors disclosed herein may comprise one of the small-sized Cas9 variants described as follows, or a Cas9 variant thereof having 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 any reference small-sized Cas9 protein. Exemplary small-sized Cas9 variants include, but are not limited to, SaCas9 and LbCas l2a.

[0210] In some embodiments, the base editors described herein may also comprise

Cas l2a/Cpfl (dCpfl) variants that may be used as a guide nucleotide sequence- programmable DNA-binding protein domain. The Cas l2a/Cpfl protein has a RuvC-like endonuclease domain that is similar to the RuvC domain of Cas9 but does not have a HNH endonuclease domain, and the N-terminal of Cpfl does not have the alpha-helical recognition lobe of Cas9. It was shown in Zetsche et al., Cell, 163, 759-771, 2015 (which is incorporated herein by reference) that, the RuvC-like domain of Cpfl is responsible for cleaving both

DNA strands and inactivation of the RuvC-like domain inactivates Cpfl nuclease activity.

[0211] Additional exemplary Cas9 equivalent protein sequences can include the following: _ _

napDNAbps that recognize non-canonical PAM sequences

[0212] In some embodiments, the napDNAbp is a nucleic acid programmable DNA binding protein that does not require a canonical (NGG) PAM sequence. In some embodiments, the napDNAbp is an argonaute protein. One example of such a nucleic acid programmable DNA binding protein is an Argonaute protein from Natronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guided endonuclease. NgAgo binds 5' phosphorylated ssDNA of ~24 nucleotides (gDNA) to guide it to its target site and will make DNA double-strand breaks at the gDNA site. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer-adjacent motif (PAM). Using a nuclease inactive NgAgo (dNgAgo) can greatly expand the bases that may be targeted. The characterization and use of NgAgo have been described in Gao et al, Nat Biotechnol., 2016 Jul;34(7):768-73. PubMed RMP3: 27136078; Swarts et al., Nature. 507(7491) (2014):258-61; and Swarts et al., Nucleic Acids Res. 43(10) (2015):5120-9, each of which is incorporated herein by reference.

[0213] In some embodiments, the disclosure provides napDNAbp domains that comprise SpCas9 variants that recognize and work best with NRRH, NRCH, and NRTH PAMs. See PCT Application No. PCT/US2019/47996, incorporated by reference herein. In some embodiments, the disclosed base editors comprise a napDNAbp domain selected from SpCas9-NRRH, SpCas9-NRTH, and SpCas9-NRCH.

[0214] In some embodiments, the disclosed base editors comprise a napDNAbp domain that has a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to SpCas9-NRRH. In some embodiments, the disclosed base editors comprise a napDNAbp domain that comprises SpCas9-NRRH. The SpCas9-NRRH has an amino acid sequence as presented in SEQ ID NO: 176 (underligned residues are mutated relative to SpCas9, as set forth in SEQ ID NO: 141): MDKKY S IGLDIGTNS VGW A VITDEYKVPS KKFKVLGNTDRHS IKKNLIGALLFDS GE

T AE ATRLKRT ARRR YTRRKNRIC YLQEIF S NEM AKVDDS FFHRLEES FLVEEDKKHE

RHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIE G

DLNPDN S D VDKLFIQLV QT YN QLFEENPIN AS G VD AKAILS ARLS KS RRLENLIAQLP

GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA

DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDEHHQDLTLLKALVRQQLP E

KYKEIFFDQS KN GY AGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR

TFDN GIIPHQIHLGELHAILRRQGDFYPFLKDNREKIEKILTFRIPYY V GPLARGNSRFA

WMTRKS EETFTPWNFEE V VDKG AS AQS FIERMTNFDKNLPNEK VLPKHS LL YE YFT V

YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKffiCFD

SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIE ERL

KTYAHLFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN

FMQLIHDDS LTFKEDIQKAQV S GQGDS LHEHIANLAGSPAIKKGILQT VKVVDELVK

VMGGHKPENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQL

QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK

NRGKS DN VPS EE V VKKMKN YWRQLLN AKLIT QRKFDNLTKAERGGLS ELD KAGFIK

RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV

REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK

AT AKYFF Y S NIMNFFKTEITLAN GEIRKRPLIETN GET GEIVWDKGRDF AT VRKVLS M

PQ VNIVKKTE V QTGGFS KES ILPKGN S DKLIARKKD WDPKKY GGFN S PT A AY S VLV V

AKVEKGKS KKLKS VKELLGITIMERS S FEKNPIGFLE AKG YKE VKKDLIIKLPKY S LFE

LENGRKRMLASAGVLHKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ

HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNL GV

PAAFKYFDTTIDKKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO:

176).

[0215] In some embodiments, the disclosed base editors comprise a napDNAbp domain that has a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to SpCas9-NRCH. In some embodiments, the disclosed base editors comprise a napDNAbp domain that comprises SpCas9-NRCH. The SpCas9-NRCH has an amino acid sequence as presented in SEQ ID NO: 177 (underlined residues are mutated relative to SpCas9):

MDKKY S IGLDIGTNS VGW A VITDEYKVPS KKFKVLGNTDRHS IKKNLIGALLFDS GE T AE ATRLKRT ARRR YTRRKNRIC YLQEIF S NEM AKVDDS FFHRLEES FLVEEDKKHE RHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDN S D VDKLFIQLV QT YN QLFEENPIN AS GVD AKAILS ARLS KS RRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDEHHQDLTLLKALVRQQLPE

KYKEIFFDQS KN GY AGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR

TFDN GIIPHQIHLGELHAILRRQGDFYPFLKDNREKIEKILTFRIPYY V GPLARGNSRFA

WMTRKS EETErPWNFEE V VDKG AS AQS FIERMTNFDKNLPNEK VLPKHS LL YE YFT V

YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD

SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIE ERL

KTYAHLFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN

FMQLIHDDS LTFKEDIQKAQV S GQGDS LHEHIANLAGSPAIKKGILQT VKVVDELVK

VMGGHKPENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQL

QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK

NRGKS DN VPS EE V VKKMKN YWRQLLN AKLIT QRKFDNLTKAERGGLS ELD KAGFIK

RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV

REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK

AT AKYFF Y S NIMNFFKTEITLAN GEIRKRPLIETN GET GEIVWDKGRDF AT VRKVLS M

PQ VNIVKKTE V QTGGFS KES ILPKGN S DKLIARKKD WDPKKY GGFN S PT V AYS VLV V

AKVEKGKS KKLKS VKELLGITIMERS S FEKNPIDFLE AKG YKE VKKDLIIKLPKY S LFE

LENGRKRMLASAGVLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ

HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNL GA

PAAFKYFDTTINRKQYNTTKEVLDATLIRQSITGLYETRIDLSQLGGD (SEQ ID NO:

177).

[0216] In some embodiments, the disclosed base editors comprise a napDNAbp domain that has a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to SpCas9-NRTH. In some embodiments, the disclosed base editors comprise a napDNAbp domain that comprises SpCas9-NRTH. The SpCas9-NRTH has an amino acid sequence as presented in SEQ ID NO: 178 (underligned residues are mutated relative to SpCas9):

MDKKY S IGLDIGTNS VGW A VITDEYKVPS KKFKVLGNTDRHS IKKNLIGALLFDS GE T AE ATRLKRT ARRR YTRRKNRIC YLQEIF S NEM AKVDDS FFHRLEES FLVEEDKKHE RHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDN S D VDKLFIQLV QT YN QLFEENPIN AS G VD AKAILS ARLS KS RRLENLIAQLP GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYA DLFLAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDEHHQDLTLLKALVRQQLPE KYKEIFFDQS KN GY AGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR TFDN GIIPHQIHLGELHAILRRQGDFYPFLKDNREKIEKILTFRIPYY V GPLARGNSRFA WMTRKS EETITPWNFEE V VDKG AS AQS FIERMTNFDKNLPNEK VLPKHS LL YE YFT V YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL

KTYAHLFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN

FMQLIHDDS LTFKEDIQKAQV S GQGDS LHEHIANLAGSPAIKKGILQT VKVVDELVK

VMGGHKPENIVIEMAREN QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQL

QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK

NRGKS DN VPS EE V VKKMKN YWRQLLN AKLIT QRKFDNLTKAERGGLS ELD KAGFIK

RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV

REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK

AT AKYFF Y S NIMNFFKTEITLAN GEIRKRPLIETN GET GEIVWDKGRDF AT VRKVLS M

PQ VNIVKKTE V QTGGFS KES ILPKGN S DKLIARKKD WDPKKY GGFN S PT V AYS VLV V

AKVEKGKS KKLKS VKELLGITIMERS S FEKNPIGFLE AKG YKE VKKDLIIKLPKY S LFE

LEN GRKRMLAS AS VLHKGNELALPS KY VNFLYLAS H YEKLKGS S EDNKQKQLF VEQ

HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNL GA

S A AFKYFDTTIGRKL YT S TKE VLD ATLIHQS IT GLYETRIDLS QLGGD (SEQ ID NO:

178).

[0217] In other embodiments, the napDNAbp of any of the disclosed base editors comprises a Cas9 derived from a Streptococcus macacae, e.g., Streptococcus macacae NCTC 11558, or SmacCas9, or a variant thereof. In some embodiments, the napDNAbp comprises a hybrid variant of SmacCas9 that incorporates an SpCas9 domain with the SmacCas9 domain and is known as Spy-macCas9, or a variant thereof. In some embodiments, the napDNAbp comprises a hybrid variant of SmacCas9 that incorporates an increased nucleolytic variant of an SpCas9 (iSpy Cas9) domain and is known as iSpy-macCas9. Relative to Spymac-Cas9, iSpyMac-Cas9 contains two mutations, R221K and N394K, that were identified by deep mutational scans of Spy Cas9 that raise modification rates of the protein on most targets. See Jakimo et ah, bioRxiv, A Cas9 with Complete PAM Recognition for Adenine Dinucleotides (Sep 2018), herein incorporated by reference. Jakimo et al. showed that the hybrids Spy- macCas9 and iSpy-macCas9 recognize a short 5'-NAA-3' PAM and recognized all evaluated adenine dinucleotide PAM sequences and posseseds robust editing efficiency in human cells. Liu et al. engineered base editors containing Spy-mac Cas9, and demonstrated that cytidine and base editors containing Spymac domains can induce efficient C-to-T and A-to-G conversions in vivo. In addition, Liu et al. suggested that the PAM scope of Spy-mac Cas9 may be 5 '-T AAA-3', rather than 5'-NAA-3' as reported by Jakimo et al. See Liu et al. Cell Discovery (2019) 5:58, herein incorporated by reference.

[0218] In some embodiments, the disclosed base editors comprise a napDNAbp domain that has a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to iSpyMac-Cas9. In some embodiments, the disclosed base editors comprise a napDNAbp domain that comprises iSpyMac-Cas9. The iSpyMac-Cas9 has an amino acid sequence as presented in SEQ ID NO: 179 (R221K and N394K mutations are underlined):

DKKY S IGLDIGTN S V GW A VITDE YKVPS KKFKVLGNTDRHS IKKNLIG ALLFDS GET A

EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH

PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGD L

NPDN S D VDKLFIQLV QT YN QLFEENPIN AS G VD AKAILS ARLS KS RKLENLIAQLPGE

KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADL

FLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEK Y

KEIFFDQS KN GY AGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLKREDLLRKQRTF

DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AW

MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVY

NELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS

VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEE RL

KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN

FMQLIHDDS LTFKEDIQKAQV S GQGDS LHEHIANLAGSPAIKKGILQT VKVVDELVK

VMGRHKPENIVIEM AREN QTTQKGQKNSRERMKRIEEGIKELGS QILKEHP VENTQL

QNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK

NRGKS DN VPS EE V VKKMKN YWRQLLN AKLIT QRKFDNLTKAERGGLS ELD KAGFIK

RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV

REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK

AT AKYFF Y S NIMNFFKTEITLAN GEIRKRPLIETN GET GEIVWDKGRDF AT VRKVLS M

PQ VNIVKKTEIQT V GQN GGLFDDNPKS PLE VTPS KLVPLKKELNPKKY GG Y QKPTT A

YPVLLITDTKQLIPISVMNKKQFEQNPVKFLRDRGYQQVGKNDFIKLPKYTLVDIGD G

IKRLW ASS KEIHKGN QLV V S KKS QILLYH AHHLDS DLS ND YLQNHN QQFD VLFNEIIS

FS KKC KLGKEHIQKIEN V Y S NKKN S AS IEELAES FIKLLGFTQLG AT S PFNFLG VKLN Q

KQYKGKKD YILPCTEGTLIRQS ITGLYETRVDLS KIGED (SEQ ID NO: 179).

[0219] In other embodiments, the napDNAbp of any of the disclosed base editors is a prokaryotic homolog of an Argonaute protein. Prokaryotic homologs of Argonaute proteins are known and have been described, for example, in Makarova K., el al,“Prokaryotic homologs of Argonaute proteins are predicted to function as key components of a novel system of defense against mobile genetic elements”, Biol Direct. 2009 Aug 25;4:29. doi:

10.1186/1745-6150-4-29, the entire contents of which is hereby incorporated by reference. In some embodiments, the napDNAbp is a Marinitoga piezophila Argunaute (MpAgo) protein.

The CRISPR-associated Marinitoga piezophila Argunaute (MpAgo) protein cleaves single- stranded target sequences using 5'-phosphorylated guides. The 5' guides are used by all known Argonautes. The crystal structure of an MpAgo-RNA complex shows a guide strand binding site comprising residues that block 5' phosphate interactions. This data suggests the evolution of an Argonaute subclass with noncanonical specificity for a 5'-hydroxylated guide.

See, e.g., Kaya el al,“A bacterial Argonaute with noncanonical guide RNA specificity”,

Proc Natl Acad Sci USA. 2016 Apr 12;113(15):4057-62, the entire contents of which are hereby incorporated by reference). It should be appreciated that other argonaute proteins may be used, and are within the scope of this disclosure.

[0220] In some embodiments, the napDNAbp is a single effector of a microbial CRISPR-Cas system. Single effectors of microbial CRISPR-Cas systems include, without limitation, Cas9, Cpfl, C2cl, C2c2, and C2c3. Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multisubunit effector complexes, while Class 2 systems have a single protein effector. For example, Cas9 and Cpfl are Class 2 effectors. In addition to Cas9 and Cpfl, three distinct Class 2 CRISPR-Cas systems (C2cl, C2c2, and C2c3) have been described by Shmakov et ah,“Discovery and Functional

Characterization of Diverse Class 2 CRISPR Cas Systems”, Mol. Cell, 2015 Nov 5; 60(3): 385-397, the entire contents of which is hereby incorporated by reference. Effectors of two of the systems, C2cl and C2c3, contain RuvC-like endonuclease domains related to Cpfl. A third system, C2c2 contains an effector with two predicated HEPN RNase domains.

Production of mature CRISPR RNA is tracrRNA-independent, unlike production of CRISPR RNA by C2cl. C2cl depends on both CRISPR RNA and tracrRNA for DNA cleavage.

Bacterial C2c2 has been shown to possess a unique RNase activity for CRISPR RNA maturation distinct from its RNA-activated single- stranded RNA degradation activity. These RNase functions are different from each other and from the CRISPR RNA-processing behavior of Cpfl. See, e.g., East-Seletsky, et ah,“Two distinct RNase activities of CRISPR- C2c2 enable guide-RNA processing and RNA detection”, Nature , 2016 Oct

13;538(7624):270-273, the entire contents of which are hereby incorporated by reference. In vitro biochemical analysis of C2c2 in Leptotrichia shahii has shown that C2c2 is guided by a single CRISPR RNA and can be programed to cleave ssRNA targets carrying complementary protospacers. Catalytic residues in the two conserved HEPN domains mediate cleavage.

Mutations in the catalytic residues generate catalytically inactive RNA-binding proteins. See e.g., Abudayyeh et al.,“C2c2 is a single-component programmable RNA-guided RNA- targeting CRISPR effector”, Science, 2016 Aug 5; 353(6299), the entire contents of which are hereby incorporated by reference. [0221] The crystal structure of Alicyclobaccillus acidoterrastris C2cl (AacC2cl) has been reported in complex with a chimeric single-molecule guide RNA (sgRNA). See e.g., Liu el al,“C2cl-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism”,

Mol. Cell , 2017 Jan 19;65(2):310-322, the entire contents of which are hereby incorporated by reference. The crystal structure has also been reported in Alicyclobacillus acidoterrestris

C2cl bound to target DNAs as ternary complexes. See e.g., Yang el al.,“P AM-dependent

Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease”, Cell, 2016

Dec 15; 167(7): 1814-1828, the entire contents of which are hereby incorporated by reference.

Catalytically competent conformations of AacC2cl, both with target and non-target DNA strands, have been captured independently positioned within a single RuvC catalytic pocket, with C2cl -mediated cleavage resulting in a staggered seven-nucleotide break of target DNA.

Structural comparisons between C2cl ternary complexes and previously identified Cas9 and

Cpfl counterparts demonstrate the diversity of mechanisms used by CRISPR-Cas9 systems.

[0222] In some embodiments, the napDNAbp may be a C2cl, a C2c2, or a C2c3 protein. In some embodiments, the napDNAbp is a C2cl protein. In some embodiments, the napDNAbp is a C2c2 protein. In some embodiments, the napDNAbp is a C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least

90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least

97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring C2cl,

C2c2, or C2c3 protein. In some embodiments, the napDNAbp is a naturally-occurring C2cl,

C2c2, or C2c3 protein.

[0223] 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 base editors 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“editing window” or a“target 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 base editors 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.

[0224] For example, a napDNAbp domain with altered PAM specificity, such as a domain with at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with wild type Francisella novicida Cpfl (SEQ ID NO: 180) (D917, E1006, and D1255), which has the following amino acid sequence:

[0225] An additional napDNAbp domain with altered PAM specificity, such as a domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with wild type Geobacillus thermodenitrificans Cas9 (SEQ ID NO: 181), which has the following amino acid sequence:

[0226] In some embodiments, the nucleic acid programmable DNA binding protein

(napDNAbp) is a nucleic acid programmable DNA binding protein that does not require a canonical (NGG) PAM sequence. In some embodiments, the napDNAbp is an argonaute protein. One example of such a nucleic acid programmable DNA binding protein is an Argonaute protein from Natronobacterium gregoryi (NgAgo). NgAgo is a ssDNA-guided endonuclease. NgAgo binds 5' phosphorylated ssDNA of ~24 nucleotides (gDNA) to guide it to its target site and will make DNA double-strand breaks at the gDNA site. In contrast to Cas9, the NgAgo-gDNA system does not require a protospacer-adjacent motif (PAM).

Using a nuclease inactive NgAgo (dNgAgo) can greatly expand the bases that may be targeted. The characterization and use of NgAgo have been described in Gao et al, Nat Biotechnol, 34(7): 768-73 (2016), PubMed PMID: 27136078; Swarts et al., Nature,

507(7491): 258-61 (2014); and Swarts et al., Nucleic Acids Res. 43(10) (2015): 5120-9, each of which is incorporated herein by reference. The sequence of Natronobacterium gregoryi Argonaute is provided in SEQ ID NO: 182.

[0227] The disclosed base editors may comprise a napDNAbp domain having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with wild type Natronobacterium gregoryi Argonaute (SEQ ID NO: 182), which has the following amino acid sequence:

RLPITT AY ADQASTHATKGYLV QTGAFESNV GFL (SEQ ID NO: 182)

Cas9 circular permutants

[0228] In various embodiments, the base editors disclosed herein may comprise a circular permutant of Cas9.

[0229] The term“circularly permuted Cas9” or“circular permutant” of Cas9 or“CP-Cas9”) refers to any Cas9 protein, or variant thereof, that occurs or has been modify to engineered as a circular permutant variant, which means the N-terminus and the C-terminus of a Cas9 protein (e.g., a wild type Cas9 protein) have been topically rearranged. Such circularly permuted Cas9 proteins, or variants thereof, retain the ability to bind DNA when complexed with a guide RNA (gRNA). See, Oakes el al,“Protein Engineering of Cas9 for enhanced function,” Methods Enzymol, 2014, 546: 491-511 and Oakes et al,“CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification,” Cell , January 10, 2019, 176: 254-267, and Huang, T.P. et al. Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors. Nat. Biotechnol. 37, 626-631 (2019). each of are incorporated herein by reference. Reference is also made to International Application No. PCT/US2019/47996, filed August 23, 2019, herein incorporated by reference. The instant disclosure contemplates any previously known CP-Cas9 or use a new CP-Cas9 so long as the resulting circularly permuted protein retains the ability to bind DNA when complexed with a guide RNA (gRNA).

[0230] Any of the Cas9 proteins described herein, including any variant, ortholog, or naturally occurring Cas9 or equivalent thereof, may be reconfigured as a circular permutant variant.

[0231] In various embodiments, the circular permutants of Cas9 may have the following structure:

N-terminus-[original C-terminus] - [optional linker] - [original N-terminus]-C-terminus.

[0232] As an example, the present disclosure contemplates the following circular permutants of canonical S. pyogenes Cas9 (1368 amino acids of UniProtKB - Q99ZW2 (CAS9_STRP1) (numbering is based on the amino acid position in SEQ ID NO: 141):

N -terminu s- [ 1268 - 1368 ] - [optional linker] -[1-1267] -C- terminu s ;

N -terminu s- [ 1168 - 1368 ] - [optional linker] -[1-1167] -C- terminu s ;

N -terminu s- [ 1068 - 1368 ] - [optional linker] -[1-1067] -C- terminu s ;

N-terminus- [968- 1368] - [optional linker] - [ 1 -967] -C-terminus;

N -terminu s- [868-1368] - [optional linker] - [l-867]-C -terminu s ;

N -terminu s- [768-1368] - [optional linker] - [ 1 -767 ] -C -terminu s ;

N-terminus- [668- 1368] - [optional linker] - [ 1 -667] -C-terminus;

Ill N -terminu s- [568-1368] - [optional linker] - [l-567]-C -terminu s ;

N -terminu s- [468-1368] - [optional linker] - [l-467]-C -terminu s ;

N -terminu s- [368-1368] - [optional linker] - [l-367]-C -terminu s ;

N -terminu s- [268-1368] - [optional linker] - [l-267]-C -terminu s ;

N -terminu s- [168-1368] - [optional linker] - [1-167]-C -terminu s ;

N-terminus-[68-1368]-[optional linker]-[l-67]-C-terminus; or

N-terminus-[10-1368]-[optional linker]-[l-9]-C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc).

[0233] In particular embodiments, the circular permuant Cas9 has the following structure (based on S. pyogenes Cas9 (1368 amino acids of UniProtKB - Q99ZW2 (CAS9_STRP1) (numbering is based on the amino acid position in SEQ ID NO: 141):

N -terminu s- [102-1368] - [optional linker] - [1-101]-C -terminu s ;

N-terminus-[1028-1368]-[optional linker]-[l-1027]-C-terminus;

N -terminus- [1041-1368] - [optional linker] -[1-1043] -C-terminus ;

N-terminus-[1249-1368]-[optional linker]-[l-1248]-C-terminus; or

N-terminus-[1300-1368]-[optional linker]-[l-1299]-C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc).

[0234] In still other embodiments, the circular permuant Cas9 has the following structure (based on S. pyogenes Cas9 (1368 amino acids of UniProtKB - Q99ZW2 (CAS9_STRP1) (numbering is based on the amino acid position in SEQ ID NO: 141):

N -terminu s- [103-1368] - [optional linker] - [ 1 - 102] -C -terminu s ;

N-terminus-[1029-1368]-[optional linker]-[l-1028]-C-terminus;

N -terminu s- [ 1042- 1368 ] - [optional linker] -[1-1041] -C- terminu s ;

N-terminus-[1250-1368]-[optional linker]-[l-1249]-C-terminus; or

N-terminus-[1301-1368]-[optional linker]-[l-1300]-C-terminus, or the corresponding circular permutants of other Cas9 proteins (including other Cas9 orthologs, variants, etc.).

[0235] In some embodiments, the circular permutant can be formed by linking a C-terminal fragment of a Cas9 to an N-terminal fragment of a Cas9, either directly or by using a linker, such as an amino acid linker. In some embodiments, The C-terminal fragment may correspond to the C-terminal 95% or more of the amino acids of a Cas9 (e.g., amino acids about 1300-1368), or the C-terminal 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or more of a Cas9. The N-terminal portion may correspond to the N-terminal 95% or more of the amino acids of a Cas9 (e.g., amino acids about 1-1300), or the N-terminal 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%,

50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or more of a Cas9 (e.g., of SEQ

ID NO: 141).

[0236] In some embodiments, the circular permutant can be formed by linking a C-terminal fragment of a Cas9 to an N-terminal fragment of a Cas9, either directly or by using a linker, such as an amino acid linker. In some embodiments, the C-terminal fragment that is rearranged to the N-terminus, includes or corresponds to the C-terminal 30% or less of the amino acids of a Cas9 (e.g., amino acids 1012-1368 of SEQ ID NO: 141). In some embodiments, the C-terminal fragment that is rearranged to the N-terminus, includes or corresponds to the C-terminal 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,

2%, or 1% of the amino acids of a Cas9 (e.g., the Cas9 of SEQ ID NO: 141). In some embodiments, the C-terminal fragment that is rearranged to the N-terminus, includes or corresponds to the C-terminal 410 residues or less of a Cas9 (e.g., the Cas9 of SEQ ID NO: 141). In some embodiments, the C-terminal portion that is rearranged to the N-terminus, includes or corresponds to the C-terminal 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140,

130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 residues of a Cas9 (e.g., the Cas9 of SEQ ID NO: 141). In some embodiments, the C-terminal portion that is rearranged to the N- terminus, includes or corresponds to the C-terminal 357, 341, 328, 120, or 69 residues of a Cas9 (e.g., the Cas9 of SEQ ID NO: 141).

[0237] In other embodiments, circular permutant Cas9 variants may be defined as a topological rearrangement of a Cas9 primary structure based on the following method, which is based on S. pyogenes Cas9 of SEQ ID NO: 141: (a) selecting a circular permutant (CP) site corresponding to an internal amino acid residue of the Cas9 primary structure, which dissects the original protein into two halves: an N-terminal region and a C-terminal region; (b) modifying the Cas9 protein sequence (e.g., by genetic engineering techniques) by moving the original C-terminal region (comprising the CP site amino acid) to preceed the original N- terminal region, thereby forming a new N-terminus of the Cas9 protein that now begins with the CP site amino acid residue. The CP site can be located in any domain of the Cas9 protein, including, for example, the helical-II domain, the RuvCIII domain, or the CTD domain. For example, the CP site may be located (relative the S. pyogenes Cas9 of SEQ ID NO: 141) at original amino acid residue 181, 199, 230, 270, 310, 1010, 1016, 1023, 1029, 1041, 1247, 1249, or 1282. Thus, once relocated to the N-terminus, original amino acid 181, 199, 230, 270, 310, 1010, 1016, 1023, 1029, 1041, 1247, 1249, or 1282 would become the new N-terminal amino acid. Nomenclature of these CP-Cas9 proteins may be referred to as

Cas9-CP181, Cas9-CP199, Cas9-CP230, Cas9-CP270, Cas9-CP310, Cas9-CP1010, Cas9-

CP1016, Cas9-CP1023, Cas9-CP1029, Cas9-CP1041, Cas9-CP1247, Cas9-CP1249, and

Cas9-CP1282, respectively. This description is not meant to be limited to making CP variants from SEQ ID NO: 141, but may be implemented to make CP variants in any Cas9 sequence, either at CP sites that correspond to these positions, or at other CP sites entireley.

This description is not meant to limit the specific CP sites in any way. Virtually any CP site may be used to form a CP-Cas9 variant.

[0238] Exemplary CP-Cas9 amino acid sequences, based on the wild-type SpCas9 of SEQ ID NO: 141, are provided below in which linker sequences are indicated by underlining and optional methionine (M) residues are indicated in bold. It should be appreciated that the disclosure provides CP-Cas9 sequences that do not include a linker sequence or that include different linker sequences. It should be appreciated that CP-Cas9 sequences may be based on Cas9 sequences other than that of SEQ ID NO: 141 and any examples provided herein are not meant to be limiting. Exemplary CP-Cas9 sequences are as follows:

[0239] The Cas9 circular permutants that may be useful in the base editor constructs described herein. Exemplary C-terminal fragments of Cas9, based on the Cas9 of SEQ ID NO: 141, which may be rearranged to an N-terminus of Cas9, are provided below. It should be appreciated that such C-terminal fragments of Cas9 are exemplary and are not meant to be limiting. These exemplary CP-Cas9 fragments have the following sequences:

Cas9 variants with modified PAM specificities

[0240] The base editors of the present disclosure may also comprise Cas9 variants with modified PAM specificities. Some aspects of this disclosure provide Cas9 proteins that exhibit activity on a target sequence that does not comprise the canonical PAM (5'-NGG-3', where N is A, C, G, or T) at its 3 '-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5'-NGG-3' PAM sequence at its 3 '-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5 -NNG- 3' PAM sequence at its 3 '-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5'-NNA-3' PAM sequence at its 3 '-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5'-NNC-3' PAM sequence at its 3 '-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5 -NNT-3' PAM sequence at its 3'-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5 -NGT-3' PAM sequence at its 3'-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5 -NGA-3' PAM sequence at its 3'-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5 -NGC-3' PAM sequence at its 3'-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5'- NAA-3' PAM sequence at its 3 -end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5 -NAC-3' PAM sequence at its 3 '-end. In some embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5 -NAT-3' PAM sequence at its 3 -end. In still other embodiments, the Cas9 protein exhibits activity on a target sequence comprising a 5 -NAG-3' PAM sequence at its 3 -end.

[0241] In some embodiments, the disclosed base editors comprise a napDNAbp domain comprising a SpCas9-NG, which has a PAM that corresponds to NGN. In some

embodiments, the disclosed base editors comprise a napDNAbp domain comprising a SpCas9-KKH, which has a PAM that corresponds to NNNRRT (SEQ ID NO: 116).

[0242] It should be appreciated that any of the amino acid mutations described herein, (e.g., A262T) from a first amino acid residue (e.g., A) to a second amino acid residue (e.g., T) may also include mutations from the first amino acid residue to an amino acid residue that is similar to (e.g., conserved) the second amino acid residue. For example, mutation of an amino acid with a hydrophobic side chain (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan) may be a mutation to a second amino acid with a different hydrophobic side chain (e.g., alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan). For example, a mutation of an alanine to a threonine (e.g., a A262T mutation) may also be a mutation from an alanine to an amino acid that is similar in size and chemical properties to a threonine, for example, serine. As another example, mutation of an amino acid with a positively charged side chain (e.g., arginine, histidine, or lysine) may be a mutation to a second amino acid with a different positively charged side chain (e.g., arginine, histidine, or lysine). As another example, mutation of an amino acid with a polar side chain (e.g., serine, threonine, asparagine, or glutamine) may be a mutation to a second amino acid with a different polar side chain (e.g., serine, threonine, asparagine, or glutamine). Additional similar amino acid pairs include, but are not limited to, the following: phenylalanine and tyrosine; asparagine and glutamine; methionine and cysteine; aspartic acid and glutamic acid; and arginine and lysine. The skilled artisan would recognize that such conservative amino acid substitutions will likely have minor effects on protein structure and are likely to be well tolerated without compromising function. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to a threonine may be an amino acid mutation to a serine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to an arginine may be an amino acid mutation to a lysine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to an isoleucine, may be an amino acid mutation to an alanine, valine, methionine, or leucine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to a lysine may be an amino acid mutation to an arginine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to an aspartic acid may be an amino acid mutation to a glutamic acid or asparagine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to a valine may be an amino acid mutation to an alanine, isoleucine, methionine, or leucine. In some embodiments, any amino of the amino acid mutations provided herein from one amino acid to a glycine may be an amino acid mutation to an alanine. It should be appreciated, however, that additional conserved amino acid residues would be recognized by the skilled artisan and any of the amino acid mutations to other conserved amino acid residues are also within the scope of this disclosure.

[0243] In some embodiments, the present disclosure may utilize any of the Cas9 variants disclosed in the SEQUENCES section herein.

[0244] In some embodiments, the Cas9 protein comprises a combination of mutations that exhibit activity on a target sequence comprising a 5 -NAA-3' PAM sequence at its 3 -end. In some embodiments, the combination of mutations are present in any one of the clones listed in Table 1. In some embodiments, the combination of mutations are conservative mutations of the clones listed in Table 1. In some embodiments, the Cas9 protein comprises the combination of mutations of any one of the Cas9 clones listed in Table 1.

[0245] Table 1: NAA PAM Clones

[0246] In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 1. In some embodiments, the Cas9 protein comprises an amino acid sequence that is 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 protein as provided by any one of the variants of Table 1.

[0247] In some embodiments, the Cas9 protein exhibits an increased activity on a target sequence that does not comprise the canonical PAM (5'-NGG-3') at its 3' end as compared to Streptococcus pyogenes Cas9 as provided by SEQ ID NO: 141. In some embodiments, the Cas9 protein exhibits an activity on a target sequence having a 3' end that is not directly adjacent to the canonical PAM sequence (5'-NGG-3') that is at least 5-fold increased as compared to the activity of Streptococcus pyogenes Cas9 as provided by SEQ ID NO: 141 on the same target sequence. In some embodiments, the Cas9 protein exhibits an activity on a target sequence that is not directly adjacent to the canonical PAM sequence (5'-NGG-3') that is at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold increased as compared to the activity of

Streptococcus pyogenes as provided by SEQ ID NO: 141 on the same target sequence. In some embodiments, the 3' end of the target sequence is directly adjacent to an AAA, GAA, CAA, or TAA sequence. In some embodiments, the Cas9 protein comprises a combination of mutations that exhibit activity on a target sequence comprising a 5 -NAC-3' PAM sequence at its 3 '-end. In some embodiments, the combination of mutations are present in any one of the clones listed in Table 2. In some embodiments, the combination of mutations are conservative mutations of the clones listed in Table 2. In some embodiments, the Cas9 protein comprises the combination of mutations of any one of the Cas9 clones listed in Table 2.

[0248] Table 2: NAC PAM Clones Mutations from wild-type SpCas9 (e.g., SEQ ID NO: 141)

[0249] In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of a Cas9 protein as provided by any one of the variants of Table 2. In some embodiments, the Cas9 protein comprises an amino acid sequence that is 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 protein as provided by any one of the variants of Table 2.

[0250] In some embodiments, the Cas9 protein comprises a combination of mutations that exhibit activity on a target sequence comprising a 5'-NAT-3' PAM sequence at its 3 '-end. In some embodiments, the combination of mutations are present in any one of the clones listed in Table 3. In some embodiments, the combination of mutations are conservative mutations of the clones listed in Table 3. In some embodiments, the Cas9 protein comprises the combination of mutations of any one of the Cas9 clones listed in Table 3.

[0251] Table 3: NAT PAM Clones

[0252] The above description of various napDNAbps which can be used in connection with the presently disclose base editors is not meant to be limiting in any way. The base editors may comprise the canonical SpCas9, or any ortholog Cas9 protein, or any variant Cas9 protein— including any naturally occurring variant, mutant, or otherwise engineered version of Cas9— that is known or which can be made or evolved through a directed evolutionary or otherwise mutagenic process. In various embodiments, the Cas9 or Cas9 varants have a nickase activity, i.e., only cleave of strand of the target DNA sequence. In other

embodiments, the Cas9 or Cas9 variants have inactive nucleases, i.e., are“dead” Cas9 proteins. Other variant Cas9 proteins that may be used are those having a smaller molecular weight than the canonical SpCas9 (e.g., for easier delivery) or having modified or rearranged primary amino acid structure (e.g., the circular permutant formats). The base editors described herein may also comprise Cas9 equivalents, including Casl2a/Cpfl and Casl2b proteins which are the result of convergent evolution. The napDNAbps used herein (e.g., SpCas9, Cas9 variant, or Cas9 equivalents) may also may also contain various modifications that alter/enhance their PAM specifities. Lastly, the application contemplates any Cas9, Cas9 variant, or Cas9 equivalent which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% sequence identity to a reference Cas9 sequence, such as a references SpCas9 canonical sequences or a reference Cas9 equivalent (e.g., Casl2a/Cpfl).

[0253] In a particular embodiment, the Cas9 variant having expanded PAM capabilities is SpCas9 (H840A) VRQR, or SpCas9-VRQR. In some embodiments, the disclosed base editors comprise a napDNAbp domain that has a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to SpCas9-VRQR. In some embodiments, the disclosed base editors comprise a napDNAbp domain that comprises SpCas9-VRQR. The SpCas9-

VRQR comprises the following amino acid sequence (with the V, R, Q, R substitutions relative to the SpCas9 (H840A) of SEQ ID NO: 193 show, in bold underline. In addition, the methionine residue in SpCas9 (H840) was removed for SpCas9 (H840A) VRQR):

[0254] In another particular embodiment, the Cas9 variant having expanded PAM

capabilities is SpCas9 (H840A) VRER, having the following amino acid sequence (with the V, R, E, R substitutions relative to the SpCas9 (H840A) of SEQ ID NO: 194 are shown in bold underline . In addition, the methionine residue in SpCas9 (H840) was removed for SpCas9 (H840A) VRER):

[0255] In addition, any available methods may be utilized to obtain or construct a variant or mutant Cas9 protein. The term“mutation,” as used herein, refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)). Mutations can include a variety of categories, such as single base polymorphisms, microduplication regions, indel, and inversions, and is not meant to be limiting in any way. Mutations can include“loss-of-function” mutations which is the normal result of a mutation that reduces or abolishes a protein activity. Most loss-of-function mutations are recessive, because in a heterozygote the second chromosome copy carries an unmutated version of the gene coding for a fully functional protein whose presence compensates for the effect of the mutation. Mutations also embrace“gain-of-function” mutations, which is one which confers an abnormal activity on a protein or cell that is otherwise not present in a normal condition. Many gain-of-function mutations are in regulatory sequences rather than in coding regions, and can therefore have a number of consequences. For example, a mutation might lead to one or more genes being expressed in the wrong tissues, these tissues gaining functions that they normally lack. Because of their nature, gain-of-function mutations are usually dominant.

[0256] Mutations can be introduced into a reference Cas9 protein using site-directed mutagenesis. Older methods of site-directed mutagenesis known in the art rely on sub cloning of the sequence to be mutated into a vector, such as an M13 bacteriophage vector, that allows the isolation of single-stranded DNA template. In these methods, one anneals a mutagenic primer (i.e., a primer capable of annealing to the site to be mutated but bearing one or more mismatched nucleotides at the site to be mutated) to the single-stranded template and then polymerizes the complement of the template starting from the 3 ' end of the mutagenic primer. The resulting duplexes are then transformed into host bacteria and plaques are screened for the desired mutation. More recently, site-directed mutagenesis has employed

PCR methodologies, which have the advantage of not requiring a single-stranded template. In addition, methods have been developed that do not require sub-cloning. Several issues must be considered when PCR-based site-directed mutagenesis is performed. First, in these methods it is desirable to reduce the number of PCR cycles to prevent expansion of undesired mutations introduced by the polymerase. Second, a selection must be employed in order to reduce the number of non-mutated parental molecules persisting in the reaction. Third, an extended-length PCR method is preferred in order to allow the use of a single PCR primer set. And fourth, because of the non-template-dependent terminal extension activity of some thermostable polymerases it is often necessary to incorporate an end-polishing step into the procedure prior to blunt-end ligation of the PCR-generated mutant product.

[0257] Any of the references noted above which relate to napDNAbp domains are hereby incorporated by reference in their entireties, if not already stated so.

Exemplary Fusion Proteins

[0258] Some aspects of the disclosure provide fusion proteins comprising a napDNAbp domain ( e.g . an nCas9 domain) and an adenosine deaminase domain. The adenosine deaminase domain may comprise a single deaminase enzyme, two deaminase enzymes, or more than two deaminase enzymes. In some embodiments, the adenosine deaminase domain comprises a single adenosine deaminase enzyme. In some embodiments, the adenosine deaminase domain comprises two adenosine deaminases, e.g., a heterodimer of adenosine deaminases. In still other embodiments, the fusion protein is an ancestrally reconstructed adenine base editor.

[0259] The present disclosure provides three newly discovered mutations to TadA 7.10 (SEQ ID NO: 96) (the TadA* used in ABEmax) that yield an adenosine deaminase mutant that, when connected to catalytically inactive TadA (e.g. TadA(E59A)) within the adenosine deaminase domain of a fusion protein, confer reduced off-target effects. These three mutations comprise substitutions at amino acid residues R47, V106, and N108. The fusion proteins of the present disclosure comprise one or more adenosine deaminases having at least one amino acid substitution at R47, V106, or N108. In other embodiments, the fusion proteins may comprise one or more adenosine deaminases having two or more such substitutions in combination. In some embodiments, the fusion proteins comprise adenosine deaminases comprising comprises a sequence with at least 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 96 (TadA 7.10), wherein any sequence variation may only occur in amino acid positions other than R47, V106, or N108 of SEQ ID NO: 96.

In other words, these fusion protein embodiments must contain amino acid substitutions at

R47, V106, or N108 of SEQ ID NO: 96.

[0260] It should be appreciated that these three mutations ( i.e ., R47, V106, or N108 of SEQ ID NO: 96) may be introduced into other adenosine deaminases, such as S. aureus TadA (saTadA), or other adenosine deaminases (e.g., bacterial adenosine deaminases), such as those sequences provided below. It would be apparent to the skilled artisan how to identify amino acid residues from other adenosine deaminases that are homologous to the mutated residues in TadA 7.10. Thus, any of the mutations identified in TadA 7.10 may be made in other adenosine deaminases that have homologous amino acid residues.

[0261] In particular embodiments, any of the fusion proteins of the disclosure comprise the sequence of SEQ ID NO: 217 or SEQ ID NO: 216. In other embodiments, any of the fusion proteins of the disclosure comprise the sequence of SEQ ID NO: 221. In other embodiments, any of the fusion proteins of the disclosure comprise a sequence selected from SEQ ID NOs: 222-225. In other embodiments, any of the fusion proteins of the disclosure comprises the sequence of SEQ ID NO: 226. In other embodiments, any of the fusion proteins of the disclosure comprise the sequence of SEQ ID NOs: 227 or 228.

[0262] Exemplary fusion proteins comprise sequences that are at least least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the following amino acid sequences (for the purposes of clarity, the adenosine deaminase domain is shown in Bold; mutations of the ecTadA deaminase domain are shown in Bold underlining; the XTEN linker is shown in italics ; and NLS is shown in underlined italics ):

ABEmax, or ABE7.10

MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR

HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVV

FGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQE

IKAQKKAQSSTDSGGSSGGSSGSETPGrSESATPESSGGSSGGSSEVEFSHEYWMR

HALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQ

GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSL

MDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSG

GSSGGSSGSETPGrSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVP S

KKFKVLGNTDRHS IKKNLIGALLFDS GET AE ATRLKRT ARRRYTRRKNRIC YLQEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV

DSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIOLVOTYNOLFEENP

IN AS G VD AK AILS ARLS KS RRLENLIAOLPGEKKN GLF GNLIALS LGLTPNFKS NFDL

AEDAKLOLSKDTYDDDLDNLLAOIGDOYADLFLAAKNLSDAILLSDILRVNTEITK

APLSASMIKRYDEHHODLTLLKALVROOLPEKYKEIFFDOSKNGYAGYIDGGASOE

EFYKFIKPILEKMDGTEELLVKLNREDLLRKORTFDNGSIPHOIHLGELHAILRROE D

FYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKG

ASAOSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS

GEOKKAIVDLLFKTNRKVTVKOLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL

LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKOLKRR

RYTGWGRLSRKLINGIRDKOSGKTILDFLKSDGFANRNFMOLIHDDSLTFKEDIOKA

OVSGOGDSLHEHIANLAGSPAIKKGILOTVKVVDELVKVMGRHKPENIVIEMAREN

OTTOKGOKNSRERMKRIEEGIKELGSOILKEHPVENTOLONEKLYLYYLONGRDM

YVDOELDINRLSDYDVDHIVPOSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKK

MKNYWROLLNAKLITORKFDNLTKAERGGLSELDKAGFIKROLVETROITKHVAOI

LDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFOFYKVREINNYHHAHDAYL

N A V V GT ALIKKYPKLES EFV Y GD YKV YD VRKMIAKS EOEIGKAT AKYFFY S NIMNF

FKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPOVNIVKKTEVOT

GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKL

KS VKELLGITIMERS S FEKNPIDFLE AKG YKE VKKDLIIKLPKY S LFELEN GRKRMLA

SAGELOKGNELALPSKYVNFLYLASHYEKLKGSPEDNEOKOLFVEOHKHYLDEIIE

OISEFSKRVILADANLDKVLSAYNKHRDKPIREOAENIIHLFTLTNLGAPAAFKYFD T

TIDRKRYTSTKEVLDATLIHOSITGLYETRIDLSOLGGDSGGS KKKRKV (SEQ

ID NO: 215)

ABEmax(TadA E59A)

MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR

HDPTAHAAIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVV

FGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQE

IKAQKKAQSSTOSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEYEESHEYWMR

HALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQ

GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSL

MDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSG

GSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAYYTDEYKYP S

KKFKVLGNTDRHS IKKNLIGALLFDS GET AE ATRLKRT ARRRYTRRKNRIC YLOEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV

DSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIOLVOTYNOLFEENP

IN AS G VD AK AILS ARLS KS RRLENLIAOLPGEKKN GLF GNLIALS LGLTPNFKS NFDL

AEDAKLOLSKDTYDDDLDNLLAOIGDOYADLFLAAKNLSDAILLSDILRVNTEITK

APLSASMIKRYDEHHODLTLLKALVROOLPEKYKEIFFDOSKNGYAGYIDGGASOE

EFYKFIKPILEKMDGTEELLVKLNREDLLRKORTFDNGSIPHOIHLGELHAILRROE D

FYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKG

ASAOSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS

GEOKKAIVDLLFKTNRKVTVKOLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL

LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKOLKRR

RYTGWGRLSRKLINGIRDKOSGKTILDFLKSDGFANRNFMOLIHDDSLTFKEDIOKA

OVSGOGDSLHEHIANLAGSPAIKKGILOTVKVVDELVKVMGRHKPENIVIEMAREN

OTTOKGOKNSRERMKRIEEGIKELGSOILKEHPVENTOLONEKLYLYYLONGRDM

YVDOELDINRLSDYDVDHIVPOSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKK

MKNYWROLLNAKLITORKFDNLTKAERGGLSELDKAGFIKROLVETROITKHVAOI

LDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFOFYKVREINNYHHAHDAYL

N A V V GT ALIKKYPKLES EFV Y GD YKV YD VRKMIAKS EOEIGKAT AKYFFY S NIMNF

FKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPOVNIVKKTEVOT

GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKL

KS VKELLGITIMERS S FEKNPIDFLE AKG YKE VKKDLIIKLPKY S LFELEN GRKRMLA

SAGELOKGNELALPSKYVNFLYLASHYEKLKGSPEDNEOKOLFVEOHKHYLDEIIE

OISEFSKRVILADANLDKVLSAYNKHRDKPIREOAENIIHLFTLTNLGAPAAFKYFD T

TIDRKRYTSTKEVLDATLIHOSITGLYETRIDLSOLGGDSGGS KKKRKV (SEQ

ID NO: 216)

ABEmax(TadA E59A, TadA* V106W) [ ABEmaxAW ]

MSEVEFSHEYWMRHALTLAKRA WDEREVPVGA VLVHNNRVIGEGWNRPIGRHDPTAHAA

IMALRQGGLVM QNYRLIDA TLYVTEEPCVM CA GAMIHSRIGR WF GARDAKT GAA GSEMD

VLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGSSGGSS GSET

PGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNR VIGE

GWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIG RVV

FG WRNAKT GAA GSEMD VEHYP GMNHR VEITEGILADE CAAEECYFFRMPR Q VFNA QKKA

OSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVI TDEYKV

PSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLOE IFSNE

MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDST DKA DLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIOLVOTYNOLFEENPINASGVDAK AI

LSARLSKSRRLFNLIA OLPGF KKNGLFGNUA LSLGLFPNFKSNFDLA FDA KLOLSKDTYD

DDLDNLLAOIGDOYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHH ODLTL

LKALVROOLPEKYKEIFFDOSKNGYAGYIDGGASOEEFYKFIKPILEKMDGTEELLV KLNR

EDLLRKORTFDNGSIPHOIHLGELHAILRROEDFYPFLKDNREKIEKILTFRIPYYV GPLAR

GNSRFAWMTRKSEETITPWNFEEWDKGASAOSFIERMTNFDKNLPNEKVLPKHSLLY EY

FTVYNELTKVKYVTEGMRKPAFLSGEOKKAIVDLLFKTNRKVTVKOLKEDYFKKIEC FDSV

EISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYA

HLFDDKVMKOLKRRRYTGWGRLSRKLINGIRDKOSGKTILDFLKSDGFANRNFMOLI HD

DSLTFKEDIOKAOVSGOGDSLHEHIANLAGSPAIKKGILOTVKVVDELVKVMGRHKP ENIV

IEMARENOTTOKGOKNSRERMKRIEEGIKELGSOILKEHPVENTOLONEKLYLYYLO NGR

DMYVDOELDINRLSDYDVDHIVPOSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVK KMK

NYWROLLNAKLITORKFDNLTKAERGGLSELDKAGFIKROLVETROITKHVAOILDS RMNT

KYDENDKLIREVKVITLKSKLVSDFRKDFOFYKVREINNYHHAHDA YLNA WGTALIKKYP

KLESEFVYGD YKVYD VRKMIAKSE OEIGKA TAKYFFYSNIMNFFKTEITLANGEIRKRPLIET

NGET GEIVWDKGRDFA TVRKVLSMPO VNIVKKTEVOTGGFSKESILPKRNSDKLIARKKD

WDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFL EAKG

YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELOKGNELALPSKYVNFLYLASHYE KLKG

SPEDNE OKOLFVE OHKHYLDEIIE OISEFSKR VILADANLDKVLSA YNKHRDKPIREOAENI

IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHOSITGLYETRIDLSOLG GDSG

GSPKKKRKV (SEQ ID NO: 217)

ABEmax(TadA E59A, TadA* V106Q)

MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR

HDPTAHAAIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVV

FGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQE

IKAQKKAQSSTOSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEYEFSHEYWMR

HALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQ

GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGQRNAKTGAAGSL

MDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSG

GSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSlGLAlGYNSYGWAYnDEYKYPS

KKFKVLGNTDRHS IKKNLIGALLFDS GET AE ATRLKRT ARRRYTRRKNRIC YLOEIF

SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV

DSTDKADFRFIYFAFAHMIKFRGHFFIEGDFNPDNSDVDKFFIOFVOTYNOFFEENP

IN AS G VD AK AIFS ARES KS RRFENFIAOFPGEKKN GFF GNFIAFS FGFTPNFKS NFDF AEDAKLOLSKDTYDDDLDNLLAOIGDOYADLFLAAKNLSDAILLSDILRVNTEITK

APLSASMIKRYDEHHODLTLLKALVROOLPEKYKEIFFDOSKNGYAGYIDGGASOE

EFYKFIKPIFEKMDGTEEFFVKFNREDFFRKORTFDNGSIPHOIHFGEFHAIFRROE D

FYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKG

ASAOSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS

GEOKKAIVDFFFKTNRKVTVKOFKEDYFKKIECFDSVEISGVEDRFNASFGTYHDF

LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKOLKRR

RYTGWGRFSRKFINGIRDKOSGKTIFDFFKSDGFANRNFMOFIHDDSFTFKEDIOKA

OVSGOGDSFHEHIANFAGSPAIKKGIFOTVKVVDEFVKVMGRHKPENIVIEMAREN

OTTOKGOKNSRERMKRIEEGIKEFGSOIFKEHPVENTOFONEKFYFYYFONGRDM

YVDOEFDINRFSDYDVDHIVPOSFFKDDSIDNKVFTRSDKNRGKSDNVPSEEVVKK

MKNYWROFFNAKFITORKFDNFTKAERGGFSEFDKAGFIKROFVETROITKHVAOI

FDSRMNTKYDENDKFIREVKVITFKSKFVSDFRKDFOFYKVREINNYHHAHDAYF

N A V V GT AFIKKYPKFES EFV Y GD YKV YD VRKMIAKS EOEIGKAT AKYFFY S NIMNF

FKTEITFANGEIRKRPFIETNGETGEIVWDKGRDFATVRKVFSMPOVNIVKKTEVOT

GGFSKESIFPKRNSDKFIARKKDWDPKKYGGFDSPTVAYSVFVVAKVEKGKSKKF

KS VKEFFGITIMERS S FEKNPIDFFE AKG YKE VKKDFIIKFPKY S FFEFEN GRKRMFA

SAGEFOKGNEFAFPSKYVNFFYFASHYEKFKGSPEDNEOKOFFVEOHKHYFDEIIE

OISEFSKRVIFADANFDKVFSAYNKHRDKPIREOAENIIHFFTFTNFGAPAAFKYFD T

TIDRKRYTSTKEVLDATLIHOSITGLYETRIDLSOLGGDSGGS/YOOG^y (SEQ

ID NO: 218)

ABEmax(TadA E59A, TadA* V106F)

MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR

HDPTAHAAIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVV

FGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQE

IKAQKKAQSSTOSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEYEESHEYWMR

HALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQ

GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGFRNAKTGAAGSL

MDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSG

GSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAYYTDEYKYP S KKFKVFGNTDRHS IKKNFIGAFFFDS GET AE ATRFKRT ARRRYTRRKNRIC YFOEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV DSTDKADFRFIYFAFAHMIKFRGHFFIEGDFNPDNSDVDKFFIOFVOTYNOFFEENP IN AS G VD AK AILS ARLS KS RRLENLIAOLPGEKKN GLF GNLIALS LGLTPNFKS NFDL AEDAKLOLSKDTYDDDLDNLLAOIGDOYADLFLAAKNLSDAILLSDILRVNTEITK

APLSASMIKRYDEHHODLTLLKALVROOLPEKYKEIFFDOSKNGYAGYIDGGASOE

EFYKFIKPIFEKMDGTEEFFVKFNREDFFRKORTFDNGSIPHOIHFGEFHAIFRROE D

FYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKG

ASAOSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS

GEOKKAIVDFFFKTNRKVTVKOFKEDYFKKIECFDSVEISGVEDRFNASFGTYHDF

LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKOLKRR

RYTGWGRFSRKFINGIRDKOSGKTIFDFFKSDGFANRNFMOFIHDDSFTFKEDIOKA

OVSGOGDSFHEHIANFAGSPAIKKGIFOTVKVVDEFVKVMGRHKPENIVIEMAREN

OTTOKGOKNSRERMKRIEEGIKEFGSOIFKEHPVENTOFONEKFYFYYFONGRDM

YVDOEFDINRFSDYDVDHIVPOSFFKDDSIDNKVFTRSDKNRGKSDNVPSEEVVKK

MKNYWROFFNAKFITORKFDNFTKAERGGFSEFDKAGFIKROFVETROITKHVAOI

FDSRMNTKYDENDKFIREVKVITFKSKFVSDFRKDFOFYKVREINNYHHAHDAYF

N A V V GT AFIKKYPKFES EFV Y GD YKV YD VRKMIAKS EOEIGKAT AKYFFY S NIMNF

FKTEITFANGEIRKRPFIETNGETGEIVWDKGRDFATVRKVFSMPOVNIVKKTEVOT

GGFSKESIFPKRNSDKFIARKKDWDPKKYGGFDSPTVAYSVFVVAKVEKGKSKKF

KS VKEFFGITIMERS S FEKNPIDFFE AKG YKE VKKDFIIKFPKY S FFEFEN GRKRMFA

SAGEFOKGNEFAFPSKYVNFFYFASHYEKFKGSPEDNEOKOFFVEOHKHYFDEIIE

OISEFSKRVIFADANFDKVFSAYNKHRDKPIREOAENIIHFFTFTNFGAPAAFKYFD T

TIDRKRYTSTKEVLDATLIHOSITGLYETRIDLSOLGGDSGGS/YOOG^y (SEQ

ID NO: 219)

ABEmax(TadA E59A, TadA* V106M)

MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR

HDPTAHAAIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVV

FGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQE

IKAQKKAQSSTOSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEYEESHEYWMR

HALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQ

GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGMRNAKTGAAGSL

MDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSG

GSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAYYTDEYKYP S KKFKVFGNTDRHS IKKNFIGAFFFDS GET AE ATRFKRT ARRRYTRRKNRIC YFOEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV DSTDKADFRFIYFAFAHMIKFRGHFFIEGDFNPDNSDVDKFFIOFVOTYNOFFEENP IN AS G VD AK AILS ARLS KS RRLENLIAOLPGEKKN GLF GNLIALS LGLTPNFKS NFDL AEDAKLOLSKDTYDDDLDNLLAOIGDOYADLFLAAKNLSDAILLSDILRVNTEITK

APLSASMIKRYDEHHODLTLLKALVROOLPEKYKEIFFDOSKNGYAGYIDGGASOE

EFYKFIKPIFEKMDGTEEFFVKFNREDFFRKORTFDNGSIPHOIHFGEFHAIFRROE D

FYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKG

ASAOSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS

GEOKKAIVDFFFKTNRKVTVKOFKEDYFKKIECFDSVEISGVEDRFNASFGTYHDF

LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKOLKRR

RYTGWGRFSRKFINGIRDKOSGKTIFDFFKSDGFANRNFMOFIHDDSFTFKEDIOKA

OVSGOGDSFHEHIANFAGSPAIKKGIFOTVKVVDEFVKVMGRHKPENIVIEMAREN

OTTOKGOKNSRERMKRIEEGIKEFGSOIFKEHPVENTOFONEKFYFYYFONGRDM

YVDOEFDINRFSDYDVDHIVPOSFFKDDSIDNKVFTRSDKNRGKSDNVPSEEVVKK

MKNYWROFFNAKFITORKFDNFTKAERGGFSEFDKAGFIKROFVETROITKHVAOI

FDSRMNTKYDENDKFIREVKVITFKSKFVSDFRKDFOFYKVREINNYHHAHDAYF

N A V V GT AFIKKYPKFES EFV Y GD YKV YD VRKMIAKS EOEIGKAT AKYFFY S NIMNF

FKTEITFANGEIRKRPFIETNGETGEIVWDKGRDFATVRKVFSMPOVNIVKKTEVOT

GGFSKESIFPKRNSDKFIARKKDWDPKKYGGFDSPTVAYSVFVVAKVEKGKSKKF

KS VKEFFGITIMERS S FEKNPIDFFE AKG YKE VKKDFIIKFPKY S FFEFEN GRKRMFA

SAGEFOKGNEFAFPSKYVNFFYFASHYEKFKGSPEDNEOKOFFVEOHKHYFDEIIE

OISEFSKRVIFADANFDKVFSAYNKHRDKPIREOAENIIHFFTFTNFGAPAAFKYFD T

TIDRKRYTSTKEVLDATLIHOSITGLYETRIDLSOLGGDSGGS/YOOG^y (SEQ

ID NO: 220)

ABEmax(TcidA E59A, TadA*N108W)

MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR

HDPTAHAAIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVV

FGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQE

IKAQKKAQSSTOSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEYEESHEYWMR

HALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQ

GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRWAKTGAAGSL

MDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSG

GSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAYYTDEYKYP S KKFKVFGNTDRHS IKKNFIGAFFFDS GET AE ATRFKRT ARRRYTRRKNRIC YFOEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV DSTDKADFRFIYFAFAHMIKFRGHFFIEGDFNPDNSDVDKFFIOFVOTYNOFFEENP IN AS G VD AK AILS ARLS KS RRLENLIAOLPGEKKN GLF GNLIALS LGLTPNFKS NFDL AEDAKLOLSKDTYDDDLDNLLAOIGDOYADLFLAAKNLSDAILLSDILRVNTEITK

APLSASMIKRYDEHHODLTLLKALVROOLPEKYKEIFFDOSKNGYAGYIDGGASOE

EFYKFIKPIFEKMDGTEEFFVKFNREDFFRKORTFDNGSIPHOIHFGEFHAIFRROE D

FYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKG

ASAOSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS

GEOKKAIVDFFFKTNRKVTVKOFKEDYFKKIECFDSVEISGVEDRFNASFGTYHDF

LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKOLKRR

RYTGWGRFSRKFINGIRDKOSGKTIFDFFKSDGFANRNFMOFIHDDSFTFKEDIOKA

OVSGOGDSFHEHIANFAGSPAIKKGIFOTVKVVDEFVKVMGRHKPENIVIEMAREN

OTTOKGOKNSRERMKRIEEGIKEFGSOIFKEHPVENTOFONEKFYFYYFONGRDM

YVDOEFDINRFSDYDVDHIVPOSFFKDDSIDNKVFTRSDKNRGKSDNVPSEEVVKK

MKNYWROFFNAKFITORKFDNFTKAERGGFSEFDKAGFIKROFVETROITKHVAOI

FDSRMNTKYDENDKFIREVKVITFKSKFVSDFRKDFOFYKVREINNYHHAHDAYF

N A V V GT AFIKKYPKFES EFV Y GD YKV YD VRKMIAKS EOEIGKAT AKYFFY S NIMNF

FKTEITFANGEIRKRPFIETNGETGEIVWDKGRDFATVRKVFSMPOVNIVKKTEVOT

GGFSKESIFPKRNSDKFIARKKDWDPKKYGGFDSPTVAYSVFVVAKVEKGKSKKF

KS VKEFFGITIMERS S FEKNPIDFFE AKG YKE VKKDFIIKFPKY S FFEFEN GRKRMFA

SAGEFOKGNEFAFPSKYVNFFYFASHYEKFKGSPEDNEOKOFFVEOHKHYFDEIIE

OISEFSKRVIFADANFDKVFSAYNKHRDKPIREOAENIIHFFTFTNFGAPAAFKYFD T

TIDRKRYTSTKEVLDATLIHOSITGLYETRIDLSOLGGDSGGS/YOOG^y (SEQ

ID NO: 221)

ABEmax(TadA E59A, TadA* R47Q)

MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR

HDPTAHAAIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVV

FGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQE

IKAQKKAQSSTOSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEYEESHEYWMR

HALTLAKRARDEREVPVGAVLVLNNRVIGEGWNQAIGLHDPTAHAEIMALRQ

GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSL

MDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSG

GSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAYYTDEYKYP S KKFKVFGNTDRHS IKKNFIGAFFFDS GET AE ATRFKRT ARRRYTRRKNRIC YFOEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV DSTDKADFRFIYFAFAHMIKFRGHFFIEGDFNPDNSDVDKFFIOFVOTYNOFFEENP IN AS G VD AK AILS ARLS KS RRLENLIAOLPGEKKN GLF GNLIALS LGLTPNFKS NFDL AEDAKLOLSKDTYDDDLDNLLAOIGDOYADLFLAAKNLSDAILLSDILRVNTEITK

APLSASMIKRYDEHHODLTLLKALVROOLPEKYKEIFFDOSKNGYAGYIDGGASOE

EFYKFIKPIFEKMDGTEEFFVKFNREDFFRKORTFDNGSIPHOIHFGEFHAIFRROE D

FYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKG

ASAOSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS

GEOKKAIVDFFFKTNRKVTVKOFKEDYFKKIECFDSVEISGVEDRFNASFGTYHDF

LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKOLKRR

RYTGWGRFSRKFINGIRDKOSGKTIFDFFKSDGFANRNFMOFIHDDSFTFKEDIOKA

OVSGOGDSFHEHIANFAGSPAIKKGIFOTVKVVDEFVKVMGRHKPENIVIEMAREN

OTTOKGOKNSRERMKRIEEGIKEFGSOIFKEHPVENTOFONEKFYFYYFONGRDM

YVDOEFDINRFSDYDVDHIVPOSFFKDDSIDNKVFTRSDKNRGKSDNVPSEEVVKK

MKNYWROFFNAKFITORKFDNFTKAERGGFSEFDKAGFIKROFVETROITKHVAOI

FDSRMNTKYDENDKFIREVKVITFKSKFVSDFRKDFOFYKVREINNYHHAHDAYF

N A V V GT AFIKKYPKFES EFV Y GD YKV YD VRKMIAKS EOEIGKAT AKYFFY S NIMNF

FKTEITFANGEIRKRPFIETNGETGEIVWDKGRDFATVRKVFSMPOVNIVKKTEVOT

GGFSKESIFPKRNSDKFIARKKDWDPKKYGGFDSPTVAYSVFVVAKVEKGKSKKF

KS VKEFFGITIMERS S FEKNPIDFFE AKG YKE VKKDFIIKFPKY S FFEFEN GRKRMFA

SAGEFOKGNEFAFPSKYVNFFYFASHYEKFKGSPEDNEOKOFFVEOHKHYFDEIIE

OISEFSKRVIFADANFDKVFSAYNKHRDKPIREOAENIIHFFTFTNFGAPAAFKYFD T

TIDRKRYTSTKEVLDATLIHOSITGLYETRIDLSOLGGDSGGS/YOOG^y (SEQ

ID NO: 222)

ABEmax(TadA E59A, TadA* R47F)

MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR

HDPTAHAAIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVV

FGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQE

IKAQKKAQSSTOSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEYEESHEYWMR

HALTLAKRARDEREVPVGAVLVLNNRVIGEGWNFAIGLHDPTAHAEIMALRQ

GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSL

MDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSG

GSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAYYTDEYKYP S KKFKVFGNTDRHS IKKNFIGAFFFDS GET AE ATRFKRT ARRRYTRRKNRIC YFOEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV DSTDKADFRFIYFAFAHMIKFRGHFFIEGDFNPDNSDVDKFFIOFVOTYNOFFEENP IN AS G VD AK AILS ARLS KS RRLENLIAOLPGEKKN GLF GNLIALS LGLTPNFKS NFDL AEDAKLOLSKDTYDDDLDNLLAOIGDOYADLFLAAKNLSDAILLSDILRVNTEITK

APLSASMIKRYDEHHODLTLLKALVROOLPEKYKEIFFDOSKNGYAGYIDGGASOE

EFYKFIKPIFEKMDGTEEFFVKFNREDFFRKORTFDNGSIPHOIHFGEFHAIFRROE D

FYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKG

ASAOSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS

GEOKKAIVDFFFKTNRKVTVKOFKEDYFKKIECFDSVEISGVEDRFNASFGTYHDF

LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKOLKRR

RYTGWGRFSRKFINGIRDKOSGKTIFDFFKSDGFANRNFMOFIHDDSFTFKEDIOKA

OVSGOGDSFHEHIANFAGSPAIKKGIFOTVKVVDEFVKVMGRHKPENIVIEMAREN

OTTOKGOKNSRERMKRIEEGIKEFGSOIFKEHPVENTOFONEKFYFYYFONGRDM

YVDOEFDINRFSDYDVDHIVPOSFFKDDSIDNKVFTRSDKNRGKSDNVPSEEVVKK

MKNYWROFFNAKFITORKFDNFTKAERGGFSEFDKAGFIKROFVETROITKHVAOI

FDSRMNTKYDENDKFIREVKVITFKSKFVSDFRKDFOFYKVREINNYHHAHDAYF

N A V V GT AFIKKYPKFES EFV Y GD YKV YD VRKMIAKS EOEIGKAT AKYFFY S NIMNF

FKTEITFANGEIRKRPFIETNGETGEIVWDKGRDFATVRKVFSMPOVNIVKKTEVOT

GGFSKESIFPKRNSDKFIARKKDWDPKKYGGFDSPTVAYSVFVVAKVEKGKSKKF

KS VKEFFGITIMERS S FEKNPIDFFE AKG YKE VKKDFIIKFPKY S FFEFEN GRKRMFA

SAGEFOKGNEFAFPSKYVNFFYFASHYEKFKGSPEDNEOKOFFVEOHKHYFDEIIE

OISEFSKRVIFADANFDKVFSAYNKHRDKPIREOAENIIHFFTFTNFGAPAAFKYFD T

TIDRKRYTSTKEVLDATLIHOSITGLYETRIDLSOLGGDSGGS/YOOG^y (SEQ

ID NO: 223)

ABEmax(TcidA E59A, TadA* R47W)

MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR

HDPTAHAAIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVV

FGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQE

IKAQKKAQSSTOSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEYEESHEYWMR

HALTLAKRARDEREVPVGAVLVLNNRVIGEGWNWAIGLHDPTAHAEIMALRQ

GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSL

MDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSG

GSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAYYTDEYKYP S KKFKVFGNTDRHS IKKNFIGAFFFDS GET AE ATRFKRT ARRRYTRRKNRIC YFOEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV DSTDKADFRFIYFAFAHMIKFRGHFFIEGDFNPDNSDVDKFFIOFVOTYNOFFEENP IN AS G VD AK AILS ARLS KS RRLENLIAOLPGEKKN GLF GNLIALS LGLTPNFKS NFDL AEDAKLOLSKDTYDDDLDNLLAOIGDOYADLFLAAKNLSDAILLSDILRVNTEITK

APLSASMIKRYDEHHODLTLLKALVROOLPEKYKEIFFDOSKNGYAGYIDGGASOE

EFYKFIKPIFEKMDGTEEFFVKFNREDFFRKORTFDNGSIPHOIHFGEFHAIFRROE D

FYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKG

ASAOSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS

GEOKKAIVDFFFKTNRKVTVKOFKEDYFKKIECFDSVEISGVEDRFNASFGTYHDF

LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKOLKRR

RYTGWGRFSRKFINGIRDKOSGKTIFDFFKSDGFANRNFMOFIHDDSFTFKEDIOKA

OVSGOGDSFHEHIANFAGSPAIKKGIFOTVKVVDEFVKVMGRHKPENIVIEMAREN

OTTOKGOKNSRERMKRIEEGIKEFGSOIFKEHPVENTOFONEKFYFYYFONGRDM

YVDOEFDINRFSDYDVDHIVPOSFFKDDSIDNKVFTRSDKNRGKSDNVPSEEVVKK

MKNYWROFFNAKFITORKFDNFTKAERGGFSEFDKAGFIKROFVETROITKHVAOI

FDSRMNTKYDENDKFIREVKVITFKSKFVSDFRKDFOFYKVREINNYHHAHDAYF

N A V V GT AFIKKYPKFES EFV Y GD YKV YD VRKMIAKS EOEIGKAT AKYFFY S NIMNF

FKTEITFANGEIRKRPFIETNGETGEIVWDKGRDFATVRKVFSMPOVNIVKKTEVOT

GGFSKESIFPKRNSDKFIARKKDWDPKKYGGFDSPTVAYSVFVVAKVEKGKSKKF

KS VKEFFGITIMERS S FEKNPIDFFE AKG YKE VKKDFIIKFPKY S FFEFEN GRKRMFA

SAGEFOKGNEFAFPSKYVNFFYFASHYEKFKGSPEDNEOKOFFVEOHKHYFDEIIE

OISEFSKRVIFADANFDKVFSAYNKHRDKPIREOAENIIHFFTFTNFGAPAAFKYFD T

TIDRKRYTSTKEVLDATLIHOSITGLYETRIDLSOLGGDSGGS/YOOG^y (SEQ

ID NO: 224)

ABEmax(TadA E59A, TadA* R47M)

MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR

HDPTAHAAIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVV

FGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQE

IKAQKKAQSSTOSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEYEESHEYWMR

HALTLAKRARDEREVPVGAVLVLNNRVIGEGWNMAIGLHDPTAHAEIMALRQ

GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSL

MDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSG

GSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAYYTDEYKYP S KKFKVFGNTDRHS IKKNFIGAFFFDS GET AE ATRFKRT ARRRYTRRKNRIC YFOEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV DSTDKADFRFIYFAFAHMIKFRGHFFIEGDFNPDNSDVDKFFIOFVOTYNOFFEENP IN AS G VD AK AILS ARLS KS RRLENLIAOLPGEKKN GLF GNLIALS LGLTPNFKS NFDL AEDAKLOLSKDTYDDDLDNLLAOIGDOYADLFLAAKNLSDAILLSDILRVNTEITK

APLSASMIKRYDEHHODLTLLKALVROOLPEKYKEIFFDOSKNGYAGYIDGGASOE

EFYKFIKPIFEKMDGTEEFFVKFNREDFFRKORTFDNGSIPHOIHFGEFHAIFRROE D

FYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKG

ASAOSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS

GEOKKAIVDFFFKTNRKVTVKOFKEDYFKKIECFDSVEISGVEDRFNASFGTYHDF

LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKOLKRR

RYTGWGRFSRKFINGIRDKOSGKTIFDFFKSDGFANRNFMOFIHDDSFTFKEDIOKA

OVSGOGDSFHEHIANFAGSPAIKKGIFOTVKVVDEFVKVMGRHKPENIVIEMAREN

OTTOKGOKNSRERMKRIEEGIKEFGSOIFKEHPVENTOFONEKFYFYYFONGRDM

YVDOEFDINRFSDYDVDHIVPOSFFKDDSIDNKVFTRSDKNRGKSDNVPSEEVVKK

MKNYWROFFNAKFITORKFDNFTKAERGGFSEFDKAGFIKROFVETROITKHVAOI

FDSRMNTKYDENDKFIREVKVITFKSKFVSDFRKDFOFYKVREINNYHHAHDAYF

N A V V GT AFIKKYPKFES EFV Y GD YKV YD VRKMIAKS EOEIGKAT AKYFFY S NIMNF

FKTEITFANGEIRKRPFIETNGETGEIVWDKGRDFATVRKVFSMPOVNIVKKTEVOT

GGFSKESIFPKRNSDKFIARKKDWDPKKYGGFDSPTVAYSVFVVAKVEKGKSKKF

KS VKEFFGITIMERS S FEKNPIDFFE AKG YKE VKKDFIIKFPKY S FFEFEN GRKRMFA

SAGEFOKGNEFAFPSKYVNFFYFASHYEKFKGSPEDNEOKOFFVEOHKHYFDEIIE

OISEFSKRVIFADANFDKVFSAYNKHRDKPIREOAENIIHFFTFTNFGAPAAFKYFD T

TIDRKRYTSTKEVLDATLIHOSITGLYETRIDLSOLGGDSGGS/YOOG^y (SEQ

ID NO: 225)

ABEmax(TadA E59Q, TadA* V106W) [ABEmaxQW]

MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR

HDPTAHAQIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVV

FGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQE

IKAQKKAQSSTOSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEYEFSHEYWMR

HALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQ

GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNAKTGAAGSL

MDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSG

GSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSlGLAlGTNSYGWAYnDEYKYPS KKFKVFGNTDRHS IKKNFIGAFFFDS GET AE ATRFKRT ARRRYTRRKNRIC YFOEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV DSTDKADFRFIYFAFAHMIKFRGHFFIEGDFNPDNSDVDKFFIOFVOTYNOFFEENP IN AS G VD AK AILS ARLS KS RRLENLIAOLPGEKKN GLF GNLIALS LGLTPNFKS NFDL AEDAKLOLSKDTYDDDLDNLLAOIGDOYADLFLAAKNLSDAILLSDILRVNTEITK

APLSASMIKRYDEHHODLTLLKALVROOLPEKYKEIFFDOSKNGYAGYIDGGASOE

EFYKFIKPIFEKMDGTEEFFVKFNREDFFRKORTFDNGSIPHOIHFGEFHAIFRROE D

FYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKG

ASAOSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS

GEOKKAIVDFFFKTNRKVTVKOFKEDYFKKIECFDSVEISGVEDRFNASFGTYHDF

LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKOLKRR

RYTGWGRFSRKFINGIRDKOSGKTIFDFFKSDGFANRNFMOFIHDDSFTFKEDIOKA

OVSGOGDSFHEHIANFAGSPAIKKGIFOTVKVVDEFVKVMGRHKPENIVIEMAREN

OTTOKGOKNSRERMKRIEEGIKEFGSOIFKEHPVENTOFONEKFYFYYFONGRDM

YVDOEFDINRFSDYDVDHIVPOSFFKDDSIDNKVFTRSDKNRGKSDNVPSEEVVKK

MKNYWROFFNAKFITORKFDNFTKAERGGFSEFDKAGFIKROFVETROITKHVAOI

FDSRMNTKYDENDKFIREVKVITFKSKFVSDFRKDFOFYKVREINNYHHAHDAYF

N A V V GT AFIKKYPKFES EFV Y GD YKV YD VRKMIAKS EOEIGKAT AKYFFY S NIMNF

FKTEITFANGEIRKRPFIETNGETGEIVWDKGRDFATVRKVFSMPOVNIVKKTEVOT

GGFSKESIFPKRNSDKFIARKKDWDPKKYGGFDSPTVAYSVFVVAKVEKGKSKKF

KS VKEFFGITIMERS S FEKNPIDFFE AKG YKE VKKDFIIKFPKY S FFEFEN GRKRMFA

SAGEFOKGNEFAFPSKYVNFFYFASHYEKFKGSPEDNEOKOFFVEOHKHYFDEIIE

OISEFSKRVIFADANFDKVFSAYNKHRDKPIREOAENIIHFFTFTNFGAPAAFKYFD T

TIDRKRYTSTKEVLDATLIHOSITGLYETRIDLSOLGGDSGGS/YOOG^y (SEQ

ID NO: 226)

ABEmax(TadA E59A, TadA*N108W, R47Q)

MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGR

HDPTAHAAIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVV

FGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQE

IKAQKKAQSSTOSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEYEESHEYWMR

HALTLAKRARDEREVPVGAVLVLNNRVIGEGWNQAIGLHDPTAHAEIMALRQ

GGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRWAKTGAAGSL

MDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSG

GSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAYYTDEYKYP S KKFKVFGNTDRHS IKKNFIGAFFFDS GET AE ATRFKRT ARRRYTRRKNRIC YFOEIF SNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV DSTDKADFRFIYFAFAHMIKFRGHFFIEGDFNPDNSDVDKFFIOFVOTYNOFFEENP IN AS G VD AK AILS ARLS KS RRLENLIAOLPGEKKN GLF GNLIALS LGLTPNFKS NFDL AEDAKLOLSKDTYDDDLDNLLAOIGDOYADLFLAAKNLSDAILLSDILRVNTEITK

APLSASMIKRYDEHHODLTLLKALVROOLPEKYKEIFFDOSKNGYAGYIDGGASOE

EFYKFIKPIFEKMDGTEEFFVKFNREDFFRKORTFDNGSIPHOIHFGEFHAIFRROE D

FYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKG

ASAOSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS

GEOKKAIVDFFFKTNRKVTVKOFKEDYFKKIECFDSVEISGVEDRFNASFGTYHDF

LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKOLKRR

RYTGWGRFSRKFINGIRDKOSGKTIFDFFKSDGFANRNFMOFIHDDSFTFKEDIOKA

OVSGOGDSFHEHIANFAGSPAIKKGIFOTVKVVDEFVKVMGRHKPENIVIEMAREN

OTTOKGOKNSRERMKRIEEGIKEFGSOIFKEHPVENTOFONEKFYFYYFONGRDM

YVDOEFDINRFSDYDVDHIVPOSFFKDDSIDNKVFTRSDKNRGKSDNVPSEEVVKK

MKNYWROFFNAKFITORKFDNFTKAERGGFSEFDKAGFIKROFVETROITKHVAOI

FDSRMNTKYDENDKFIREVKVITFKSKFVSDFRKDFOFYKVREINNYHHAHDAYF

N A V V GT AFIKKYPKFES EFV Y GD YKV YD VRKMIAKS EOEIGKAT AKYFFY S NIMNF

FKTEITFANGEIRKRPFIETNGETGEIVWDKGRDFATVRKVFSMPOVNIVKKTEVOT

GGFSKESIFPKRNSDKFIARKKDWDPKKYGGFDSPTVAYSVFVVAKVEKGKSKKF

KS VKEFFGITIMERS S FEKNPIDFFE AKG YKE VKKDFIIKFPKY S FFEFEN GRKRMFA

SAGEFOKGNEFAFPSKYVNFFYFASHYEKFKGSPEDNEOKOFFVEOHKHYFDEIIE

OISEFSKRVIFADANFDKVFSAYNKHRDKPIREOAENIIHFFTFTNFGAPAAFKYFD T

TIDRKRYTSTKEVFDATFIHOSITGFYETRIDFSOFGGDSGGS/YOOG^y (SEQ

ID NO: 227)

ABEmax(TadA E59A, TadA*V106W, R47Q)

MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPT AHAAIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRWFGARDAKT GAA GSLMD VLHHPGMNHR VEITEGILADECAALLSDFFRMRRQEIKA QKKA QSS TD SGGSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREV PVGA VLVLNNR VIGEGWNQAIGLHDPTAHAEIMALRQGGLVMQNYRLIDA TLYVTF EPCVMCA GAMIHSRIGR WFGWRNAKTGAA GSLMD VLHYPGMNHR VEITE GILAD ECAALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGG SDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE AT RLKRTARRRYTRRKNRICYLOEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI V DEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDK L

F!OL VOTYNOLEEENPINA SG VDA KA 1LSA RLSKSRRLENLIA OLPGE KKNGLFGNUA LSLG LTPNFKSNFDLA EDA KLOLSKDTYDDDLDNLLA QIC, DO YA DLELAA KNLSDA ILLSDILR V NTEITKAPLSASMIKRYDEHHODLTLLKALVROOLPEKYKEIFFDOSKNGYAGYIDGGAS O

EEFYKFIKPILEKMDGTEELLVKLNREDLLRKORTFDNGSIPHOIHLGELHAILRRO EDFY

PFEKDNREKIEKILTFRIP YYVGPLARGNSRFA WMTRKSEETITP WNFEEVVDKGASA OS FI

ERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEOKKAIV DLL

FKTNRKVTVKOLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDN EENE

DILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKOLKRRRYTGWGRLSRKLINGI RDKO

SGKTILDFLKSDGFANRNFMOLIHDDSLTFKEDIOKAOVSGOGDSLHEHIANLAGSP AIKK

GILOTVKVVDELVKVMGRHKPENIVIEMARENOTTOKGOKNSRERMKRIEEGIKELG SOIL

KEHPVENTOLONEKLYLYYLONGRDMYVDOELDINRLSDYDVDHIVPOSFLKDDSID NKV

LTRSDKNR GKSDNVPSEEVVKKMKNYWR OEENAKEIT ORKFDNLTKAERGGESEEDKA G

EIKROL VETROITKH WA OILDSRMNTK YDENDKLIREVKVITLKSKL VSDERKDEOE YK VRE

INNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEOEIGKATA KYFF

YSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPOVNIV KKTEV

OTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKL KSV

KELLGITIMERSSE EKN P IDE LEAKGY KEV KKDUIKLP KYSLE ELENGRKRMLASAGELOK

GNELA LPSK YVNEL YLASH YEKLKGSPEDNEOKOLEVEOHKH YLDEIIEOISEESKR VILA D

ANLDKVLSAYNKHRDKPIREOAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKE VLDA

TEIHOSITGL YETRIDES OEGGDS GGSPKKKRKV (SEQ ID NO: 228)

Fusion protein architectures

[0263] As provided above, exemplary aspects of the disclosure provide fusion proteins comprising a Cas9 domain and an adenosine deaminase domain. The Cas9 domain may be any of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) provided herein. In some embodiments, any of the Cas9 domains or Cas9 proteins (e.g., dCas9 or nCas9) provided herein may be fused with any of the adenosine deaminases provided herein. In some embodiments, the adenosine deaminase domain comprises a single adenosine deaminase enzyme. In other embodiments, the adenosine deaminase domain comprises two adenosine deaminases, e.g., a heterodimer of adenosine deaminases.

[0264] In some embodiments, the fusion proteins comprising adenosine deaminases and a napDNAbp (e.g., Cas9 domain) do not include a linker sequence. In some embodiments, a linker is present between the adenosine deaminases and/or between an adenosine deaminase and the napDNAbp. In some embodiments, the“]-[” used in the general architecture above indicates the presence of an optional linker. In some embodiments, an adenosine deaminase and the napDNAbp are fused via any of the linkers provided herein, and the adenosine deaminases are fused to each other via any of the linkers provided herein. For example, in some embodiments the adenosine deaminases and the napDNAbp are fused via any of the linkers provided below in the section entitled“Linkers”.

[0265] In some embodiments, the fusion proteins provided herein further comprise one or more nuclear targeting sequences, for example, a nuclear localization sequence (NLS). In some embodiments, a NLS comprises an amino acid sequence that facilitates the importation of a protein, that comprises an NLS, into the cell nucleus ( e.g ., by nuclear transport). In some embodiments, any of the fusion proteins provided herein further comprise a nuclear localization sequence (NLS). In certain embodiments, any of the base editors comprise two NLSs. In some embodiments, one or more of the NLSs are bipartite NLSs (“bpNLS”). In certain embodiments, the disclosed base editors comprise two bipartite NLSs. In some embodiments, the disclosed base editors comprise more than two bipartite NLSs.

[0266] 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 C-terminus of the napDNAbp. In some embodiments, the NLS is fused to the N-terminus of the adenosine deaminase. In some embodiments, the NLS is fused to the C-terminus of the adenosine deaminase. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker. In some embodiments, the NLS comprises an amino acid sequence of any one of the NLS sequences provided or referenced herein. In some embodiments, the NLS comprises an amino acid sequence as set forth in SEQ ID NO:

117 or SEQ ID NO: 118. In some embodiments, the NLS comprises an amino acid sequence as set forth in SEQ ID NO: 114 or SEQ ID NO: 115. Additional nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al, PCT/EP2000/011690, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, a NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 117), MDSLLMNRRKFLY QFKNVRWAKGRRETYLC (SEQ ID NO: 118), KRT ADGS EFES PKKKRKV (SEQ ID NO: 114), or KRT ADGS EFEPKKKRKV (SEQ ID NO: 115).

[0267] In some embodiments, the fusion proteins provided herein do not comprise a linker.

In some embodiments, a linker is present between one or more of the domains or proteins (e.g., adenosine deaminase, napDNAbp, and/or NLS). In some embodiments, the“]-[” used in the general architecture above indicates the presence of an optional linker.

[0268] In some embodiments, the general architecture of exemplary fusion proteins with a first adenosine deaminase, a second adenosine deaminase, and a napDNAbp comprises any one of the following structures, where NLS is a nuclear localization sequence ( e.g ., any NLS provided herein), Nt is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein.

[0269] In some embodiments, the general architecture of exemplary fusion proteins comprising a first adenosine deaminase, a second adenosine deaminase, and a napDNAbp. Nth- [first adenosine deaminase] -[second adenosine deaminase] -[napDNAbp] -COOH;

NH ₂ -[first adenosine deaminase]-[napDNAbp]-[second adenosine deaminase] -COOH;

N¾- [napDNAbp] -[first adenosine deaminase] -[second adenosine deaminase] -COOH;

NH ₂ -[second adenosine deaminase] -[first adenosine deaminase]-[napDNAbp]-COOH;

N¾- [second adenosine deaminase] -[napDNAbp] -[first adenosine deaminase] -COOH;

N¾- [napDNAbp] -[second adenosine deaminase] -[first adenosine deaminase] -COOH;

[0270] In particular embodiments, the disclosure provides a fusion protein comprising the architecture NH ₂ -[first adenosine deaminase]-[second adenosine deaminase]-[napDNAbp]- [NLS]-COOH.

[0271] Exemplary fusion proteins comprising a first adenosine deaminase, a second adenosine deaminase, a napDNAbp, and an NLS, where NLS is a nuclear localization sequence (e.g., any NLS provided herein).

N¾- [NLS] -[first adenosine deaminase] -[second adenosine deaminase] -[napDNAbp] -COOH; NH ₂ -[first adenosine deaminase] -[NLS] -[second adenosine deaminase] -[napDNAbp] -COOH; NH ₂ -[first adenosine deaminase] -[second adenosine deaminase]-[NLS]-[napDNAbp]-COOH; NH ₂ -[first adenosine deaminase] -[second adenosine deaminase]-[napDNAbp]-[NLS]-COOH; N¾- [NLS] -[first adenosine deaminase] -[napDNAbp] -[second adenosine deaminase] -COOH; NH ₂ -[first adenosine deaminase]-[NLS]-[napDNAbp]-[second adenosine deaminase] -COOH; NH ₂ -[first adenosine deaminase]-[napDNAbp]-[NLS]-[second adenosine deaminase]-COOH; NH ₂ -[first adenosine deaminase]-[napDNAbp]-[second adenosine deaminase]-[NLS]-COOH; NH ₂ -[NLS]-[napDNAbp]-[first adenosine deaminase]-[second adenosine deaminase] -COOH; NH ₂ -[napDNAbp]-[NLS]-[first adenosine deaminase]-[second adenosine deaminase] -COOH; N¾- [napDNAbp] -[first adenosine deaminase]-[NLS]-[second adenosine deaminase]-COOH; N¾- [napDNAbp] -[first adenosine deaminase] -[second adenosine deaminase]-[NLS]-COOH; N¾- [NLS] -[second adenosine deaminase] -[first adenosine deaminase] -[napDNAbp] -COOH; NH ₂ -[second adenosine deaminase] -[NLS] -[first adenosine deaminase] -[napDNAbp] -COOH; NH ₂ -[second adenosine deaminase] -[first adenosine deaminase]-[NLS]-[napDNAbp]-COOH; NH ₂ -[second adenosine deaminase] -[first adenosine deaminase]-[napDNAbp]-[NLS]-COOH; N¾- [NLS] -[second adenosine deaminase]-[napDNAbp]-[first adenosine deaminase] -COOH; NH ₂ -[second adenosine deaminase]-[NLS]-[napDNAbp]-[first adenosine deaminase]-COOH; NH2-[second adenosine deaminase]-[napDNAbp]-[NLS]-[first adenosine deaminase] -COOH;

NH2-[second adenosine deaminase] -[napDNAbp]- [first adenosine deaminase]-[NLS]-COOH;

NH2-[NLS]-[napDNAbp]-[second adenosine deaminase] -[first adenosine deaminase] -COOH;

NH2-[napDNAbp]-[NLS]-[second adenosine deaminase] -[first adenosine deaminase] -COOH;

N¾- [napDNAbp] -[second adenosine deaminase] -[NLS]- [first adenosine deaminase] -COOH; or

N¾- [napDNAbp] -[second adenosine deaminase] -[first adenosine deaminase]-[NLS]-COOH.

[0272] In some embodiments, the fusion proteins provided herein do not comprise a linker.

In some embodiments, a linker is present between one or more of the domains or proteins ( e.g ., first adenosine deaminase, second adenosine deaminase, napDNAbp, and/or NLS). In some embodiments, the“]-[” used in the general architecture above indicates the presence of an optional linker.

[0273] It should be appreciated that the fusion proteins of the present disclosure may comprise one or more additional domains, such as one or more hetereologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the base editor components). A disclosed fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. 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.

[0274] Examples of protein domains that may be fused to a base editor or component thereof (e.g., the napDNAbp domain, the nucleobase modification domain, or the NLS domain) include, without limitation, epitope tags, and reporter gene sequences. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta- glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). A base editor may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP 16 protein fusions. Additional domains that may form part of a base editor are described in US Patent Publication No. 2011/0059502, published March 10, 2011 and incorporated herein by reference in its entirety.

[0275] In an aspect of the disclosure, a reporter gene which includes, but is not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol

acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP), may be introduced into a cell to encode a gene product which serves as a marker by which to measure the alteration or modification of expression of the gene product. In certain embodiments of the disclosure the gene product is luciferase. In a further embodiment of the disclosure the expression of the gene product is decreased.

[0276] 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..

Linkers

[0277] In some embodiments of the disclosed adenine base editors, linkers may be used to link any of the protein or protein domains described herein. The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length. In certain

embodiments, the linker is a polypeptide or based on amino acids. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta- alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises amino acids. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may include functionalized moieties to facilitate attachment of a nucleophile

(e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

[0278] In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein). In some embodiments, the linker is a bond ( e.g., a covalent bond), an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is

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, 31, 32, 33, 34, 35-40, 40-45, 45-50, 50-60, 60-70,

70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, a linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 111), which may also be referred to as the XTEN linker. In some embodiments, the linker is 32 amino acids in length. In some embodiments, the linker comprises the amino acid sequence

(SGGS) ₂ -SGSETPGTSESATPES-(SGGS) ₂ (SEQ ID NO: 112), which may also be referred to as (SGGS) ₂ -XTEN-(SGGS) ₂ (SEQ ID NO: 112). In some embodiments, the linker comprises the amino acid sequence, wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, a linker comprises the amino acid sequence SGGS (SEQ ID NO: 113). In some embodiments, a linker comprises (SGGS) _n (SEQ ID NO: 229), (GGGS) _n (SEQ ID NO:

230), (GGGGS) _n (SEQ ID NO: 231), (G)„ (SEQ ID NO: 232), (EAAAK)„ (SEQ ID NO:

233), (SGGS)„-SGSETPGTSESATPES-(SGGS)„ (SEQ ID NO: 234), (GGS)n (SEQ ID NO:

235), SGSETPGTSESATPES (SEQ ID NO: 236), or (XP)„ (SEQ ID NO: 237) 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, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

13, 14, or 15. In some embodiments, a linker comprises SGSETPGTSESATPES (SEQ ID

NO: 236), and SGGS (SEQ ID NO: 229). In some embodiments, a linker comprises

SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 238). In some embodiments, a linker comprises SGGSSGGSSGS ETPGT S ES ATPES SGGSSGGS (SEQ ID NO: 239). In some embodiments, a linker comprises

GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PS EGS APGT S TEPS EGS APGT S ES ATPES GPGS EP AT S GGS GGS (SEQ ID NO: 240). In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 241).

In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence

SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 242). In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence

SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGS SGGS (SEQ ID NO: 243). In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence

PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA P GTSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 244). It should be appreciated that any of the linkers provided herein may be used to link a first adenosine deaminase and a second adenosine deaminase; an adenosine deaminase ( e.g ., a first or a second adenosine deaminase) and a napDNAbp; a napDNAbp and an NLS; or an adenosine deaminase (e.g., a first or a second adenosine deaminase) and an NLS.

[0279] In some embodiments, any of the fusion proteins provided herein, comprise an adenosine deaminase and a napDNAbp that are fused to each other via a linker. In some embodiments, any of the fusion proteins provided herein, comprise a first adenosine deaminase and a second adenosine deaminase that are fused to each other via a linker. In some embodiments, any of the fusion proteins provided herein, comprise an NLS, which may be fused to an adenosine deaminase (e.g., a first and/or a second adenosine deaminase) and a nucleic acid programmable DNA binding protein (napDNAbp). Various linker lengths and flexibilities between an adenosine deaminase (e.g., an engineered ecTadA) and a napDNAbp (e.g., a Cas9 domain), and/or between a first adenosine deaminase and a second adenosine deaminase may be employed (e.g., ranging from flexible linkers of the form of SEQ ID NOs: 229-245 (see, e.g., Guilinger JP, Thompson DB, Liu DR. Fusion of catalytically inactive Cas9 to Fokl 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 (SEQ ID NO: 237)) in order to achieve the optimal length for deaminase activity for the specific application. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS) _n (SEQ ID NO: 245) motif, wherein n is 1,

3, or 7. In some embodiments, the adenosine deaminase and the napDNAbp, and/or the first adenosine deaminase and the second adenosine deaminase of any of the fusion proteins provided herein are fused via a linker comprising an amino acid sequence selected from SEQ ID NOs: 229-245. In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker is 32 amino acids in length. In some embodiments, the linker is 32 amino acids in length. In some embodiments, the linker comprises the amino acid sequence

(SGGS) ₂ -SGSETPGTSESATPES-(SGGS) ₂ (SEQ ID NO: 112), which may also be referred to as (SGGS) ₂ -XTEN-(SGGS) ₂ (SEQ ID NO: 112). In some embodiments, the linker comprises the amino acid sequence, wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker is 92 amino acids in length.

Reduced Off -Tar get Effects

Reduced RNA Editing Effects

[0280] In some aspects, provided herein are adenine base editors and methods of editing DNA by contacting DNA with any of these disclosed base editors that generate (or cause) reduced off-target effects.. In some embodiments, the base editors are evolved or engineered to have reduced RNA editing effects. The term“RNA editing effects,” as used herein, refers to the introduction of modifications ( e.g . deaminations) of nucleotides within cellular RNA, e.g., messenger RNA (mRNA). An important goal of DNA base editing efficiency is the modification (e.g. deamination) of a specific nucleotide within DNA, without introducing modifications of similar nucleotides within RNA. RNA editing effects are“low” or “reduced” when a detected mutation is introduced into RNA molecules at a frequency of 0.3% or less. RNA editing may be measured by techniques known in the art, including high- throughput screening of sequencing reads and RNA-seq. The effects of RNA editing on the function of a protein translated from the edited mRNA transcript may be predicted by use of the SIFT algorithm, which bases predictions on sequence homology and the physical properties of amino acids.

[0281] The present disclosure further provides methods of administering the disclosed adenine base editors wherein the method yields reduced off-target effects, such as low RNA editing effects. In some embodiments, the methods induce (or yield, provide or cause) an average adenosine (A) to inosine (I) (A-to-I) editing frequency in cellular mRNA transcripts of 0.3% or less. In some embodiments, the methods induce (or cause) an average adenosine (A) to inosine (I) (A-to-I) actual and/or consistent editing frequencies in RNA of about 0.3% or less. The methods may induce actual or average A-to-I editing frequencies in RNA of about 0.5% or less, 0.4% or less, 0.35% or less, 0.3% or less, 0.25% or less, 0.2% or less, 0.15% or less, 0.12% or less, 0.1% or less, 0.08% or less, 0.075% or less, 0.06% or less, 0.05% or less, 0.04% or less, or 0.01% or less. In particular embodiments, the methods and base editors used therein induce an average A-to-I editing frequency of 0.068%.

[0282] In some embodiments, the methods induce (or provide or cause) an average adenosine

(A) to inosine (I) (A-to-I) editing frequency across the mRNA transcriptome of a human cell

(e.g. an HEK293 cell) of about 0.2% or less. The methods may induce actual or average A- to-I transcriptome-wide editing frequencies in RNA of about 0.5% or less, 0.4% or less,

0.35% or less, 0.25% or less, 0.2% or less, 0.15% or less, 0.12% or less, 0.1% or less, 0.08% or less, 0.075% or less, 0.06% or less, 0.05% or less, 0.04% or less, or 0.01% or less. In particular embodiments, the methods induce a human mRNA transcriptome-wide average A- to-I editing frequency of 0.14%.

[0283] In some aspects, the methods induce average overall magnitudes of detectable A-to-I edits among 182 total adenosines analyzed among three mRNA transcripts of 50 edits or less. The methods may induce magnitudes of A-to-I edits in this substrate of about 40 edits or less, 35 edits or less, 30 edits or less, 25 edits or less, 20 edits or less, or 15 edits or less. In a particular embodiment, the methods induce magnitudes of 26 edits or less.

[0284] In some aspects, the methods induce average overall magnitudes of detectable A-to-I edits among the transcriptome of a human cell of 65,000 edits or less. The methods may induce magnitudes of A-to-I edits in this substrate of about 70,000 edits or less, 62,000 edits or less, 60,000 edits or less, 58,000 edits or less, 57,750 edits or less, 57,500 edits or less, 57,000 edits or less, 56,500 edits or less, 56,000 edits or less, 55,500 edits or less, 55,000 edits or less, or 54,000 edits or less. In a particular embodiment, the methods induce transcriptome-wide magnitudes of 57,700 edits or less.

Reduced off-target DNA Editing and Indel Frequencies

[0285] Some aspects of the disclosure are based on the recognition that any of the adenine base editors provided herein are capable of modifying a specific DNA 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 DNA substrate. 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 adenine base editors that efficiently modify (e.g. mutate or deaminate) a specific nucleotide within a DNA, without generating a large number of insertions or deletions ( i.e ., indels) in the nucleic acid (while at the same time having lower RNA editing effects than existing adenine base editors).

[0286] 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 (e.g. deamination). In some embodiments, the intended mutation is a mutation associated with a disease or disorder. In some embodiments, the intended mutation is a adenine (A) to guanine (G) point mutation associated with a disease or disorder. In some embodiments, the intended mutation is a thymine (T) to cytosine (C) point mutation associated with a disease or disorder. In some embodiments, the intended mutation is a adenine (A) to guanine (G) point mutation within the coding region of a gene. In some embodiments, the intended mutation is a thymine (T) to cytosine (C) point mutation within the coding region of a gene. In some embodiments, the intended mutation is a deamination 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 eliminates a stop codon comprising the nucleic acid sequence 5'-TAG-3',

5'-TAA-3', or 5'-TGA-3'.

[0287] In some embodiments, the intended mutation is a deamination that alters the regulatory sequence of a gene (e.g., a gene promotor or gene repressor). In some

embodiments, the intended mutation is a deamination introduced into the gene promoter or gene repressor. In particular embodiments, the deamination introduced into the gene promoter (or gene repressor) leads to a decrease in the transcription of a gene operably linked to the gene promoter (or gene repressor). In other embodiments, the deamination leads to an increase in the transcription of a gene operably linked to the gene promoter (or gene repressor).

[0288] In some embodiments, the intended mutation is a deamination that alters the splicing of a gene. Accordingly, in some embodiments, the intended deamination results in the introduction of a splice site in a gene. In other embodiments, the intended deamination results in the removal of a splice site.

[0289] In certain embodiments, any of the adenine 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, to calculate indel frequencies, sequencing reads are scanned for exact matches to two 10-bp sequences that flank both sides of a window in which indels might occur. If no exact matches are located, the read is excluded from analysis. If the length of this indel window exactly matches the reference sequence the read is classified as not containing an indel. If the indel window is two or more bases longer or shorter than the reference sequence, then the sequencing read is classified as an insertion or deletion, respectively.

[0290] In some embodiments, the adenine base editors provided herein are capable of limiting formation of indels in a region of a DNA substrate. 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 an adenine base editor.

[0291] 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 m utati o n s : u n i n t c n dcd 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 m utati o n s : u n i n t c n dcd 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. 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.

Guide sequences (e.g., guide RNAs) [0292] The present disclosure further provides guide RNAs for use in accordance with the disclosed methods of editing. The disclosure provides guide RNAs that are designed to recognize target sequences. Such gRNAs may be designed to have guide sequences (or

“spacers”) having complementarity to a protospacer within the target sequence. Guide RNAs are also provided for use with one or more of the disclosed fusion proteins, e.g., in the disclosed methods of editing a nucleic acid molecule. Such gRNAs may be designed to have guide sequences having complementarity to a protospacer within a target sequence to be edited, and to have backbone sequences that interact specifically with the napDNAbp domains of any of the disclosed base editors, such as Cas9 nickase domains of the disclosed base editors.

[0293] The disclosure further provides methods for editing a target nucleic acid molecule, e.g., a single nucleobase within a genome, with an adenine base editor described herein (e.g., in the form of an evolved base editor as described herein, or a vector or construct encoding same), e.g. editing of cellular mRNA. Such methods involve transducing (e.g., via transfection) cells with a plurality of complexes each comprising a fusion protein (e.g., a fusion protein comprising a Cas9 nickase (nCas9) domain and an adenosine deaminase domain) and a gRNA molecule. In some embodiments, the gRNA is bound to the

napDNAbp domain (e.g., nCas9 domain) of the fusion protein. In some embodiments, each gRNA comprises a guide sequence of at least 10 contiguous nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides) that is complementary to a target sequence. In certain embodiments, the methods involve the transfection of nucleic acid constructs (e.g., plasmids) that each (or together) encode the components of a complex of fusion protein and gRNA molecule.

[0294] In other aspects, the present specification provides complexes comprising the adenine base editors described herein and a gRNA bound to the Cas9 domain of the fusion protein, such as a single guide RNA. The guide RNA may be 15-100 nucleotides in length and comprise a sequence of at least 10, at least 15, or at least 20 contiguous nucleotides that is complementary to a target nucleotide sequence. The guide RNA may comprise a sequence of

15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to a target nucleotide sequence.

[0295] In various embodiments, the disclosed ABEs may be complexed, bound, or otherwise associated with (e.g., via any type of covalent or non-covalent bond) one or more guide sequences, i.e., the sequence which becomes associated or bound to the base editor and directs its localization to a specific target sequence having complementarity to the guide sequence or a portion thereof. The particular design embodiments of a guide sequence will depend upon the nucleotide sequence of a genomic target site of interest (i.e., the desired site to be edited) and the type of napDNAbp (e.g., type of Cas protein) present in the base editor, among other factors, such as PAM sequence locations, percent G/C content in the target sequence, the degree of microhomology regions, secondary structures, etc.

[0296] In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a napDNAbp (e.g., a Cas9, Cas9 homolog, or Cas9 variant) to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%,

85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the

Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the

Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X,

BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP

(available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17,

18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.

[0297] In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide sequence to direct sequence- specific binding of a base editor to a target sequence may be assessed by any suitable assay. For example, the components of a base editor, including the guide sequence to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of a base editor disclosed herein, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a base editor, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art.

[0298] A guide sequence may be selected to target any target sequence. In some

embodiments, the target sequence is a sequence within a genome of a cell. Exemplary target sequences include those that are unique in the target genome. For example, for the S.

pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGG (SEQ ID NO: 196) where

NNNNNNNNNNNNXGG (N is A, G, T, or C; and X can be anything) (SEQ ID NO: 197) has a single occurrence in the genome. A unique target sequence in a genome may include an

S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGG (SEQ

ID NO: 198) where NNNNNNNNNNNXGG (N is A, G, T, or C; and X can be anything)

(SEQ ID NO: 199) has a single occurrence in the genome. For the S. thermophilus

CRISPRlCas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW (SEQ ID NO: 200) where

NNNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T)

(SEQ ID NO: 201) has a single occurrence in the genome. A unique target sequence in a genome may include an S. thermophilus CRISPR 1 Cas9 target site of the form

MMMMMMMMMNNNNNNNNNNNXXAGAAW (SEQ ID NO: 202) where

NNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T)

(SEQ ID NO: 203) has a single occurrence in the genome. For the S. pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form

MMMMMMMMNNNNNNNNNNNNXGGXG (SEQ ID NO: 204) where

NNNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) (SEQ ID NO:

205) has a single occurrence in the genome. A unique target sequence in a genome may include an S. pyogenes Cas9 target site of the form

MMMMMMMMMNNNNNNNNNNNXGGXG (SEQ ID NO: 206) where

NNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) (SEQ ID NO: 207) has a single occurrence in the genome. In each of these sequences“M” may be A, G, T, or C, and need not be considered in identifying a sequence as unique.

[0299] In some embodiments, a guide sequence is selected to reduce the degree of secondary structure within the guide sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker & Stiegler {Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online Webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see, e.g., A. R. Gruber el al., 2008, Cell 106(1): 23-24; and PA Carr & GM Church, 2009, Nature Biotechnology 27(12): 1151- 62). Additional algorithms may be found in Chuai, G. el al, DeepCRISPR: optimized CRISPR guide RNA design by deep learning , Genome Biol. 19:80 (2018), and U.S.

application Ser. No. 61/836,080 and U.S. Patent No. 8,871,445, issued October 28, 2014, the entireties of each of which are incorporated herein by reference. [0300] In general, a tracr mate sequence includes any sequence that has sufficient

complementarity with a tracr sequence to promote one or more of: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a complex at a target sequence, wherein the complex comprises the tracr mate sequence hybridized to the tracr sequence. In general, degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the tracr sequence or tracr mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and tracr mate sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the tracr sequence and tracr mate sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin. Preferred loop forming sequences for use in hairpin structures are four nucleotides in length, and most preferably have the sequence GAAA. However, longer or shorter loop sequences may be used, as may alternative sequences. The sequences preferably include a nucleotide triplet (for example, AAA), and an additional nucleotide (for example C or G). Examples of loop forming sequences include CAAA and AAAG. In an embodiment of the disclosure, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In certain embodiments, the transcript has two, three, four or five hairpins. In a further embodiment of the disclosure, the transcript has at most five hairpins.

In some embodiments, the single transcript further includes a transcription termination sequence; preferably this is a polyT sequence, for example six T nucleotides. Further non limiting examples of single polynucleotides comprising a guide sequence, a tracr mate sequence, and a tracr sequence are as follows (listed 5' to 3'), where“N” represents a base of a guide sequence, the first block of lower case letters represent the tracr mate sequence, and the second block of lower case letters represent the tracr sequence, and the final poly-T sequence represents the transcription terminator:

(1) NNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagata aggctt

catgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTT TT (SEQ ID NO: 208);

(2)

NNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggc ttcatgccgaaatca acaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 209); (3)

NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagat aaggcttcatgccgaa atca acaccctgtcattttatggcagggtgtTTTTT (SEQ ID NO: 210); (4)

NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtc cgttatcaacttga aaa agtggcaccgagtcggtgcTTTTTT (SEQ ID NO: 211); (5)

NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtc cgttatcaactt gaa aaagtgTTTTTTT (SEQ ID NO: 212); and (6)

NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtc cgttatcaTTT TT TTT (SEQ ID NO: 213). In some embodiments, sequences (1) to (3) are used in combination with Cas9 from S. thermophilus CRISPR1. In some embodiments, sequences (4) to (6) are used in combination with Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a separate transcript from a transcript comprising the tracr mate sequence.

[0301] It will be apparent to those of skill in the art that in order to target any of the fusion proteins comprising a Cas9 domain and a methyltransferase, 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 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:base editing enzyme/domain fusion protein.

[0302] In some embodiments, the guide RNAs for use in accordance with the disclosed methods of editing comprise a backbone structure that is recognized by an S. pyogenes Cas9 protein or domain, such as an SpCas9 domain of the disclosed base editors. The backbone structure recognized by an SpCas9 protein may comprise the sequence 5'-[guide sequence]- guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaagu ggcaccgagucggugcuuu uu-3' (SEQ ID NO: 214), wherein the guide sequence comprises a sequence that is complementary to the protospacer of the target sequence. See U.S. Publication No.

2015/0166981, published June 18, 2015, the disclosure of which is incorporated by reference herein. The guide sequence is typically 20 nucleotides long.

[0303] In other embodiments, the guide RNAs for use in accordance with the disclosed methods of editing comprise a backbone structure that is recognized by an S. aureus Cas9 protein. The backbone structure recognized by an SaCas9 protein may comprise the sequence 5 '-[guide sequence] - guuuuaguacucuguaaugaaaauuacagaaucuacuaaaacaaggcaaaaugccguguu uaucucgucaacuuguugg cgagauuuuuuu-3' (SEQ ID NO: 195). [0304] The sequences of suitable guide RNAs for targeting the disclosed fusion proteins to specific genomic target sites will be apparent to those of skill in the art based on the instant disclosure. Such suitable guide RNA sequences typically comprise guide sequences that are complementary to a nucleic sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Some exemplary guide RNA sequences suitable for targeting any of the provided fusion proteins to specific target sequences are provided herein.

Additional guide sequences are are well known in the art and can be used with the base editors described herein. Additional exemplary guide sequences are disclosed in, for example, Jinek M., et al., Science 337:816-821(2012); Mali P, Esvelt KM & Church GM

(2013) Cas9 as a versatile tool for engineering biology, Nature Methods , 10, 957-963; Li JF et al, (2013) Multiplex and homologous recombination-mediated genome editing in

Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9, Nature Biotechnology ,

31, 688-691; Hwang, W.Y. et al., Efficient genome editing in zebrafish using a CRISPR-Cas system, Nature Biotechnology 31, 227-229 (2013); Cong L et al., (2013) Multiplex genome engineering using CRIPSR/Cas systems, Science, 339, 819-823; Cho SW et al, (2013)

Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease,

Nature Biotechnology, 31, 230-232; 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 Acid Res. (2013); Briner AE et al., (2014) Guide RNA functional modules direct Cas9 activity and orthogonality, Mol

Cell, 56, 333-339, the entire contents of each of which are herein incorporated by reference.

[0305] The disclosure further relates in various aspects to methods of making the disclosed improved adenine base editors by various modes of manipulation that include but are not limited to codon optimization to achieve greater expression levels in a cell, and the use of nuclear localization sequences (NLS)s, preferably at least two NLSs, to increase the localization of the expressed base editors into a cell nucleus.

Methods for making fusion proteins

[0306] The disclosure further relates in various aspects to methods of making the disclosed fusion proteins by various modes of manipulation that include, but are not limited to, codon optimization to achieve greater expression levels in a cell, and the use of nuclear localization sequences (NLSs), preferably at least two NLSs, e.g., two bipartite NLSs, to increase the localization of the expressed fusion proteins into a cell nucleus.

[0307] The fusion proteins contemplated herein can include modifications that result in increased expression, for example, through codon optimization. [0308] In some embodiments, the base editors (or a component thereof) is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20,

25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the“Codon Usage Database”, and these tables can be adapted in a number of ways. See Nakamura, Y., el al.“Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In some

embodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.

[0309] In other embodiments, the base editors of the disclosure have improved expression (as compared to non-modified or state of the art counterpart editors) as a result of ancestral sequence reconstruction analysis. Ancestral sequence reconstruction (ASR) is the process of analyzing modern sequences within an evolutionary/phylogenetic context to infer the ancestral sequences at particular nodes of a tree. These ancient sequences are most often then synthesized, recombinantly expressed in laboratory microorganisms or cell lines, and then characterized to reveal the ancient properties of the extinct biomolecules 2, 3, 4, 5, 6. This process has produced tremendous insights into the mechanisms of molecular adaptation and functional divergence7. Despite such insights, a major criticism of ASR is the general inability to benchmark accuracy of the implemented algorithms. It is difficult to benchmark ASR for many reasons. Notably, genetic material is not preserved in fossils on a long enough time scale to satisfy most ASR studies (many millions to billions of years ago), and it is not yet physically possible to travel back in time to collect samples. Reference is made to Cai et al,“Reconstruction of ancestral protein sequences and its applications,” BMC Evolutionary

Biology 2004, 4:33 and Zakas et al,“Enhancing the pharmaceutical properties of protein drugs by ancestral sequence reconstruction,” Nature Biotechnology , 35, pp. 35-37 (2017), each of which are incorporated herein by reference.

[0310] There are many software packages available which can perform ancestral state reconstruction. Generally, these software packages have been developed and maintained through the efforts of scientists in related fields and released under free software licenses.

The following list is not meant to be a comprehensive itemization of all available packages, but provides a representative sample of the extensive variety of packages that implement methods of ancestral reconstruction with different strengths and features: PAML

(Phylogenetic Analysis by Maximum Likelihood, available at

//abacus. gene.ucl.ac.uk/software/paml.html ), BEAST (Bayesian evolutionary analysis by sampling trees, available at //www.beast2.org/wiki/index.php/Main_Page), and Diversitree (FitzJohn RG, 2012. Diversitree: comparative phylogenetic analyses of diversification in R. Methods in Ecology and Evolution), and HyPHy (Hypothesis testing using phylogenies, available at //hyphy.org/w/index.php/Main_Page).

[0311] The Examples demonstrate one embodiment for using ASR to increase overall expression of base editors disclosed herein.

[0312] The above description is meant to be non-limiting with regard to making base editors having increased expression, and thereby increase editing efficiencies.

Vectors

[0313] Several embodiments of the making and using the base editors of the disclosure relate to vector systems comprising one or more vectors encoding the improved adenine base editors. Vectors may be designed to clone and/or express the adenine base editors of the disclosure. Vectors may also be designed to transfect the adenine base editors of the disclosure into one or more cells, e.g., a target diseased eukaryotic cell for treatment with the base editor systems and methods disclosed herein.

[0314] Vectors may be designed for expression of base editor transcripts (e.g. nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, base editor transcripts may be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press. San Diego, Calif. (1990). Alternatively, expression vectors encoding one or more adenine base editors described herein may be transcribed and translated in vitro , for example using T7 promoter regulatory sequences and T7 polymerase.

[0315] Vectors may be introduced and propagated in a prokaryotic cells. In some

embodiments, a prokaryote is used to amplify copies of a vector to be introduced into a eukaryotic cell or as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (e.g. amplifying a plasmid as part of a viral vector packaging system). In some embodiments, a prokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism. Expression of proteins in prokaryotes is most often carried out in

Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins.

[0316] Fusion expression vectors also may be used to express the adenine base editors of the disclosure. Such vectors generally add a number of amino acids to a protein encoded therein, such as to the amino terminus of the recombinant protein. Such fusion vectors may serve one or more purposes, such as: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the

recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Example fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAF (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[0317] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al, (1988) Gene 69:301-315) and pET l id (Studier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

[0318] In some embodiments, a vector drives protein expression in insect cells using baculovims expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al, 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

[0319] In some embodiments, a vector is capable of driving expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC

(Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2,

cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring

Harbor, N.Y., 1989.

[0320] In some embodiments, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue- specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver- specific; Pinkert, et al, 1987. Genes Dev. 1: 268-277), lymphoid- specific promoters (Calame and Eaton, 1988. Adv. Immunol 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Baneiji, et al, 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron- specific promoters (e.g., the neurofilament promoter; Byme and Ruddle, 1989. Proc. Natl Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al, 1985. Science 230: 912-916), and mammary gland- specific promoters (e.g., milk whey promoter, U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the a-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).

Methods of Editing A Target Nucleobase Pair, Methods of Treatment, and Uses for the ABEs

[0321] Some embodiments of the disclosure provide methods for editing 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 an adenosine deaminase domain) and a guide nucleic acid (e.g., gRNA), wherein the target region comprises a targeted nucleobase pair. As a result of embodiments of these methods, strand separation of said target region is induced, a first nucleobase of said target nucleobase pair in a single strand of the target region is converted to a second nucleobase, and no more than one strand of said target region is cut (or nicked), wherein a third nucleobase complementary to the first nucleobase base is replaced by a fourth nucleobase complementary to the second nucleobase.

[0322] In other aspects, the present disclosure provides for methods of making the adenine base editors described herein, as well as methods of using the adenine base editors or nucleic acid molecules encoding the adenine base editors in applications including editing a nucleic acid molecule, e.g., a genome. In certain embodiments, methods of engineering the adenine base editors to have reduced RNA editing effects while retaining excellent DNA editing efficiency involve mutagenesis. In certain embodiments, following the successful mutagenesis of the one or more components of the adenine base editor (e.g., one or more adenosine deaminases), methods of making the base editors comprise recombinant protein expression methodologies known to one of ordinary skill in the art.

[0323] In some embodiments, the first nucleobase is an adenine. In some embodiments, the second nucleobase is a deaminated adenine, or hypoxanthine. In some embodiments, the third nucleobase is a thymine (of the target A:T base pair). In some embodiments, the fourth nucleobase is a cytosine. In some embodiments, the method further comprises replacing the second nucleobase with a fifth nucleobase (guanine) that is complementary to the fourth nucleobase, thereby generating an intended edited base pair (e.g., A:T to G:C). In some embodiments, at least 5% of the intended base pairs are edited.

[0324] In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the intended base paires are edited. In various embodiments, the disclosed editing methods result in a DNA base editing efficiency of at least about 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99%. The step of contacting may result in in a DNA base editing efficiency of at least about 51%, 52%, 53%, 54%, 55%, 56% or 57%. In particular, the step of contacting results in base editing efficiencies of greater than 54%. In certain

embodiments, base editing efficiencies of 99% may be realized.

[0325] In some embodiments, the disclosed editing methods result in an actual or average off-target DNA editing frequency of about 2.0% or less, 1.75% or less, 1.5% or less, 1.2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.75% or less, 0.7% or less, 0.65% or less, or 0.6% or less. In a particular embodiment, the methods result in an actual or average off- target DNA editing frequence of 0.79+0.18%.

[0326] 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 intended edited base pair is upstream of a PAM site. In some embodiments, the intended edited base pair is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. In some embodiments, the intended edited 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 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 adenine base editors provided herein. In some embodiments, a target window is a deamination window.

[0327] 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 (e.g., A:T target base pair), b) converting a first nucleobase (e.g., the A base) of said target nucleobase pair in a single strand of the target region to a second nucleobase (e.g., hypoxanthine), c) cutting (or nicking) 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 (cytosine) that is complementary to the second nucleobase, and the second nucleobase is replaced with a fifth nucleobase that is complementary to the fourth nucleobase, thereby generating an intended edited base pair, wherein the efficiency of generating the intended edited base pair is at least 5%.

[0328] 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 adenine. In some embodiments, the second nucleobase is not G, C, A, or T. In some embodiments, the second base is hypoxanthine. In some embodiments, the base editor inhibits base excision repair of the edited strand. In some embodiments, the base editor protects (e.g., from base excision repair) or binds the non-edited strand. In some embodiments, the base editor comprises UGI activity. In some embodiments, the base editor comprises a catalytically inactive inosine- specific nuclease. In some embodiments, the base editor comprises nickase activity. [0329] In another embodiment, the disclosure provides editing methods comprising contacting a DNA, or RNA molecule with any of the adenine base editors provided herein, and with at least one guide nucleic acid ( e.g ., guide RNA), wherein the guide nucleic acid,

( e.g ., guide RNA) is about 15-100 nucleotides long and comprises a sequence of at least 10 contiguous nucleotides that is complementary to a target sequence. In some embodiments, the 3' end of the target sequence is immediately adjacent to a canonical PAM sequence

(NGG). In some embodiments, the 3' end of the target sequence is not immediately adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is immediately adjacent to an AGC, GAG, TTT, GTG, or CAA sequence.

[0330] 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 fusion protein (e.g., comprising an adenosine deaminase and a Cas9 domain), or the complex, results in a correction of the point mutation. In some embodiments, the target DNA sequence comprises a G to A point mutation associated with a disease or disorder, and wherein the deamination of the mutant A base results in a sequence that is not associated with a disease or disorder. In some embodiments, the target DNA sequence encodes a protein, and the point mutation is in a codon and results in a change in the amino acid encoded by the mutant codon as compared to the wild-type codon. In some embodiments, the deamination of the mutant A results in a change of the amino acid encoded by the mutant codon. In some embodiments, the deamination of the mutant A results in the codon encoding the wild-type amino acid. In some embodiments, the 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 phenylketonuria, von Willebrand disease (vWD), a neoplastic disease associated with a mutant PTEN or BRCA1, or Li-Fraumeni syndrome. A list of exemplary diseases and disorders that may be treated using the base editors described herein is shown in Table 7.

Table 7 includes the target gene, the mutation to be corrected, the related disease and the nucleotide sequence of the associated protospacer and PAM.

Table 7 - List of exemplary diseases that may be treated using the base editors described herein. The Adenine to be edited in the protospacer is indicated by underlining and the PAM is indicated in bold.

[0331] Some embodiments provide methods for targeted editing using the adenine base editors provided herein. In some embodiments, the base editors are used to introduce a point mutation into a nucleic acid by deaminating a target nucleobase, e.g., an A 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 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.

[0332] In some embodiments, the purpose of the methods provided herein is to restore the function of a dysfunctional gene via genome editing. The nucleobase editing proteins provided herein can be validated for gene editing-based human therapeutics in vitro, e.g., by correcting a disease-associated mutation in human cell culture. It will be understood by the skilled artisan that the nucleobase editing proteins provided herein, e.g., the fusion proteins comprising a nucleic acid programmable DNA binding protein (e.g., Cas9) and an adenosine deaminase domain may be used to correct any single point G to A or C to T mutation. In the first case, deamination of the mutant A to I corrects the mutation, and in the latter case, deamination of the A that is base-paired with the mutant T, followed by a round of replication, corrects the mutation. Exemplary point mutations that may be corrected are listed in Tables 1.

[0333] 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 a nucleic acid programmable DNA binding protein and an adenosine deaminase domain also have applications in“reverse” gene therapy, where certain gene functions are purposely suppressed or abolished. In these cases, site-specifically mutating residues that lead to inactivating mutations in a protein, or mutations that inhibit function of the protein may be used to abolish or inhibit protein function

Methods of treatment

[0334] The instant disclosure provides methods for the treatment of a subject diagnosed with a disease associated with or caused by a point mutation that may be corrected by a 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 point mutation as described above, an effective amount of an adenosine deaminase fusion protein that corrects the point mutation or introduces a deactivating mutation into a 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 may 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.

[0335] 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 may be corrected by deaminase-mediated gene editing. Some such diseases are described herein, and additional suitable diseases that may 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:

2-methyl-3-hydroxybutyric aciduria; 3 beta-Hydroxysteroid dehydrogenase deficiency; 3-

Methylglutaconic aciduria; 3-Oxo-5 alpha-steroid delta 4-dehydrogenase deficiency; 46, XY sex reversal, type 1, 3, and 5; 5-Oxoprolinase deficiency; 6-pymvoyl-tetrahydropterin synthase deficiency; Aarskog syndrome; Aase syndrome; Achondrogenesis type 2;

Achromatopsia 2 and 7; Acquired long QT syndrome; Acrocallosal syndrome, Schinzel type;

Acrocapitofemoral dysplasia; Acrodysostosis 2, with or without hormone resistance;

Acroerythrokeratoderma; Acromicric dysplasia; Acth-independent macronodular adrenal hyperplasia 2; Activated PI3K-delta syndrome; Acute intermittent porphyria; deficiency of

Acyl-CoA dehydrogenase family, member 9; Adams-Oliver syndrome 5 and 6; Adenine phosphoribosyltransferase deficiency; Adenylate kinase deficiency; hemolytic anemia due to

Adenylosuccinate lyase deficiency; Adolescent nephronophthisis; Renal-hepatic-pancreatic dysplasia; Meckel syndrome type 7; Adrenoleukodystrophy; Adult junctional epidermolysis bullosa; Epidermolysis bullosa, junctional, localisata variant; Adult neuronal ceroid lipofuscinosis; Adult neuronal ceroid lipofuscinosis; Adult onset ataxia with oculomotor apraxia; ADULT syndrome; Afibrinogenemia and congenital Afibrinogenemia; autosomal recessive Agammaglobulinemia 2; Age-related macular degeneration 3, 6, 11, and 12;

Aicardi Goutieres syndromes 1, 4, and 5; Chilbain lupus 1; Alagille syndromes 1 and 2;

Alexander disease; Alkaptonuria; Allan-Hemdon-Dudley syndrome; Alopecia universalis congenital; Alpers encephalopathy; Alpha- 1- antitrypsin deficiency; autosomal dominant, autosomal recessive, and X-linked recessive Alport syndromes; Alzheimer disease, familial,

3, with spastic paraparesis and apraxia; Alzheimer disease, types, 1, 3, and 4;

hypocalcification type and hypomaturation type, IIA1 Amelogenesis imperfecta;

Aminoacylase 1 deficiency; Amish infantile epilepsy syndrome; Amyloidogenic transthyretin amyloidosis; Amyloid Cardiomyopathy, Transthyretin-related; Cardiomyopathy;

Amyotrophic lateral sclerosis types 1, 6, 15 (with or without frontotemporal dementia), 22

(with or without frontotemporal dementia), and 10; Frontotemporal dementia with TDP43 inclusions, TARDBP-related; Andermann syndrome; Andersen Tawil syndrome; Congenital long QT syndrome; Anemia, nonspherocytic hemolytic, due to G6PD deficiency; Angelman syndrome; Severe neonatal-onset encephalopathy with microcephaly; susceptibility to Autism, X-linked 3; Angiopathy, hereditary, with nephropathy, aneurysms, and muscle cramps; Angiotensin i-converting enzyme, benign serum increase; Aniridia, cerebellar ataxia, and mental retardation; Anonychia; Antithrombin III deficiency; Antley-Bixler syndrome with genital anomalies and disordered steroidogenesis; Aortic aneurysm, familial thoracic 4,

6, and 9; Thoracic aortic aneurysms and aortic dissections; Multisystemic smooth muscle dysfunction syndrome; Moyamoya disease 5; Aplastic anemia; Apparent mineralocorticoid excess; Arginase deficiency; Arginino succinate lyase deficiency; Aromatase deficiency;

Arrhythmogenic right ventricular cardiomyopathy types 5, 8, and 10; Primary familial hypertrophic cardiomyopathy; Arthrogryposis multiplex congenita, distal, X-linked;

Arthrogryposis renal dysfunction cholestasis syndrome; Arthrogryposis, renal dysfunction, and cholestasis 2; Asparagine synthetase deficiency; Abnormality of neuronal migration;

Ataxia with vitamin E deficiency; Ataxia, sensory, autosomal dominant; Ataxia- telangiectasia syndrome; Hereditary cancer-predisposing syndrome; Atransferrinemia; Atrial fibrillation, familial, 11, 12, 13, and 16; Atrial septal defects 2, 4, and 7 (with or without atrioventricular conduction defects); Atrial standstill 2; Atrioventricular septal defect 4;

Atrophia bulborum hereditaria; ATR-X syndrome; Auriculocondylar syndrome 2;

Autoimmune disease, multisystem, infantile-onset; Autoimmune lymphoproliferative syndrome, type la; Autosomal dominant hypohidrotic ectodermal dysplasia; Autosomal dominant progressive external ophthalmoplegia with mitochondrial DNA deletions 1 and 3;

Autosomal dominant torsion dystonia 4; Autosomal recessive centronuclear myopathy;

Autosomal recessive congenital ichthyosis 1, 2, 3, 4A, and 4B; Autosomal recessive cutis laxa type IA and IB; Autosomal recessive hypohidrotic ectodermal dysplasia syndrome;

Ectodermal dysplasia l ib; hypohidrotic/hair/tooth type, autosomal recessive; Autosomal recessive hypophosphatemic bone disease; Axenfeld-Rieger syndrome type 3; Bainbridge-

Ropers syndrome; Bannayan-Riley-Ruvalcaba syndrome; PTEN hamartoma tumor syndrome; Baraitser-Winter syndromes 1 and 2; Barakat syndrome; Bardet-Biedl syndromes

1, 11, 16, and 19; Bare lymphocyte syndrome type 2, complementation group E; Bartter syndrome antenatal type 2; Bartter syndrome types 3, 3 with hypocalciuria , and 4; Basal ganglia calcification, idiopathic, 4; Beaded hair; Benign familial hematuria; Benign familial neonatal seizures 1 and 2; Seizures, benign familial neonatal, 1, and/or myokymia; Seizures,

Early infantile epileptic encephalopathy 7; Benign familial neonatal-infantile seizures;

Benign hereditary chorea; Benign scapuloperoneal muscular dystrophy with cardiomyopathy;

Bemard-Soulier syndrome, types A1 and A2 (autosomal dominant); Bestrophinopathy, autosomal recessive; beta Thalassemia; Bethlem myopathy and Bethlem myopathy 2; Bietti crystalline corneoretinal dystrophy; Bile acid synthesis defect, congenital, 2; Biotinidase deficiency; Birk Barel mental retardation dysmorphism syndrome; Blepharophimosis, ptosis, and epicanthus inversus; Bloom syndrome; Borjeson-Forssman-Lehmann syndrome; Boucher

Neuhauser syndrome; Brachydactyly types A1 and A2; Brachydactyly with hypertension;

Brain small vessel disease with hemorrhage; Branched-chain ketoacid dehydrogenase kinase deficiency; Branchiootic syndromes 2 and 3; Breast cancer, early-onset; Breast-ovarian cancer, familial 1, 2, and 4; Brittle cornea syndrome 2; Brody myopathy; Bronchiectasis with or without elevated sweat chloride 3; Brown- Vialetto- Van laere syndrome and Brown-

Vialetto-Van Laere syndrome 2; Brugada syndrome; Brugada syndrome 1; Ventricular fibrillation; Paroxysmal familial ventricular fibrillation; Brugada syndrome and Brugada syndrome 4; Long QT syndrome; Sudden cardiac death; Bull eye macular dystrophy;

Stargardt disease 4; Cone-rod dystrophy 12; Bullous ichthyosiform erythroderma; Burn-

Mckeown syndrome; Candidiasis, familial, 2, 5, 6, and 8; Carbohydrate-deficient

glycoprotein syndrome type I and II; Carbonic anhydrase VA deficiency, hyperammonemia due to; Carcinoma of colon; Cardiac arrhythmia; Long QT syndrome, LQT1 subtype;

Cardioencephalomyopathy, fatal infantile, due to cytochrome c oxidase deficiency;

Cardiofaciocutaneous syndrome; Cardiomyopathy; Danon disease; Hypertrophic

cardiomyopathy; Left ventricular noncompaction cardiomyopathy; Carnevale syndrome;

Carney complex, type 1; Carnitine acylcamitine translocase deficiency; Carnitine

palmitoyltransferase I , II, II (late onset), and II (infantile) deficiency; Cataract 1, 4, autosomal dominant, autosomal dominant, multiple types, with microcornea, coppock-like, juvenile, with microcomea and glucosuria, and nuclear diffuse nonprogressive;

Catecholaminergic polymorphic ventricular tachycardia; Caudal regression syndrome; Cd8 deficiency, familial; Central core disease; Centromeric instability of chromosomes 1,9 and 16 and immunodeficiency; Cerebellar ataxia infantile with progressive external ophthalmoplegi and Cerebellar ataxia, mental retardation, and dysequilibrium syndrome 2; Cerebral amyloid angiopathy, APP-related; Cerebral autosomal dominant and recessive arteriopathy with subcortical infarcts and leukoencephalopathy; Cerebral cavernous malformations 2;

Cerebrooculofacioskeletal syndrome 2; Cerebro-oculo-facio- skeletal syndrome;

Cerebroretinal microangiopathy with calcifications and cysts; Ceroid lipofuscinosis neuronal

2, 6, 7, and 10; Ch\xc3\xa9diak-Higashi syndrome , Chediak-Higashi syndrome, adult type;

Charcot-Marie-Tooth disease types IB, 2B2, 2C, 2F, 21, 2U (axonal), 1C (demyelinating), dominant intermediate C, recessive intermediate A, 2A2, 4C, 4D, 4H, IF, IVF, and X;

Scapuloperoneal spinal muscular atrophy; Distal spinal muscular atrophy, congenital nonprogressive; Spinal muscular atrophy, distal, autosomal recessive, 5; CHARGE association; Childhood hypophosphatasia; Adult hypophosphatasia; Cholecystitis; Progressive familial intrahepatic cholestasis 3; Cholestasis, intrahepatic, of pregnancy 3;

Cholestanol storage disease; Cholesterol monooxygenase (side-chain cleaving) deficiency;

Chondrodysplasia Blomstrand type; Chondrodysplasia punctata 1, X-linked recessive and 2

X-linked dominant; CHOPS syndrome; Chronic granulomatous disease, autosomal recessive cytochrome b-positive, types 1 and 2; Chudley-McCullough syndrome; Ciliary dyskinesia, primary, 7, 11, 15, 20 and 22; Citrullinemia type I; Citrullinemia type I and II; Cleidocranial dysostosis; C-like syndrome; Cockayne syndrome type A, ; Coenzyme Q10 deficiency, primary 1, 4, and 7; Coffin Siris/Intellectual Disability; Coffin-Lowry syndrome; Cohen syndrome, ; Cold-induced sweating syndrome 1; COLE-CARPENTER SYNDROME 2;

Combined cellular and humoral immune defects with granulomas; Combined d-2- and 1-2- hydroxyglutaric aciduria; Combined malonic and methylmalonic aciduria; Combined oxidative phosphorylation deficiencies 1, 3, 4, 12, 15, and 25; Combined partial and complete

17-alpha-hydroxylase/ 17, 20-lyase deficiency; Common variable immunodeficiency 9;

Complement component 4, partial deficiency of, due to dysfunctional cl inhibitor;

Complement factor B deficiency; Cone monochromatism; Cone-rod dystrophy 2 and 6;

Cone-rod dystrophy amelogenesis imperfecta; Congenital adrenal hyperplasia and Congenital adrenal hypoplasia, X-linked; Congenital amegakaryocytic thrombocytopenia; Congenital aniridia; Congenital central hypoventilation; Hirschsprung disease 3; Congenital contractural arachnodactyly; Congenital contractures of the limbs and face, hypotonia, and developmental delay; Congenital disorder of glycosylation types IB, ID, 1G, 1H, 1 J, IK, IN, IP, 2C, 2J,

2K, Urn; Congenital dyserythropoietic anemia, type I and II; Congenital ectodermal dysplasia of face; Congenital erythropoietic porphyria; Congenital generalized lipodystrophy type 2;

Congenital heart disease, multiple types, 2; Congenital heart disease; Interrupted aortic arch;

Congenital lipomatous overgrowth, vascular malformations, and epidermal nevi; Non-small cell lung cancer; Neoplasm of ovary; Cardiac conduction defect, nonspecific; Congenital microvillous atrophy; Congenital muscular dystrophy; Congenital muscular dystrophy due to partial LAMA2 deficiency; Congenital muscular dystrophy-dystroglycanopathy with brain and eye anomalies, types A2, A7, A8, Al l, and A14; Congenital muscular dystrophy- dystroglycanopathy with mental retardation, types B2, B3, B5, and B15; Congenital muscular dystrophy-dystroglycanopathy without mental retardation, type B5; Congenital muscular hypertrophy-cerebral syndrome; Congenital myasthenic syndrome, acetazolamide- responsive; Congenital myopathy with fiber type disproportion; Congenital ocular coloboma;

Congenital stationary night blindness, type 1A, IB, 1C, IE, IF, and 2A; Coproporphyria;

Cornea plana 2; Comeal dystrophy, Fuchs endothelial, 4; Comeal endothelial dystrophy type

2; Comeal fragility keratoglobus, blue sclerae and joint hypermobility; Cornelia de Lange syndromes 1 and 5; Coronary artery disease, autosomal dominant 2; Coronary heart disease;

Hyperalphalipoproteinemia 2; Cortical dysplasia, complex, with other brain malformations 5 and 6; Cortical malformations, occipital; Corticosteroid-binding globulin deficiency;

Corticosterone methyloxidase type 2 deficiency; Costello syndrome; Cowden syndrome 1;

Coxa plana; Craniodiaphyseal dysplasia, autosomal dominant; Craniosynostosis 1 and 4;

Craniosynostosis and dental anomalies; Creatine deficiency, X-linked; Crouzon syndrome;

Cryptophthalmos syndrome; Cryptorchidism, unilateral or bilateral; Cushing symphalangism;

Cutaneous malignant melanoma 1; Cutis laxa with osteodystrophy and with severe pulmonary, gastrointestinal, and urinary abnormalities; Cyanosis, transient neonatal and atypical nephropathic; Cystic fibrosis; Cystinuria; Cytochrome c oxidase i deficiency;

Cytochrome-c oxidase deficiency ; D-2-hydroxyglutaric aciduria 2; Darier disease, segmental; Deafness with labyrinthine aplasia microtia and microdontia (LAMM); Deafness, autosomal dominant 3a, 4, 12, 13, 15, autosomal dominant nonsyndromic sensorineural 17,

20, and 65; Deafness, autosomal recessive 1A, 2, 3, 6, 8, 9, 12, 15, 16, 18b, 22, 28, 31, 44,

49, 63, 77, 86, and 89; Deafness, cochlear, with myopia and intellectual impairment, without vestibular involvement, autosomal dominant, X-linked 2; Deficiency of 2-methylbutyryl-CoA dehydrogenase; Deficiency of 3-hydroxyacyl-CoA dehydrogenase; Deficiency of alpha- mannosidase; Deficiency of aromatic-L-amino-acid decarboxylase; Deficiency of

bisphosphoglycerate mutase; Deficiency of butyryl-CoA dehydrogenase; Deficiency of ferroxidase; Deficiency of galactokinase; Deficiency of guanidinoacetate methyltransferase;

Deficiency of hyaluronoglucosaminidase; Deficiency of ribose-5-phosphate isomerase;

Deficiency of steroid 11 -beta-monooxygenase; Deficiency of UDPglucose-hexose-1- phosphate uridylyltransferase; Deficiency of xanthine oxidase; Dejerine-Sottas disease;

Charcot-Marie-Tooth disease, types ID and IVF; Dejerine-Sottas syndrome, autosomal dominant; Dendritic cell, monocyte, B lymphocyte, and natural killer lymphocyte deficiency;

Desbuquois dysplasia 2; Desbuquois syndrome; DFNA 2 Nonsyndromic Hearing Loss;

Diabetes mellitus and insipidus with optic atrophy and deafness; Diabetes mellitus, type 2, and insulin-dependent, 20; Diamond-Blackfan anemia 1, 5, 8, and 10; Diarrhea 3 (secretory sodium, congenital, syndromic) and 5 (with tufting enteropathy, congenital); Dicarboxylic aminoaciduria; Diffuse palmoplantar keratoderma, Bothnian type; Digitorenocerebral syndrome; Dihydropteridine reductase deficiency; Dilated cardiomyopathy 1A, 1AA, 1C, 1G,

IBB, 1DD, IFF, 1HH, II, IKK, IN, IS, 1Y, and 3B; Left ventricular noncompaction 3;

Disordered steroidogenesis due to cytochrome p450 oxidoreductase deficiency; Distal arthrogryposis type 2B; Distal hereditary motor neuronopathy type 2B; Distal myopathy

Markesbery-Griggs type; Distal spinal muscular atrophy, X-linked 3; Distichiasis- lymphedema syndrome; Dominant dystrophic epidermolysis bullosa with absence of skin;

Dominant hereditary optic atrophy; Donnai Barrow syndrome; Dopamine beta hydroxylase deficiency; Dopamine receptor d2, reduced brain density of; Dowling-degos disease 4; Doyne honeycomb retinal dystrophy; Malattia leventinese; Duane syndrome type 2; Dubin-Johnson syndrome; Duchenne muscular dystrophy; Becker muscular dystrophy; Dysfibrinogenemia;

Dyskeratosis congenita autosomal dominant and autosomal dominant, 3; Dyskeratosis congenita, autosomal recessive, 1, 3, 4, and 5; Dyskeratosis congenita X-linked; Dyskinesia, familial, with facial myokymia; Dysplasminogenemia; Dystonia 2 (torsion, autosomal recessive), 3 (torsion, X-linked), 5 (Dopa-responsive type ), 10, 12, 16, 25, 26 (Myoclonic);

Seizures, benign familial infantile, 2; Early infantile epileptic encephalopathy 2, 4, 7, 9, 10,

11, 13, and 14; Atypical Rett syndrome; Early T cell progenitor acute lymphoblastic leukemia; Ectodermal dysplasia skin fragility syndrome; Ectodermal dysplasia-syndactyly syndrome 1; Ectopia lentis, isolated autosomal recessive and dominant; Ectrodactyly, ectodermal dysplasia, and cleft lip/palate syndrome 3; Ehlers-Danlos syndrome type 7

(autosomal recessive), classic type, type 2 (progeroid ), hydroxylysine-deficient, type 4, type

4 variant, and due to tenascin-X deficiency; Eichsfeld type congenital muscular dystrophy;

Endocrine-cerebroosteodysplasia; Enhanced s-cone syndrome; Enlarged vestibular aqueduct syndrome; Enterokinase deficiency; Epidermodysplasia verruciformis; Epidermolysa bullosa simplex and limb girdle muscular dystrophy, simplex with mottled pigmentation, simplex with pyloric atresia, simplex, autosomal recessive, and with pyloric atresia; Epidermolytic palmoplantar keratoderma; Familial febrile seizures 8; Epilepsy, childhood absence 2, 12

(idiopathic generalized, susceptibility to) 5 (nocturnal frontal lobe), nocturnal frontal lobe type 1, partial, with variable foci, progressive myoclonic 3, and X-linked, with variable learning disabilities and behavior disorders; Epileptic encephalopathy, childhood-onset, early infantile, 1, 19, 23, 25, 30, and 32; Epiphyseal dysplasia, multiple, with myopia and conductive deafness; Episodic ataxia type 2; Episodic pain syndrome, familial, 3; Epstein syndrome; Fechtner syndrome; Erythropoietic protoporphyria; Estrogen resistance; Exudative vitreoretinopathy 6; Fabry disease and Fabry disease, cardiac variant; Factor H, VII, X, v and factor viii, combined deficiency of 2, xiii, a subunit, deficiency; Familial adenomatous polyposis 1 and 3; Familial amyloid nephropathy with urticaria and deafness; Familial cold urticarial; Familial aplasia of the vermis; Familial benign pemphigus; Familial cancer of breast; Breast cancer, susceptibility to; Osteosarcoma; Pancreatic cancer 3; Familial cardiomyopathy; Familial cold autoinflammatory syndrome 2; Familial colorectal cancer;

Familial exudative vitreoretinopathy, X-linked; Familial hemiplegic migraine types 1 and 2;

Familial hypercholesterolemia; Familial hypertrophic cardiomyopathy 1, 2, 3, 4, 7, 10, 23 and 24; Familial hypokalemia-hypomagnesemia; Familial hypoplastic, glomerulocystic kidney;

Familial infantile myasthenia; Familial juvenile gout; Familial Mediterranean fever and

Familial mediterranean fever, autosomal dominant; Familial porencephaly; Familial porphyria cutanea tarda; Familial pulmonary capillary hemangiomatosis; Familial renal glucosuria; Familial renal hypouricemia; Familial restrictive cardiomyopathy 1; Familial type

1 and 3 hyperlipoproteinemia; Fanconi anemia, complementation group E, I, N, and O;

Fanconi-Bickel syndrome; Favism, susceptibility to; Febrile seizures, familial, 11; Feingold syndrome 1; Fetal hemoglobin quantitative trait locus 1; FG syndrome and FG syndrome 4;

Fibrosis of extraocular muscles, congenital, 1, 2, 3a (with or without extraocular

involvement), 3b; Fish-eye disease; Fleck comeal dystrophy; Floating-Harbor syndrome;

Focal epilepsy with speech disorder with or without mental retardation; Focal segmental glomerulosclerosis 5; Forebrain defects; Frank Ter Haar syndrome; Borrone Di Rocco

Crovato syndrome; Frasier syndrome; Wilms tumor 1; Freeman-Sheldon syndrome;

Frontometaphyseal dysplasia land 3; Frontotemporal dementia; Frontotemporal dementia and/or amyotrophic lateral sclerosis 3 and 4; Frontotemporal Dementia Chromosome 3-

Linked and Frontotemporal dementia ubiquitin-positive; Fructose-biphosphatase deficiency;

Fuhrmann syndrome; Gamma-aminobutyric acid transaminase deficiency; Gamstorp-

Wohlfart syndrome; Gaucher disease type 1 and Subacute neuronopathic; Gaze palsy, familial horizontal, with progressive scoliosis; Generalized dominant dystrophic

epidermolysis bullosa; Generalized epilepsy with febrile seizures plus 3, type 1, type 2;

Epileptic encephalopathy Lennox-Gastaut type; Giant axonal neuropathy; Glanzmann thrombasthenia; Glaucoma 1, open angle, e, F, and G; Glaucoma 3, primary congenital, d;

Glaucoma, congenital and Glaucoma, congenital, Coloboma; Glaucoma, primary open angle, juvenile-onset; Glioma susceptibility 1; Glucose transporter type 1 deficiency syndrome;

Glucose-6-phosphate transport defect; GLUT1 deficiency syndrome 2; Epilepsy, idiopathic generalized, susceptibility to, 12; Glutamate formiminotransferase deficiency; Glutaric acidemia IIA and IIB; Glutaric aciduria, type 1; Gluthathione synthetase deficiency;

Glycogen storage disease 0 ( muscle), II (adult form), IXa2, IXc, type 1A; type II, type IV,

IV (combined hepatic and myopathic), type V, and type VI; Goldmann-Favre syndrome;

Gordon syndrome; Gorlin syndrome; Holoprosencephaly sequence; Holoprosencephaly 7;

Granulomatous disease, chronic, X-linked, variant; Granulosa cell tumor of the ovary; Gray platelet syndrome; Griscelli syndrome type 3; Groenouw comeal dystrophy type I; Growth and mental retardation, mandibulofacial dysostosis, microcephaly, and cleft palate; Growth hormone deficiency with pituitary anomalies; Growth hormone insensitivity with

immunodeficiency; GTP cyclohydrolase I deficiency; Hajdu-Cheney syndrome; Hand foot uterus syndrome; Hearing impairment; Hemangioma, capillary infantile; Hematologic neoplasm; Hemochromatosis type 1, 2B, and 3; Microvascular complications of diabetes 7;

Transferrin serum level quantitative trait locus 2; Hemoglobin H disease, nondeletional;

Hemolytic anemia, nonspherocytic, due to glucose phosphate isomerase deficiency;

Hemophagocytic lymphohistiocytosis, familial, 2; Hemophagocytic lymphohistiocytosis, familial, 3; Heparin cofactor II deficiency; Hereditary acrodermatitis enteropathica;

Hereditary breast and ovarian cancer syndrome; Ataxia-telangiectasia-like disorder;

Hereditary diffuse gastric cancer; Hereditary diffuse leukoencephalopathy with spheroids;

Hereditary factors II, IX, VIII deficiency disease; Hereditary hemorrhagic telangiectasia type

2; Hereditary insensitivity to pain with anhidrosis; Hereditary lymphedema type I; Hereditary motor and sensory neuropathy with optic atrophy; Hereditary myopathy with early respiratory failure; Hereditary neuralgic amyotrophy; Hereditary Nonpolyposis Colorectal

Neoplasms; Lynch syndrome I and II; Hereditary pancreatitis; Pancreatitis, chronic, susceptibility to; Hereditary sensory and autonomic neuropathy type IIB amd IIA; Hereditary sideroblastic anemia; Hermansky-Pudlak syndrome 1, 3, 4, and 6; Heterotaxy, visceral, 2, 4, and 6, autosomal; Heterotaxy, visceral, X-linked; Heterotopia; Histiocytic medullary reticulosis; Histiocytosis-lymphadenopathy plus syndrome; Holocarboxylase synthetase deficiency; Holoprosencephaly 2, 3,7, and 9; Holt-Oram syndrome; Homocysteinemia due to

MTHFR deficiency, CBS deficiency, and Homocystinuria, pyridoxine-responsive;

Homocystinuria-Megaloblastic anemia due to defect in cobalamin metabolism, cblE complementation type; Howel-Evans syndrome; Hurler syndrome; Hutchinson-Gilford syndrome; Hydrocephalus; Hyperammonemia, type III; Hypercholesterolaemia and

Hypercholesterolemia, autosomal recessive; Hyperekplexia 2 and Hyperekplexia hereditary;

Hyperferritinemia cataract syndrome; Hyperglycinuria; Hyperimmunoglobulin D with periodic fever; Mevalonic aciduria; Hyperimmunoglobulin E syndrome; Hyperinsulinemic hypoglycemia familial 3, 4, and 5; Hyperinsulinism-hyperammonemia syndrome;

Hyperlysinemia; Hypermanganesemia with dystonia, polycythemia and cirrhosis;

Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome; Hyperparathyroidism 1 and 2; Hyperparathyroidism, neonatal severe; Hyperphenylalaninemia, bh4-deficient, a, due to partial pts deficiency, BH4-deficient, D, and non-pku; Hyperphosphatasia with mental retardation syndrome 2, 3, and 4; Hypertrichotic osteochondrodysplasia;

Hypobetalipoproteinemia, familial, associated with apob32; Hypocalcemia, autosomal dominant 1; Hypocalciuric hypercalcemia, familial, types 1 and 3; Hypochondrogenesis;

Hypochromic microcytic anemia with iron overload; Hypoglycemia with deficiency of glycogen synthetase in the liver; Hypogonadotropic hypogonadism 11 with or without anosmia; Hypohidrotic ectodermal dysplasia with immune deficiency; Hypohidrotic X-linked ectodermal dysplasia; Hypokalemic periodic paralysis 1 and 2; Hypomagnesemia 1, intestinal; Hypomagnesemia, seizures, and mental retardation; Hypomyelinating

leukodystrophy 7; Hypoplastic left heart syndrome; Atrioventricular septal defect and common atrioventricular junction; Hypospadias 1 and 2, X-linked; Hypothyroidism, congenital, nongoitrous, 1; Hypotrichosis 8 and 12; Hypotrichosis-lymphedema- telangiectasia syndrome; I blood group system; Ichthyosis bullosa of Siemens; Ichthyosis exfoliativa; Ichthyosis prematurity syndrome; Idiopathic basal ganglia calcification 5;

Idiopathic fibrosing alveolitis, chronic form; Dyskeratosis congenita, autosomal dominant, 2 and 5; Idiopathic hypercalcemia of infancy; Immune dysfunction with T-cell inactivation due to calcium entry defect 2; Immunodeficiency 15, 16, 19, 30, 31C, 38, 40, 8, due to defect in cd3-zeta, with hyper IgM type 1 and 2, and X-Linked, with magnesium defect, Epstein-Barr vims infection, and neoplasia; Immunodeficiency-centromeric instability-facial anomalies syndrome 2; Inclusion body myopathy 2 and 3; Nonaka myopathy; Infantile convulsions and paroxysmal choreoathetosis, familial; Infantile cortical hyperostosis; Infantile GM1 gangliosidosis; Infantile hypophosphatasia; Infantile nephronophthisis; Infantile nystagmus,

X-linked; Infantile Parkinsonism-dystonia; Infertility associated with multi-tailed

spermatozoa and excessive DNA; Insulin resistance; Insulin-resistant diabetes mellitus and acanthosis nigricans; Insulin-dependent diabetes mellitus secretory diarrhea syndrome;

Interstitial nephritis, karyomegalic; Intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia congenita, and genital anomalies; Iodotyrosyl coupling defect; IRAK4 deficiency; Iridogoniodysgenesis dominant type and type 1; Iron accumulation in brain;

Ischiopatellar dysplasia; Islet cell hyperplasia; Isolated 17,20-lyase deficiency; Isolated lutropin deficiency; Isovaleryl-CoA dehydrogenase deficiency; Jankovic Rivera syndrome;

Jervell and Lange-Nielsen syndrome 2; Joubert syndrome 1, 6, 7, 9/15 (digenic), 14, 16, and

17, and Orofaciodigital syndrome xiv; Junctional epidermolysis bullosa gravis of Herlitz;

Juvenile GM>1< gangliosidosis; Juvenile polyposis syndrome; Juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome; Juvenile retinoschisis; Kabuki make-up syndrome;

Kallmann syndrome 1, 2, and 6; Delayed puberty; Kanzaki disease; Karak syndrome;

Kartagener syndrome; Kenny-Caffey syndrome type 2; Keppen-Lubinsky syndrome;

Keratoconus 1; Keratosis follicularis; Keratosis palmoplantaris striata 1; Kindler syndrome;

L-2-hydroxyglutaric aciduria; Larsen syndrome, dominant type; Lattice corneal dystrophy

Type III; Leber amaurosis; Zellweger syndrome; Peroxisome biogenesis disorders; Zellweger syndrome spectrum; Leber congenital amaurosis 11, 12, 13, 16, 4, 7, and 9; Leber optic atrophy; Aminoglycoside-induced deafness; Deafness, nonsyndromic sensorineural, mitochondrial; Left ventricular noncompaction 5; Left-right axis malformations; Leigh disease; Mitochondrial short-chain Enoyl-CoA Hydratase 1 deficiency; Leigh syndrome due to mitochondrial complex I deficiency; Leiner disease; Leri Weill dyschondrosteosis; Lethal congenital contracture syndrome 6; Leukocyte adhesion deficiency type I and III;

Leukodystrophy, Hypomyelinating, 11 and 6; Leukoencephalopathy with ataxia, with

Brainstem and Spinal Cord Involvement and Lactate Elevation, with vanishing white matter, and progressive, with ovarian failure; Leukonychia totalis; Lewy body dementia;

Lichtenstein-Knorr Syndrome; Li-Fraumeni syndrome 1; Lig4 syndrome; Limb-girdle muscular dystrophy, type IB, 2A, 2B, 2D, Cl, C5, C9, C14; Congenital muscular dystrophy- dystroglycanopathy with brain and eye anomalies, type A14 and B14; Lipase deficiency combined; Lipid proteinosis; Lipodystrophy, familial partial, type 2 and 3; Lissencephaly 1, 2

(X-linked), 3, 6 (with microcephaly), X-linked; Subcortical laminar heterotopia, X-linked;

Liver failure acute infantile; Loeys-Dietz syndrome 1, 2, 3; Long QT syndrome 1, 2, 2/9, 2/5,

(digenic), 3, 5 and 5, acquired, susceptibility to; Lung cancer; Lymphedema, hereditary, id;

Lymphedema, primary, with myelodysplasia; Lymphoproliferative syndrome 1, 1 (X-linked), and 2; Lysosomal acid lipase deficiency; Macrocephaly, macrosomia, facial dysmorphism syndrome; Macular dystrophy, vitelliform, adult-onset; Malignant hyperthermia susceptibility type 1; Malignant lymphoma, non-Hodgkin; Malignant melanoma; Malignant tumor of prostate; Mandibuloacral dysostosis; Mandibuloacral dysplasia with type A or B

lipodystrophy, atypical; Mandibulofacial dysostosis, Treacher Collins type, autosomal recessive; Mannose-binding protein deficiency; Maple syrup urine disease type 1A and type

3; Marden Walker like syndrome; Marfan syndrome; Marinesco-Sj\xc3\xb6gren syndrome;

Martsolf syndrome; Maturity-onset diabetes of the young, type 1, type 2, type 11, type 3, and type 9; May-Hegglin anomaly; MYH9 related disorders; Sebastian syndrome; McCune-

Albright syndrome; Somatotroph adenoma; Sex cord-stromal tumor; Cushing syndrome;

McKusick Kaufman syndrome; McLeod neuroacanthocytosis syndrome; Meckel-Gruber syndrome; Medium-chain acyl-coenzyme A dehydrogenase deficiency; Medulloblastoma;

Megalencephalic leukoencephalopathy with subcortical cysts land 2a; Megalencephaly cutis marmorata telangiectatica congenital; PIK3CA Related Overgrowth Spectrum;

Megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome 2; Megaloblastic anemia, thiamine-responsive, with diabetes mellitus and sensorineural deafness; Meier-Gorlin syndromes land 4; Melnick-Needles syndrome; Meningioma; Mental retardation, X-linked,

3, 21, 30, and 72; Mental retardation and microcephaly with pontine and cerebellar hypoplasia; Mental retardation X-linked syndromic 5; Mental retardation, anterior maxillary protrusion, and strabismus; Mental retardation, autosomal dominant 12, 13, 15, 24, 3, 30, 4, 5, 6, and 9; Mental retardation, autosomal recessive 15, 44, 46, and 5; Mental retardation, stereotypic movements, epilepsy, and/or cerebral malformations; Mental retardation, syndromic, Claes-Jensen type, X-linked; Mental retardation, X-linked, nonspecific, syndromic, Hedera type, and syndromic, wu type; Merosin deficient congenital muscular dystrophy; Metachromatic leukodystrophy juvenile, late infantile, and adult types;

Metachromatic leukodystrophy; Metatrophic dysplasia; Methemoglobinemia types I and 2;

Methionine adenosyltransferase deficiency, autosomal dominant; Methylmalonic acidemia with homocystinuria, ; Methylmalonic aciduria cblB type, ; Methylmalonic aciduria due to methylmalonyl-CoA mutase deficiency; METHYLMALONIC ACIDURIA, mut(0) TYPE;

Microcephalic osteodysplastic primordial dwarfism type 2; Microcephaly with or without chorioretinopathy, lymphedema, or mental retardation; Microcephaly, hiatal hernia and nephrotic syndrome; Microcephaly; Hypoplasia of the corpus callosum; Spastic paraplegia

50, autosomal recessive; Global developmental delay; CNS hypomyelination; Brain atrophy;

Microcephaly, normal intelligence and immunodeficiency; Microcephaly-capillary malformation syndrome; Microcytic anemia; Microphthalmia syndromic 5, 7, and 9;

Microphthalmia, isolated 3, 5, 6, 8, and with coloboma 6; Microspherophakia; Migraine, familial basilar; Miller syndrome; Minicore myopathy with external ophthalmoplegia;

Myopathy, congenital with cores; Mitchell-Riley syndrome; mitochondrial 3-hydroxy-3- methylglutaryl-CoA synthase deficiency; Mitochondrial complex I, II, III, III (nuclear type 2,

4, or 8) deficiency; Mitochondrial DNA depletion syndrome 11, 12 (cardiomyopathic type),

2, 4B (MNGIE type), 8B (MNGIE type); Mitochondrial DNA-depletion syndrome 3 and 7, hepatocerebral types, and 13 (encephalomyopathic type); Mitochondrial phosphate carrier and pyruvate carrier deficiency; Mitochondrial trifunctional protein deficiency; Long-chain

3-hydroxyacyl-CoA dehydrogenase deficiency; Miyoshi muscular dystrophy 1; Myopathy, distal, with anterior tibial onset; Mohr-Tranebjaerg syndrome; Molybdenum cofactor deficiency, complementation group A; Mowat-Wilson syndrome; Mucolipidosis III Gamma;

Mucopolysaccharidosis type VI, type VI (severe), and type VII; Mucopolysaccharidosis,

MPS-I-H/S, MPS-II, MPS-III-A, MPS-III-B, MPS-III-C, MPS-IV-A, MPS-IV-B; Retinitis

Pigmentosa 73; Gangliosidosis GM1 typel (with cardiac involvenment) 3; Multicentric osteolysis nephropathy; Multicentric osteolysis, nodulosis and arthropathy; Multiple congenital anomalies; Atrial septal defect 2; Multiple congenital anomalies -hypotonia- seizures syndrome 3; Multiple Cutaneous and Mucosal Venous Malformations; Multiple endocrine neoplasia, types land 4; Multiple epiphyseal dysplasia 5 or Dominant; Multiple gastrointestinal atresias; Multiple pterygium syndrome Escobar type; Multiple sulfatase deficiency; Multiple synostoses syndrome 3; Muscle AMP deaminase deficiency; Muscle eye brain disease; Muscular dystrophy, congenital, megaconial type; Myasthenia, familial infantile, 1; Myasthenic Syndrome, Congenital, 11, associated with acetylcholine receptor deficiency; Myasthenic Syndrome, Congenital, 17, 2A (slow-channel), 4B (fast-channel), and without tubular aggregates; Myeloperoxidase deficiency; MYH-associated polyposis;

Endometrial carcinoma; Myocardial infarction 1; Myoclonic dystonia; Myoclonic-Atonic

Epilepsy; Myoclonus with epilepsy with ragged red fibers; Myofibrillar myopathy 1 and

ZASP-related; Myoglobinuria, acute recurrent, autosomal recessive; Myoneural

gastrointestinal encephalopathy syndrome; Cerebellar ataxia infantile with progressive external ophthalmoplegia; Mitochondrial DNA depletion syndrome 4B, MNGIE type;

Myopathy, centronuclear, 1, congenital, with excess of muscle spindles, distal, 1, lactic acidosis, and sideroblastic anemia 1, mitochondrial progressive with congenital cataract, hearing loss, and developmental delay, and tubular aggregate, 2; Myopia 6; Myosclerosis, autosomal recessive; Myotonia congenital; Congenital myotonia, autosomal dominant and recessive forms; Nail-patella syndrome; Nance-Horan syndrome; Nanophthalmos 2; Navajo neurohepatopathy; Nemaline myopathy 3 and 9; Neonatal hypotonia; Intellectual disability;

Seizures; Delayed speech and language development; Mental retardation, autosomal dominant 31; Neonatal intrahepatic cholestasis caused by citrin deficiency; Nephrogenic diabetes insipidus, Nephrogenic diabetes insipidus, X-linked; Nephrolithiasis/osteoporosis, hypophosphatemic, 2; Nephronophthisis 13, 15 and 4; Infertility; Cerebello-oculo-renal syndrome (nephronophthisis, oculomotor apraxia and cerebellar abnormalities); Nephrotic syndrome, type 3, type 5, with or without ocular abnormalities, type 7, and type 9; Nestor-

Guillermo progeria syndrome; Neu-Laxova syndrome 1; Neurodegeneration with brain iron accumulation 4 and 6; Neuroferritinopathy; Neurofibromatosis, type land type 2;

Neurofibrosarcoma; Neurohypophyseal diabetes insipidus; Neuropathy, Hereditary Sensory,

Type IC; Neutral 1 amino acid transport defect; Neutral lipid storage disease with myopathy;

Neutrophil immunodeficiency syndrome; Nicolaides-Baraitser syndrome; Niemann-Pick disease type Cl, C2, type A, and type Cl, adult form; Non-ketotic hyperglycinemia; Noonan syndrome 1 and 4, LEOPARD syndrome 1; Noonan syndrome-like disorder with or without juvenile myelomonocytic leukemia; Normokalemic periodic paralysis, potassium-sensitive;

Norum disease; Epilepsy, Hearing Loss, And Mental Retardation Syndrome; Mental

Retardation, X-Linked 102 and syndromic 13; Obesity; Ocular albinism, type I;

Oculocutaneous albinism type IB, type 3, and type 4; Oculodentodigital dysplasia;

Odontohypophosphatasia; Odontotrichomelic syndrome; Oguchi disease; Oligodontia- colorectal cancer syndrome; Opitz G/BBB syndrome; Optic atrophy 9; Oral-facial-digital syndrome; Ornithine aminotransferase deficiency; Orofacial cleft 11 and 7, Cleft lip/palate- ectodermal dysplasia syndrome; Orstavik Lindemann Solberg syndrome; Osteoarthritis with mild chondrodysplasia; Osteochondritis dissecans; Osteogenesis imperfecta type 12, type 5, type 7, type 8, type I, type III, with normal sclerae, dominant form, recessive perinatal lethal;

Osteopathia striata with cranial sclerosis; Osteopetrosis autosomal dominant type 1 and 2, recessive 4, recessive 1, recessive 6; Osteoporosis with pseudoglioma; Oto-palato-digital syndrome, types I and II; Ovarian dysgenesis 1; Ovarioleukodystrophy; Pachyonychia congenita 4 and type 2; Paget disease of bone, familial; Pallister-Hall syndrome;

Palmoplantar keratoderma, nonepidermolytic, focal or diffuse; Pancreatic agenesis and congenital heart disease; Papillon-Lef\xc3\xa8vre syndrome; Paragangliomas 3;

Paramyotonia congenita of von Eulenburg; Parathyroid carcinoma; Parkinson disease 14, 15,

19 (juvenile-onset), 2, 20 (early-onset), 6, (autosomal recessive early-onset, and 9; Partial albinism; Partial hypoxanthine-guanine phosphoribosyltransferase deficiency; Patterned dystrophy of retinal pigment epithelium; PC-K6a; Pelizaeus-Merzbacher disease; Pendred syndrome; Peripheral demyelinating neuropathy, central dysmyelination; Hirschsprung disease; Permanent neonatal diabetes mellitus; Diabetes mellitus, permanent neonatal, with neurologic features; Neonatal insulin-dependent diabetes mellitus; Maturity-onset diabetes of the young, type 2; Peroxisome biogenesis disorder 14B, 2A, 4A, 5B, 6A, 7A, and 7B;

Perrault syndrome 4; Perry syndrome; Persistent hyperinsulinemic hypoglycemia of infancy; familial hyperinsulinism; Phenotypes; Phenylketonuria; Pheochromocytoma; Hereditary

Paraganglioma- Pheochromocytoma Syndromes; Paragangliomas 1; Carcinoid tumor of intestine; Cowden syndrome 3; Phosphoglycerate dehydrogenase deficiency;

Phosphoglycerate kinase 1 deficiency; Photosensitive trichothiodystrophy; Phytanic acid storage disease; Pick disease; Pierson syndrome; Pigmentary retinal dystrophy; Pigmented nodular adrenocortical disease, primary, 1; Pilomatrixoma; Pitt- Hopkins syndrome; Pituitary dependent hypercortisolism; Pituitary hormone deficiency, combined 1, 2, 3, and 4;

Plasminogen activator inhibitor type 1 deficiency; Plasminogen deficiency, type I; Platelet- type bleeding disorder 15 and 8; Poikiloderma, hereditary fibrosing, with tendon contractures, myopathy, and pulmonary fibrosis; Polycystic kidney disease 2, adult type, and infantile type;

Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy;

Polyglucosan body myopathy 1 with or without immunodeficiency; Polymicrogyria, asymmetric, bilateral frontoparietal; Polyneuropathy, hearing loss, ataxia, retinitis

pigmentosa, and cataract; Pontocerebellar hypoplasia type 4; Popliteal pterygium syndrome;

Porencephaly 2; Porokeratosis 8, disseminated superficial actinic type; Porphobilinogen synthase deficiency; Porphyria cutanea tarda; Posterior column ataxia with retinitis pigmentosa; Posterior polar cataract type 2; Prader-Willi-like syndrome; Premature ovarian failure 4, 5, 7, and 9; Primary autosomal recessive microcephaly 10, 2, 3, and 5; Primary ciliary dyskinesia 24; Primary dilated cardiomyopathy; Left ventricular noncompaction 6; 4,

Left ventricular noncompaction 10; Paroxysmal atrial fibrillation; Primary hyperoxaluria, type I, type, and type III; Primary hypertrophic osteoarthropathy, autosomal recessive 2;

Primary hypomagnesemia; Primary open angle glaucoma juvenile onset 1; Primary pulmonary hypertension; Primrose syndrome; Progressive familial heart block type IB;

Progressive familial intrahepatic cholestasis 2 and 3; Progressive intrahepatic cholestasis;

Progressive myoclonus epilepsy with ataxia; Progressive pseudorheumatoid dysplasia;

Progressive sclerosing poliodystrophy; Prolidase deficiency; Proline dehydrogenase deficiency; Schizophrenia 4; Properdin deficiency, X-linked; Propionic academia; Proprotein convertase 1/3 deficiency; Prostate cancer, hereditary, 2; Protan defect; Proteinuria; Finnish congenital nephrotic syndrome; Proteus syndrome; Breast adenocarcinoma;

Pseudoachondroplastic spondyloepiphyseal dysplasia syndrome; Pseudohypoaldosteronism type 1 autosomal dominant and recessive and type 2; Pseudohypoparathyroidism type 1A,

Pseudopseudohypoparathyroidism; Pseudoneonatal adrenoleukodystrophy; Pseudoprimary hyperaldosteronism; Pseudoxanthoma elasticum; Generalized arterial calcification of infancy

2; Pseudoxanthoma elasticum-like disorder with multiple coagulation factor deficiency;

Psoriasis susceptibility 2; PTEN hamartoma tumor syndrome; Pulmonary arterial

hypertension related to hereditary hemorrhagic telangiectasia; Pulmonary Fibrosis And/Or

Bone Marrow Failure, Telomere-Related, 1 and 3; Pulmonary hypertension, primary, 1, with hereditary hemorrhagic telangiectasia; Purine-nucleoside phosphorylase deficiency; Pyruvate carboxylase deficiency; Pyruvate dehydrogenase El -alpha deficiency; Pyruvate kinase deficiency of red cells; Raine syndrome; Rasopathy; Recessive dystrophic epidermolysis bullosa; Nail disorder, nonsyndromic congenital, 8; Reifenstein syndrome; Renal adysplasia;

Renal carnitine transport defect; Renal coloboma syndrome; Renal dysplasia; Renal dysplasia, retinal pigmentary dystrophy, cerebellar ataxia and skeletal dysplasia; Renal tubular acidosis, distal, autosomal recessive, with late-onset sensorineural hearing loss, or with hemolytic anemia; Renal tubular acidosis, proximal, with ocular abnormalities and mental retardation; Retinal cone dystrophy 3B; Retinitis pigmentosa; Retinitis pigmentosa 10,

11, 12, 14, 15, 17, and 19; Retinitis pigmentosa 2, 20, 25, 35, 36, 38, 39, 4, 40, 43, 45, 48, 66,

7, 70, 72; Retinoblastoma; Rett disorder; Rhabdoid tumor predisposition syndrome 2;

Rhegmatogenous retinal detachment, autosomal dominant; Rhizomelic chondrodysplasia punctata type 2 and type 3; Roberts-SC phocomelia syndrome; Robinow Sorauf syndrome;

Robinow syndrome, autosomal recessive, autosomal recessive, with brachy-syn-polydactyly;

Rothmund-Thomson syndrome; Rapadilino syndrome; RRM2B-related mitochondrial disease; Rubinstein-Taybi syndrome; Salla disease; Sandhoff disease, adult and infantil types;

Sarcoidosis, early-onset; Blau syndrome; Schindler disease, type 1; Schizencephaly;

Schizophrenia 15; Schneckenbecken dysplasia; Schwannomatosis 2; Schwartz Jampel syndrome type 1; Sclerocornea, autosomal recessive; Sclerosteosis; Secondary

hypothyroidism; Segawa syndrome, autosomal recessive; Senior-Loken syndrome 4 and 5, ;

Sensory ataxic neuropathy, dysarthria, and ophthalmoparesis; Sepiapterin reductase deficiency; SeSAME syndrome; Severe combined immunodeficiency due to ADA

deficiency, with microcephaly, growth retardation, and sensitivity to ionizing radiation, atypical, autosomal recessive, T cell-negative, B cell-positive, NK cell-negative of NK- positive; Partial adenosine deaminase deficiency; Severe congenital neutropenia; Severe congenital neutropenia 3, autosomal recessive or dominant; Severe congenital neutropenia and 6, autosomal recessive; Severe myoclonic epilepsy in infancy; Generalized epilepsy with febrile seizures plus, types 1 and 2; Severe X-linked myotubular myopathy; Short QT syndrome 3; Short stature with nonspecific skeletal abnormalities; Short stature, auditory canal atresia, mandibular hypoplasia, skeletal abnormalities; Short stature, onychodysplasia, facial dysmorphism, and hypotrichosis; Primordial dwarfism; Short-rib thoracic dysplasia 11 or 3 with or without polydactyly; Sialidosis type I and II; Silver spastic paraplegia syndrome;

Slowed nerve conduction velocity, autosomal dominant; Smith-Lemli-Opitz syndrome;

Snyder Robinson syndrome; Somatotroph adenoma; Prolactinoma; familial, Pituitary adenoma predisposition; Sotos syndrome 1 or 2; Spastic ataxia 5, autosomal recessive,

Charlevoix- Saguenay type, 1,10, or 11, autosomal recessive; Amyotrophic lateral sclerosis type 5; Spastic paraplegia 15, 2, 3, 35, 39, 4, autosomal dominant, 55, autosomal recessive, and 5A; Bile acid synthesis defect, congenital, 3; Spermatogenic failure 11, 3, and 8;

Spherocytosis types 4 and 5; Spheroid body myopathy; Spinal muscular atrophy, lower extremity predominant 2, autosomal dominant; Spinal muscular atrophy, type II;

Spinocerebellar ataxia 14, 21, 35, 40, and 6; Spinocerebellar ataxia autosomal recessive 1 and

16; Splenic hypoplasia; Spondylocarpotarsal synostosis syndrome; Spondylocheirodysplasia,

Ehlers-Danlos syndrome-like, with immune dysregulation, Aggrecan type, with congenital joint dislocations, short limb-hand type, Sedaghatian type, with cone-rod dystrophy, and

Kozlowski type; Parastremmatic dwarfism; Stargardt disease 1; Cone-rod dystrophy 3;

Stickler syndrome type 1; Kniest dysplasia; Stickler syndrome, types l(nonsyndromic ocular) and 4; Sting-associated vasculopathy, infantile-onset; Stormorken syndrome; Sturge -Weber syndrome, Capillary malformations, congenital, 1 ; Succinyl-CoA acetoacetate transferase deficiency; Sucrase-isomaltase deficiency; Sudden infant death syndrome; Sulfite oxidase deficiency, isolated; Supravalvar aortic stenosis; Surfactant metabolism dysfunction, pulmonary, 2 and 3; Symphalangism, proximal, lb; Syndactyly Cenani Lenz type;

Syndactyly type 3; Syndromic X-linked mental retardation 16; Talipes equinovarus; Tangier disease; TARP syndrome; Tay-Sachs disease, B1 variant, Gm2-gangliosidosis (adult), Gm2- gangliosidosis (adult-onset); Temtamy syndrome; Tenorio Syndrome; Terminal osseous dysplasia; Testosterone 17-beta-dehydrogenase deficiency; Tetraamelia, autosomal recessive; Tetralogy of Fallot; Hypoplastic left heart syndrome 2; Truncus arteriosus; Malformation of the heart and great vessels; Ventricular septal defect 1; Thiel-Behnke corneal dystrophy; Thoracic aortic aneurysms and aortic dissections; Marfanoid habitus; Three M syndrome 2; Thrombocytopenia, platelet dysfunction, hemolysis, and imbalanced globin synthesis;

Thrombocytopenia, X-linked; Thrombophilia, hereditary, due to protein C deficiency, autosomal dominant and recessive; Thyroid agenesis; Thyroid cancer, follicular; Thyroid hormone metabolism, abnormal; Thyroid hormone resistance, generalized, autosomal dominant; Thyrotoxic periodic paralysis and Thyrotoxic periodic paralysis 2; Thyrotropin releasing hormone resistance, generalized; Timothy syndrome; TNF receptor-associated periodic fever syndrome (TRAPS); Tooth agenesis, selective, 3 and 4; Torsades de pointes; Townes-Brocks-branchiootorenal-like syndrome; Transient bullous dermolysis of the newborn; Treacher collins syndrome 1; Trichomegaly with mental retardation, dwarfism and pigmentary degeneration of retina; Trichorhinophalangeal dysplasia type I;

Trichorhinophalangeal syndrome type 3; Trimethylaminuria; Tuberous sclerosis syndrome; Lymphangiomyomatosis; Tuberous sclerosis 1 and 2; Tyrosinase-negative oculocutaneous albinism; Tyrosinase-positive oculocutaneous albinism; Tyrosinemia type I; UDPglucose-4- epimerase deficiency; Ullrich congenital muscular dystrophy; Ulna and fibula absence of with severe limb deficiency; Upshaw-Schulman syndrome; Urocanate hydratase deficiency; Usher syndrome, types 1, IB, ID, 1G, 2A, 2C, and 2D; Retinitis pigmentosa 39; UV- sensitive syndrome; Van der Woude syndrome; Van Maldergem syndrome 2; Hennekam lymphangiectasia-lymphedema syndrome 2; Variegate porphyria; Ventriculomegaly with cystic kidney disease; Verheij syndrome; Very long chain acyl-CoA dehydrogenase deficiency; Vesicoureteral reflux 8; Visceral heterotaxy 5, autosomal; Visceral myopathy; Vitamin D-dependent rickets, types land 2; Vi tel 1i form dystrophy ; von Willebrand disease type 2M and type 3; Waardenburg syndrome type 1, 4C, and 2E (with neurologic

involvement); Klein-Waardenberg syndrome; Walker-Warburg congenital muscular dystrophy; Warburg micro syndrome 2 and 4; Warts, hypogammaglobulinemia, infections, and myelokathexis; Weaver syndrome; Weill-Marchesani syndrome 1 and 3; Weill- Marchesani-like syndrome; Weissenbacher-Zweymuller syndrome; Werdnig-Hoffmann disease; Charcot-Marie-Tooth disease; Wemer syndrome; WFSl-Related Disorders; Wiedemann- Steiner syndrome; Wilson disease; Wolfram-like syndrome, autosomal dominant; Worth disease; Van Buchem disease type 2; Xeroderma pigmentosum, complementation group b, group D, group E, and group G; X-linked agammaglobulinemia; X-linked hereditary motor and sensory neuropathy; X-linked ichthyosis with steryl-sulfatase deficiency; X-linked periventricular heterotopia; Oto-palato-digital syndrome, type I; X- linked severe combined immunodeficiency; Zimmermann-Laband syndrome and

Zimmermann-Laband syndrome 2; and Zonular pulverulent cataract 3.

[0336] In some aspects, the present disclosure provides uses of any one of the fusion proteins described herein and a guide RNA targeting this fusion protein to a target A:T base pair in a nucleic acid molecule in the manufacture of a kit for base editing, wherein the base editing comprises contacting the nucleic acid molecule with the fusion protein and guide RNA under conditions suitable for the substitution of the adenine (A) of the A:T nucleobase pair with a guanine (G). In some embodiments of these uses, the nucleic acid molecule is a double- stranded DNA molecule. In some embodiments, the step of contacting of induces separation of the double- stranded DNA at a target region. In some embodiments, the step of contacting further comprises nicking one strand of the double- stranded DNA, wherein the one strand comprises an unmutated strand that comprises the T of the target A:T nucleobase pair.

[0337] In some embodiments of the described uses, the step of contacting is performed in vitro. In other embodiments, the step of contacting is performed in vivo. In some

embodiments, the step of contacting is performed in a subject (e.g., a human subject or a non human animal subject). In some embodiments, the step of contacting is performed in a cell, such as a human or non-human animal cell.

[0338] The present disclosure also provides uses of any one of the fusion proteins described herein as a medicament. The present disclosure also provides uses of any one of the complexes of fusion proteins and guide RNAs described herein as a medicament.

Pharmaceutical compositions

[0339] Other aspects of the present disclosure relate to pharmaceutical compositions comprising any of the adenosine deaminases, fusion proteins, or the fusion protein-gRNA complexes described herein. The term“pharmaceutical composition”, as used herein, refers to a composition formulated for pharmaceutical use. In some embodiments, the

pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g. for specific delivery, increasing half-life, or other therapeutic compounds). [0340] In some embodiments, any of the fusion proteins, gRNAs, and/or complexes described herein are provided as part of a pharmaceutical composition. In some

embodiments, the pharmaceutical composition comprises any of the fusion proteins provided herein. In some embodiments, the pharmaceutical composition comprises any of the complexes provided herein. In some embodiments pharmaceutical composition comprises a gRNA, a napDNAbp-dCas9 fusion protein, and a pharmaceutically acceptable excipient. In some embodiments pharmaceutical composition comprises a gRNA, a napDNAbp-nCas9 fusion protein, and a pharmaceutically acceptable excipient. Pharmaceutical compositions may optionally comprise one or more additional therapeutically active substances.

[0341] In some embodiments, compositions provided herein are administered to a subject, for example, to a human subject, in order to effect a targeted genomic modification within the subject. In some embodiments, cells are obtained from the subject and contacted with a any of the pharmaceutical compositions provided herein. In some embodiments, cells removed from a subject and contacted ex vivo with a pharmaceutical composition are re-introduced into the subject, optionally after the desired genomic modification has been effected or detected in the cells. Methods of delivering pharmaceutical compositions comprising nucleases are known, and are described, for example, in U.S. Pat. Nos. 6,453,242; 6,503,717;

6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of all of which are incorporated by reference herein in their entireties. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals or organisms of all sorts. Modification of

pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, domesticated animals, pets, and commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys.

[0342] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient(s) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.

[0343] Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington’s The Science and Practice of

Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD,

2006; incorporated in its entirety herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof.

See also PCT application PCT/US2010/055131 (Publication No. WO/2011053982), filed

Nov. 2, 2010, incorporated in its entirety herein by reference, for additional suitable methods, reagents, excipients and solvents for producing pharmaceutical compositions comprising a nuclease. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the

pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.

[0344] As used herein, the term“pharmaceutically-acceptable carrier” means a

pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body). A pharmaceutically acceptable carrier is“acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.).

Some examples of materials which can serve as pharmaceutically-acceptable carriers include:

(1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose,

methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations.

Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the

formulation. The terms such as“excipient”,“carrier”,“pharmaceutically acceptable carrier” or the like are used interchangeably herein.

[0345] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, e.g., for gene editing. Suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and

intracerebroventricular administration.

[0346] In some embodiments, the pharmaceutical composition described herein is administered locally to a diseased site (e.g., tumor site). In some embodiments, the pharmaceutical composition described herein is administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber.

[0347] In other embodiments, the pharmaceutical composition described herein is delivered in a controlled release system. In one embodiment, a pump may be used (see, e.g., Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials may be used. (See, e.g., Medical Applications of

Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al, 1985, Science 228:190; During et al, 1989 , Ann. Neurol. 25:351;

Howard et al, 1989, J. Neurosurg. 71:105.) Other controlled release systems are discussed, for example, in Langer, supra.

[0348] In some embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human. In some embodiments, pharmaceutical composition for administration by injection are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0349] A pharmaceutical composition for systemic administration may be a liquid, e.g., sterile saline, lactated Ringer’s or Hank’s solution. In addition, the pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated.

[0350] The pharmaceutical composition may be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration. The particles may be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein. Compounds may be entrapped in“stabilized plasmid- lipid particles” (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating (Zhang Y. P. et al, Gene Ther. 1999, 6:1438-47). Positively charged lipids such as N-[l-(2,3-dioleoyloxi)propyl]-N,N,N-trimethyl-amoniummethyls ulfate, or “DOTAP,” are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g., U.S. Patent Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757; each of which is incorporated herein by reference.

[0351] The pharmaceutical composition described herein may be administered or packaged as a unit dose, for example. The term“unit dose” when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

[0352] Further, the pharmaceutical composition may be provided as a pharmaceutical kit comprising (a) a container containing a compound of the disclosure in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection. The pharmaceutically acceptable diluent may be used for reconstitution or dilution of the lyophilized compound of the disclosure. Optionally associated with such container(s) may be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0353] In another aspect, an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease described herein and may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. The active agent in the composition is a compound of the disclosure. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically- acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution.

It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

Delivery methods

[0354] In some aspects, the disclosure provides methods comprising delivering one or more polynucleotides, such as or one or more vectors as described herein, one or more transcripts thereof, and/or one or proteins transcribed therefrom, to a host cell. In some aspects, the disclosure further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells. In some embodiments, a base editor as described herein in combination with (and optionally complexed with) a guide sequence is delivered to a cell.

[0355] In some embodiments, the method of delivery provided comprises nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.

[0356] Conventional viral and non-viral based gene transfer methods may be used to introduce nucleic acids in mammalian cells or target tissues. Such methods may be used to administer nucleic acids encoding components of a base editor to cells in culture, or in a host organism. Non-viral vector delivery systems include ribonucleoprotein (RNP) complexes,

DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science

256:808-813 (1992); Nabel & Feigner, TIB TECH 11:211-217 (1993); Mitani & Caskey,

TIB TECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature

357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149- 1154 (1988); Vigne, Restorative

Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin

51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology

Doerfler and Bihm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

[0357] In certain embodiments, the method of delivery and vector provided herein is an RNP complex. RNP delivery of base editors markedly increases the DNA specificity of base editing. RNP delivery of base editors leads to decoupling of on- and off-target editing. RNP delivery ablated off-target editing at non-repetitive sites while maintaining on-target editing comparable to plasmid delivery, and greatly reduced off-target editing even at the highly repetitive VEGFA site 2. See Rees, H.A. et al., Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery, Nat. Commun.

8, 15790 (2017), which is incorporated by reference herein in its entirety.

[0358] Methods of non-viral delivery of nucleic acids include RNP complexes, include lipofection, nucleofection, electoporation, stable genome integration (e.g., piggybac), microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of

DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™, Lipofectin™ and SF

Cell Line 4D-Nucleofector X Kit™ (Lonza)). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO

91/17424; WO 91/16024. Delivery may be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration). Delivery may be achieved through the use of RNP complexes.

[0359] The preparation of lipidmucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992);

U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085,

4,837,028, and 4,946,787).

[0360] The use of RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors may be administered directly to patients {in vivo ) or they may be used to treat cells in vitro, and the modified cells may optionally be administered to patients {ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.

[0361] The tropism of a viruses can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis- acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia vims (GaLV), Simian Immuno deficiency vims (SIV), human immuno deficiency vims (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al, J. Virol. 65:2220-2224 (1991);

PCT/US 94/05700). In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated vims (“AAV”) vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Constmction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414;

Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.

4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al, J. Virol. 63:03822-3828 (1989).

[0362] Packaging cells are typically used to form vims particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and y2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper vims promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovims is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. Reference is made to US 2003/0087817, published May 8, 2003, International Patent Application No. WO 2016/205764, published December 22, 2016, International Patent Application No. WO 2018/071868, published April 19, 2018, and U.S. Patent Publication No. 2018/0127780, published May 10, 2018, the disclosures of each of which are incorporated herein by reference.

[0363] In various embodiments, the disclosed expression constructs may be engineered for delivery in one or more rAAV vectors. An rAAV as related to any of the methods and compositions provided herein may be of any serotype including any derivative or pseudotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 2/1, 2/5, 2/8, 2/9, 3/1, 3/5, 3/8, or 3/9). An rAAV may comprise a genetic load (i.e., a recombinant nucleic acid vector that expresses a gene of interest, such as a whole or split fusion protein that is carried by the rAAV into a cell) that is to be delivered to a cell. An rAAV may be chimeric.

[0364] As used herein, the serotype of an rAAV refers to the serotype of the capsid proteins of the recombinant vims. Non-limiting examples of derivatives and pseudotypes include rAAV2/l, rAAV2/5, rAAV2/8, rAAV2/9, AAV2-AAV3 hybrid, AAVrh.10, AAVhu.14, AAV3a/3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6,

AAV2.5, AAV6.2, AAV2i8, AAV-HSC15/17, AAVM41, AAV9.45, AAV6(Y445F/Y731F),

AAV2.5T, AAV-HAE1/2, AAV clone 32/83, AAVShHIO, AAV2 (Y->F), AAV8 (Y733F),

AAV2.15, AAV2.4, AAVM41, and AAVr3.45. A non-limiting example of derivatives and pseudotypes that have chimeric VP1 proteins is rAAV2/5-lVPlu, which has the genome of

AAV2, capsid backbone of AAV5 and VPlu of AAV1. Other non-limiting example of derivatives and pseudotypes that have chimeric VP1 proteins are rAAV2/5-8VPlu, rAAV2/9-lVPlu, and rAAV2/9-8VPlu.

[0365] AAV derivatives/pseudotypes, and methods of producing such

derivatives/pseudotypes are known in the art (see, e.g., Mol Ther. 2012 Apr;20(4):699-708. doi: 10.1038/mt.2011.287. Epub 2012 Jan 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan Al, Schaffer DV, Samulski RJ.). Methods for producing and using pseudotyped rAAV vectors are known in the art (see, e.g., Duan el al, J. Virol., 75:7662- 7671, 2001; Halbert et al., J. Virol., 74:1524-1532, 2000; Zolotukhin et al, Methods, 28:158- 167, 2002; and Auricchio et al., Hum. Molec. Genet., 10:3075-3081, 2001).

[0366] Methods of making or packaging rAAV particles are known in the art and reagents are commercially available (see, e.g., Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167; and U.S. Patent Publication Numbers US20070015238 and US20120322861, which are incorporated herein by reference; and plasmids and kits available from ATCC and Cell Biolabs, Inc.). For example, a plasmid comprising a gene of interest may be combined with one or more helper plasmids, e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VP1, VP2, and VP3, including a modified VP2 region as described herein), and transfected into a recombinant cells such that the rAAV particle can be packaged and subsequently purified.

[0367] In some embodiments, the fusion proteins can be divided at a split site and provided as two halves of a whole/complete fusion protein. The two halves can be delivered to cells (e.g., as expressed proteins or on separate expression vectors) and once in contact inside the cell, the two halves form the complete fusion protein through the self-splicing action of the inteins on each fusion protein half. Split intein sequences can be engineered into each of the halves of the encoded fusion protein to facilitate their transplicing inside the cell and the concomitant restoration of the complete, functioning ABE.

[0368] These split intein-based methods overcome several barriers to in vivo delivery. For example, the DNA encoding fusion proteins is larger than the recombinant AAV (rAAV) packaging limit, and so requires different solutions. One such solution is formulating the editor fused to split intein pairs that are packaged into two separate rAAV particles that, when co-delivered to a cell, reconstitute the functional editor protein. Several other special considerations to account for the unique features of base editing are described, including the optimization of second-site nicking targets and properly packaging fusion proteins into virus vectors, including lentiviruses and rAAV.

[0369] Accordingly, the disclosure provides dual rAAV vectors and dual rAAV vector particles that comprise expression constructs that encode two halves of any of the disclosed fusion proteins, wherein the encoded fusion protein is divided between the two halves at a split site. In some embodiments, the two halves may be delivered to cells (e.g., as expressed proteins or on separate expression vectors) and once in contact inside the cell, the two halves form the complete fusion protein through the self-splicing action of the inteins on each fusion protein half. Split intein sequences can be engineered into each of the halves of the encoded fusion protein to facilitate their transplicing inside the cell and the concomitant restoration of the complete, functioning ABE.

[0370] In various embodiments, the fusion proteins may be engineered as two half proteins (i.e., an ABE N-terminal half and a ABE C-terminal half) by“splitting” the whole fusion protein as a“split site.” The“split site” refers to the location of insertion of split intein sequences (i.e., the N intein and the C intein) between two adjacent amino acid residues in the fusion protein. More specifically, the“split site” refers to the location of dividing the whole fusion protein into two separate halves, wherein in each halve is fused at the split site to either the N intein or the C intein motifs. The split site can be at any suitable location in the fusion protein fusion protein, but preferably the split site is located at a position that allows for the formation of two half proteins which are appropriately sized for delivery (e.g., by expression vector) and wherein the inteins, which are fused to each half protein at the split site termini, are available to sufficiently interact with one another when one half protein contacts the other half protein inside the cell.

[0371] Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US 2003/0087817, incorporated herein by reference.

[0372] It should be appreciated that any fusion protein, e.g., any of the fusion proteins provided herein, may be introduced into the cell in any suitable way, either stably or transiently. In some embodiments, a fusion protein may be transfected into the cell. In some embodiments, the cell may be transduced or transfected with a nucleic acid construct that encodes a fusion protein. For example, a cell may be transduced (e.g., with a virus encoding a fusion protein), or transfected (e.g., with a plasmid encoding a fusion protein) with a nucleic acid that encodes a fusion protein, or the translated fusion protein. Such transduction may be a stable or transient transduction. In some embodiments, cells expressing a fusion protein or containing a fusion protein may be transduced or transfected with one or more gRNA molecules, for example when the fusion protein comprises a Cas9 (e.g., nCas9) domain. In some embodiments, a plasmid expressing a fusion protein may be introduced into cells through electroporation, transient (e.g., lipofection) and stable genome integration (e.g., piggybac) and viral transduction or other methods known to those of skill in the art.

Kits and cells

[0373] This disclosure provides kits comprising a nucleic acid construct comprising nucleotide sequences encoding the fusion proteins, gRNAs, and/or complexes described herein. Some embodiments of this disclosure provide kits comprising a nucleic acid construct comprising a nucleotide sequence encoding an adenosine deaminase-napDNAbp fusion protein capable of deaminating an adenosine in a nucleic acid molecule. In some

embodiments, the nucleotide sequence encodes any of the adenosine deaminases provided herein. In some embodiments, the nucleotide sequence comprises a heterologous promoter that drives expression of the adenosine deaminase. The nucleotide sequence may further comprise a heterologous promoter that drives expression of the gRNA, or a heterologous promoter that drives expression of the fusion protein and the gRNA.

[0374] In some embodiments, the kit further comprises an expression construct encoding a guide nucleic acid backbone, e.g., 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 nucleic acid, e.g., guide RNA backbone.

[0375] The disclosure further provides kits comprising a fusion protein as provided herein, a gRNA having complementarity to a target sequence, and one or more of the following:

cofactor proteins, buffers, media, and target cells (e.g., human cells). Kits may comprise combinations of several or all of the aforementioned components.

[0376] Some embodiments of this disclosure provide kits comprising a nucleic acid construct, comprising (a) a nucleotide sequence encoding a napDNAbp (e.g., a Cas9 domain) fused to an adenosine deaminase domain; 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 nucleic acid backbone, e.g., 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 nucleic acid, e.g., guide RNA backbone. [0377] Some embodiments of this disclosure provide cells comprising any of the fusion proteins or complexes provided herein. In some embodiments, the cells comprise nucleotide constructs that encodes any of the fusion proteins provided herein. In some embodiments, the cells comprise any of the nucleotides or vectors provided herein. In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art.

[0378] In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL- 2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A 172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293. BxPC3. C3H- 10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr -/-, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML Tl, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT- 29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYOl, LNCap, Ma-Mel 1-48, MC- 38, MCF-7, MCF-IOA, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK 11, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI- H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassus, Va.)). In some embodiments, a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences. In some embodiments, a cell transiently transfected with the components of a

CRISPR system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a CRISPR complex, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence. In some embodiments, cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells are used in assessing one or more test compounds.

[0379] In the Examples discussed below, the widespread, low-level cellular RNA editing from adenine base editors was identified, that was greatly reduced, without substantially sacrificing on-target DNA editing, by introducing the E59A or E59Q mutation into TadA and the V106W mutation in TadA*. In addition to decoupling DNA and RNA editing activities, the ABEmaxAW variant substantially reduced off-target DNA editing activity and the formation of indel byproducts. Although it is noted that even ABEmax-mediated RNA editing is both low-level (averaging 0.21% across all transcripts) and transient given the short half-life of most cellular RNAs (27, 29), the extent to which low-level RNA editing may interfere with base editing biological studies or therapeutics development efforts will depend strongly on features of the specific applications, including the duration of exposure to the base editor. It is recommended that researchers use ABEmaxAW or ABEmaxQW for adenine base editing applications that require minimizing RNA editing, off-target DNA editing, and/or indel formation.

[0380] It should be appreciated that the foregoing concepts, and additional concepts discussed below, may be arranged in any suitable combination, as the present disclosure is not limited in this respect. Further, other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non limiting embodiments when considered in conjunction with the accompanying figures.

EXAMPLES

Example 1

[0381] HEK293T cells were transfected with a plasmid expressing ABEmax and isolating genomic DNA and RNA after 48 hours. After cDNA generation from poly-adenylated cellular mRNA, high-throughput sequencing (HTS) was performed on 220- to 250-nt regions of three mRNA amplicons: CTNNB1, IP90, and RSL1D1. CTNNB1 and IP90 were chosen as two examples of abundant mRNAs in HEK293T cells, and RSL1D1 was studied because it contains a region highly homologous to the 20-nt region of E. coli tRNA ^Arg2 that is the native substrate of TadA (26). The TadA minimal substrate sequence is

GCUCGGCUACGAACCGAG (SEQ ID NO: 1), while the homologous region of RSL1D1 mRNA is agUCGGCUACGGAAuuuAG (SEQ ID NO: 2), where upper-case letters indicate sequence identity. In all three transcripts, ABEmax generated low but detectable levels of RNA editing above the endogenous level of A-to-I editing from cellular deaminases(27, 29), which was measured using a Cas9(D10A)-only control. ABEmax expression increased both the extent of A-to-I conversions throughout the transcript (FIG. 1C), measured by the number of sequenced adenosines with an A-to-I conversion frequency >0.10%, as well as the magnitude of A-to-I editing (FIG. ID), measured by the average percentage of A-to-I conversion at every sequenced adenosine. For example, ABEmax generated an average of 1.3+0.41% A-to-I conversion among all sequenced adenosines in RSL1D1 mRNA, a 22-fold increase relative to the Cas9(D10A) nickase-only control that averaged 0.060+0.010% A-to-I conversion in the same transcript. Likewise, ABEmax resulted in detectable deamination of 27+2 out of 46 adenosines sequenced in RSL1D1 mRNA, while the Cas9(D10A) nickase control resulted in detectable deamination of 7+1 (3.9-fold fewer) of these 46 adenosines (FIGs. 1C, ID). To test if RNA editing activity requires fusion with the Cas9 component of the base editor, the TadA-TadA* monomer was overexpressed in trans with Cas9(D10A) nickase or dead Cas9 (dCas9) and observed substantial RNA editing under these conditions at all three tested transcripts (FIGs. 1C, ID). This outcome confirmed that RNA editing activity arises from the unassisted binding of TadA domains to cellular RNA, and focused efforts to improve the DNA:RNA specificity of ABE on engineering these deaminases. Taken together, these results establish that the TadA-TadA* deaminase component of ABEmax mediates low levels of cellular RNA editing.

[0382] Glu 70 is a critical catalytic residue in E. coli TadA, and the TadA E70A mutant either alon e(26) or in ABE(i) has no deaminase activity. In the soluble, N-terminally truncated version of TadA(26) used in ABE(7 ), Glu 70 corresponds to Glu 59, and will be referred to as Glu 59 hereafter. To identify which TadA monomers mediate RNA editing in ABEmax, inactivating E59A mutations were introduced into either the TadA or TadA* monomer of ABEmax and RNA (FIGs. 1C, ID) and DNA (FIG. IE) editing activity of the resulting variants was measured. Installing the E59A mutation in the wild-type TadA monomer to generate ABEmax (TadA E59A) modestly reduced the average number of edited adenosines in all three tested transcripts relative to ABEmax (FIG. 1C). Despite the modest reduction in RNA editing activity associated with ABEmax(TadA E59A), ABEmax(TadA E59A) maintains high DNA base editing activity similar to that of ABEmax. ABEmax averaged 46.6+3.9% DNA editing across the seven endogenous genomic sites tested, chosen because they result in a wide range of ABEmax editing efficiencies (from 85+6.6% to 4.5+0.70%), while ABEmax(TadA E59A) averaged 41.5+5.4% DNA editing at the same sites (FIG. IE). ABEmax(TadA E59A) also displayed reduced indel formation at these seven genomic sites compared to ABEmax (FIGs. 4A-4D), from a mean of 2.3+0.39% with ABEmax to 1.1+0.24% with ABEmax(TadA E59A). These data suggest that inactivation of the catalytic domain in the wild-type TadA monomer can reduce off-target RNA editing and indel formation without substantially sacrificing on-target DNA editing efficiency.

[0383] By contrast, neither ABEmax(TadA* E59A) nor ABEmax(TadA E59A, TadA* E59A) edit RNA (FIGs. 1C, ID) or DNA (FIG. IE), with one notable exception:

ABEmax(TadA* E59A), which contains a wild-type TadA monomer but an inactivated evolved TadA* monomer, edits RSL1D1 mRNA at position 152, the adenosine that is highly homologous to that of TadA’ s native tRNAArg substrate (FIG. IF). Together, these data indicate that both wild-type TadA and TadA* in ABEmax can deaminate RNA in a Cas9- independent manner. This off-target RNA editing activity may be reduced by inactivating the wild-type TadA monomer, but residual RNA editing activity remains from TadA*, which cannot be inactivated without abolishing DNA editing activity (FIG. ID).

[0384] To test if these findings apply to many different cellular transcripts, transcriptome wide analysis was performed of HEK293T cells treated with ABEmax, ABEmax(TadA E59A), ABEmax(TadA* E59A), and ABEmax(TadA E59A, TadA* E59A). Cells were transfected with plasmids expressing the base editor and an LDLR-targeting sgRNA.

Targeting the base editors to an expressed gene mimics their typical use (3) and enables detection of the on-target U-to-C edit in the corresponding LDLR mRNA transcript during transcriptome-wide RNA-Seq as an internal positive control (FIG. 1G). Since A-to-I editing in cellular mRNA from endogenous ADAR deaminases is a common source of natural RNA editing in metazoans (27, 29), cells treated with Cas9(D10A) nickase were used only as a control to identify A-to-I RNA editing levels from endogenous cellular deaminases.

[0385] Transcriptome-wide RNA-seq data revealed that, on average, ABEmax

overexpression induced 14,959 additional high-confidence A-to-I edits compared to the Cas9 nickase-only control (FIG. 1H). Although ABEmax overexpression adds only 28% more detected A-to-I edits than the 53,334 endogenous cellular A-to-I edits observed in the Cas9 nickase-only control, these additional ABEmax-induced RNA edits were widespread throughout the transcriptome, including 10,335 transcripts not edited in the Cas9 nickase- only control samples. These data confirm that low-level RNA editing is widespread throughout the transcriptome among cells overexpressing ABEmax. [0386] RNA editing across the transcriptome was reduced by inactivating either TadA or

TadA* monomers. Catalytically inactivated ABEmax(TadA E59A, TadA* E59A) resulted in

53,917 A-to-I edits, similar to the 53,334 A-to-I edits detected in the Cas9 nickase-only control. ABEmax(TadA E59A) resulted in 12,142 more A-to-I edits than the Cas9 nickase- only control, 19% fewer additional A-to-I edits than the 14,959 mediated by ABEmax (FIG.

IH). The average A-to-I RNA editing frequency across all transcripts was 0.22% for

ABEmax, 0.19% for ABEmax(TadA E59A), and 0.13% for Cas9(D10A) nickase only (FIG.

II). Together, these findings indicate that transcriptome-wide RNA editing is modestly reduced by inactivating the wild-type TadA monomer in ABEmax.

[0387] Given the lack of an elucidated structure of ABE or of the E. coli TadA homodimer bound to RNA, the crystal structure of S. aureus TadA was used, which has high sequence homology to E. coli TadA (25), to guide the design of ABE mutants that further reduce RNA editing. Starting with ABEmax(TadA E59A), the construct with the inactivated wild-type TadA domain that shows reduced RNA editing but maintains strong DNA base editing (FIGs. 1C-1H), mutations were installed into the evolved TadA* monomer.

Example 2

[0388] Three TadA* residues were identified, predicted to interact with the RNA substrate as targets for substitutions that might impair TadA* -mediated RNA deamination. It was hypothesized that impeding the ability of TadA* to accommodate 2'-hydroxyl groups that are present in RNA, but absent in DNA, by replacing these three amino acids with larger or more hydrophobic residues (Gin, Phe, Trp, or Met) could further improve the DNA versus RNA editing specificity of ABEmax (TadA E59A). Arg 47 is predicted to form a hydrogen bond with the 2'-hydroxyl group of the substrate adenosine (FIG. 2A). Arg 47 was replaced in TadA* with Gin, Phe, Trp, or Met in an effort to abrogate this interaction. A series of ABEmax mutants was also generated with TadA* substitutions at either Asn 108 (FIG. 2B) or Val 106 (FIG. 2C), two residues that are located close to the catalytic site of TadA, and that mutated from Asp 108 and Ala 106 during the evolution of TadA*(l). Asp 108 is predicted to directly hydrogen bond with the 2 '-hydroxyl group of the uridine immediately 5' of the substrate adenosine (FIG. 2B), and replacement of Ala 106 might fill some of the space that accommodates this uridine, including its 2' hydroxyl group, with larger and more hydrophobic side chains (FIG. 2C). Asn 108 was replaced in ABEmax TadA* with Gin, Phe, Trp, Lys, or Met, and Val 106 in ABEmax TadA* with Gin, Phe, Trp, or Met, in an effort to disrupt the ability of TadA* to accommodate ribonucleotides by eliminating the possibility of forming hydrogen bonds with 2' hydroxyl groups in RNA or by steric occlusion. An additional Asn 108 Lys mutation was also tested to provide a polar side-chain that is incapable of serving as a hydrogen bond acceptor assuming protonation at physiological pH.

[0389] HEK293T cells were transfected with each of these 13 ABEmax(TadA E59A) mutants and measured the resulting on-target DNA A*T-to-G*C base editing at the seven genomic loci tested (FIG. 2D). High-throughput sequencing of regions of IP90, RSL1D1, and

CTNNB1 cDNAs was used to rapidly assess the RNA editing activities of these 13 mutants in HEK293T cells (FIGs. 2E, 2F) prior to transcriptome-wide RNA-seq analyses.

[0390] Replacing TadA* Arg 47 in ABEmax(TadA E59A) with Gin, Met, Phe or Trp maintained relatively high DNA base editing efficiency, particularly at sites where the target

A is at protospacer position 5 (counting the PAM as positions 21-23). Average editing efficiencies were reduced from a mean of 47+3.9% for ABEmax(TadA E59A) to a range of

31-41% for the four TadA* Arg 47 variants. Among the four Arg 47 mutants tested,

ABEmax(TadA E59A, TadA* R47M) and ABEmax(TadA E59A, TadA* R47Q), the most efficient variants for DNA base editing, showed little or no reduction in RNA editing activity compared to ABEmax(TadA E59A) (FIGs. 2E, 2F). The two variants in which Arg 47 was replaced with larger and more rigid hydrophobic residues, ABEmax(TadA E59A, TadA*

R47F) and ABEmax(TadA E59A, TadA* R47W), resulted in up to a 2.0-fold reduction in the number of edited adenosines in the interrogated transcripts (FIG. 2E). Unfortunately, this reduction in RNA editing was accompanied by a similar reduction in DNA editing at sgRNAs in which the target A was located at positions other than position 5 in the protospacer (FIG.

2D). These data indicate that replacing Arg 47 with Phe or Trp impairs both RNA and DNA editing and replacing this residue with Met or Gin impairs neither DNA nor RNA editing.

[0391] Mutation of TadA* Asn 108 in ABEmax(TadA E59A) generally preserved DNA base editing at sites in which the target A was at protospacer position 5, but greatly reduced DNA editing at other target sites. The most active Asn 108 mutant, ABEmax(TadA E59A, TadA*

N108K), mediated 25+0.2% average on-target DNA editing (FIG. 2D), a 1.9-fold reduction compared to ABEmax, but also exhibited the highest levels of RNA editing among the Asn

108 mutants assayed (FIG. 2E and FIG. 2F). Mutation of TadA* Asn 108 in ABEmax(TadA

E59A) to Phe, Trp, Gin or Met greatly reduced RNA editing compared to ABEmax in the three transcripts sequenced at depth to levels statistically indistinguishable from background

RNA editing observed in the Cas9(D10A)-only controls (Student’s two-tailed T test, p>0.05 for comparisons between number of edited adenosines in each transcript) (FIG. 2E).

Together, these data indicate that Asn 108 in TadA* is important for efficient DNA base editing at protospacer positions beyond the most preferred one (position 5), and is also essential for RNA editing. The ABEmax(TadA E59A, TadA* N108Q) or ABEmax(TadA E59A, TadA* N108W) variants may be useful when the target A is at protospacer position 5, and minimizing RNA editing is critical.

[0392] Substitution of TadA* Val 106 in ABEmax(TadA E59A) resulted in variants that exhibited much lower RNA editing while maintaining DNA editing levels similar to those of ABEmax(TadA E59A) and ABEmax. All four Val 106 mutants mediated effective DNA base editing across the seven genomic loci tested; the most efficient DNA base editor among these mutants was ABEmax(TadA E59A, TadA* V106W), hereafter referred to as

ABEmaxAW, which yielded an average of 36+1.4% A*T-to-G*C DNA editing (compared to 41+5.4% for ABEmax(TadA E59A), and 47+3.9% for ABEmax). ABEmaxAW exhibited both the highest level of DNA base editing and the lowest level of RNA off-target editing amongst the Val 106 mutants tested (FIGs. 2E, 2F). Analysis of the RNA isolated from cells transfected with ABEmaxAW indicated that the number of detectable A-to-I edits among the regions of the three transcripts analyzed was significantly reduced from an average of 94+8 (out of 182 total adenosines) with ABEmax to 26+10 with ABEmaxAW, similar to the background of 12+6 for Cas9 nickase alone (FIG. 2E). The average magnitude of A-to-I edits was also greatly reduced in cells treated with ABEmaxAW (0.068% average A-to-I editing frequency among 182 total adenosines) to levels approaching those observed from Cas9 nickase alone (0.041% average), a 7.2-fold reduction compared with the 0.49% average A-to- I editing frequency of ABEmax (FIG. 2F). These findings establish that ABEmaxAW greatly reduces off-target RNA editing while preserving most of the on-target DNA editing activity of ABEmax.

Example 3

[0393] The applicability of the findings was tested to other mammalian cell types. First, the DNA base editing activities of ABEmax, ABEmax(TadA E59A), ABEmaxAW, and

ABEmax (TadA E59A, TadA* N108W) in HeLa cells (FIGs. 5A-5B), and ABEmax and ABEmaxAW in U20S and K562 cells (FIGs. 6A-6F) were compared. DNA base editing efficiencies among unsorted HeLa and U20S cells were uniformly lower than in HEK293T cells (FIG. 5A), possibly due to poorer transfection or nucleofection efficiencies (75). The DNA base editing activity of ABEmaxAW relative to ABEmax and ABEmax(TadA E59A), however, generally remained similar in all three cell types (FIGs. 5A, 6A). Next, RNA editing frequencies and magnitudes were investigated in U20S and K562 cells, and it was found that compared to ABEmax, the use of ABEmaxAW greatly reduced RNA editing to levels indistinguishable from those of the Cas9(D10A) control (FIGs. 6C and 6D). Together, these data indicate that ABEmaxAW can mitigate RNA editing in multiple mammalian cell types.

[0394] The effect of longer exposure time to ABEmax or ABEmaxAW in HEK293T cells was assessed by harvesting cells 5 days post-transfection, instead of 48 hours. This change increased the average DNA base editing associated with both ABEmax and ABEmaxAW by 1.1-fold, to 52+2.7% for ABEmax and 39+1.7% with ABEmaxAW (FIGs. 7A, 7B).

Surprisingly, average RNA editing was reduced compared to the 48-hour treatment; ABEmax yielded 0.29+0.063% A-to-I average editing across the 182 adenosines sequenced (compared with 0.49+0.13% at 48 hours). The average frequency of A-to-I mutation with ABEmaxAW after 5 days (0.074+0.014%, 3.9-fold lower than that of ABEmax) remained close to the background frequency associated with Cas9(D10A) nickase alone of 0.051+0.010% (FIGs. 7C, 7D). It is believed that the steady loss (or silencing) of transfected plasmids expressing base editors, coupled with the constant degradation and replenishment of the transcriptome, may result in lower RNA editing rates at longer time points.

[0395] These TadA* mutations might further weaken the ability of ABEmax variants to bind off-target DNA sequences that are already more weakly bound by Cas9. To test this possibility, the levels of off-target DNA editing were measured by ABEmax and a subset of the ABEmax variants described above. HTS was used to assess the frequencies of off-target A*T-to-G*C base editing and indel formation at 12 known off-target sites associated with HEK site 2, HEK site 3 and HEK site 4(31 ) (FIGs. 8, 9, 10). Among these 12 off-target sites, ten had at least one adenosine within the canonical ABE editing window (from protospacer position 4 to 8)(1, 3). The mean A*T-to-G*C editing efficiency at these ten candidate off- target loci from ABEmax was 2.1+0.22%, similar to that of ABEmax(TadA E59A)

(2.0+0.28%) (FIGs. 8, 9, 10). Notably, ABEmaxAW generated an average off-target editing frequency of 0.79+0.18%, a 2.5-fold improvement compared to ABEmax(TadA E59A), and a 2.7-fold improvement relative to ABEmax. Collectively, these results indicate that mutations that reduce the tolerance of ABEmax for RNA editing also increase the DNA specificity of base editing, likely by reducing DNA binding interactions that support productive editing of off-target loci.

[0396] Notably, ABEmaxAW also generated 3.7-fold fewer indels than ABEmax at the seven on-target DNA loci tested (from an average of 2.3+0.39% with ABEmax to

0.62+0.0069% with ABEmaxAW (FIGs. 4A-4D). The reason for this reduced indel frequency is unclear, but it is hypothesized that indel formation may be dependent on the structure or activity of the wild-type TadA monomer. Consistent with this hypothesis, ABEmax(TadA E59A) also shows reduced average indel formation (1.1+0.24%) and ABEmax(TadA* E59A), which cannot perform DNA base editing, induces indels at an elevated frequency of 4.3+0.45% (FIGs. 4A-4D).

Example 4

[0397] To further illuminate the impact of V 106W in TadA* on the DNA and RNA editing activities of ABE, two additional ABEmax mutants, ABEmax(TadA E59, TadA* V106W) (ABEmaxEW), and ABEmax(TadA E59Q, TadA* V106W) (ABEmaxQW), were generated and tested (FIGs. 1 lA-1 ID). ABEmaxEW displayed slightly higher DNA on-target editing frequencies than ABEmaxAW (FIG. 11A), but also greater indel (FIG. 11B) and RNA editing frequencies (FIGs. 11C, 11D), confirming that mutation of both the wild-type and the evolved TadA monomers is required for the most effective reduction in RNA editing and indel frequencies. ABEmaxQW performed as well as or slightly better than ABEmaxAW at on-target DNA base editing (FIG. 11 A) and displayed similarly low levels of off-target RNA editing (FIGs. 11C, 11D). Consistent with observations that the wild-type TadA monomer plays a role in indel formation, both ABEmaxEW and ABEmaxQW displayed substantially higher indel frequencies than ABEmaxAW (FIG. HB).These comparisons together indicate that both inactivation of the wild-type TadA and mutation of the evolved monomer with V106W, are required to minimize off-target RNA editing, and ABEmaxQW may display higher on-target base editing efficiency at some sites than ABEmaxAW, but without the consistently lower indel frequencies of ABEmaxAW.

Example 5

[0398] Finally, RNA-Seq was performed to identify transcriptome-wide A-to-I editing frequencies associated with ABEmaxAW. Importantly, robust on-target DNA editing activity was confirmed in the RNA-seq samples treated with ABEmax, ABEmax(E59A), and ABEmaxAW by observing substantial U-to-C mutation in the LDLR mRNA, which resulted from base editing the corresponding genomic DNA site directed by the LDLR-targeting sgRNA (FIG. 2G). While the proportion of edited LDLR mRNA reads is reduced in the ABEmaxAW sample (24/90, 27%) as compared to the ABEmax (37/98, 38%) and

ABEmax(TadA E59A) (18/66, 27%), the numbers of LDLR mRNA transcripts aligned to the reference sequence are low, making precise quantitation challenging. Consistent with the above results analyzing the three test transcripts in depth, ABEmaxAW only slightly elevated the number of A-to-I edits (57,685) beyond those observed in the Cas9(D10A) nickase-only control (53,334). ABEmaxAW thus resulted in substantially fewer transcriptome edits compared to ABEmax or ABEmax(E59A) (10,608 fewer A-to-I edits than ABEmax, and 7,791 fewer than ABEmax(E59A)) (FIGs. 2G, 2H). The average A-to-I RNA editing frequency was also compared across all transcripts, and it was found that the 0.22% average

A-to-I RNA editing for ABEmax was reduced to 0.14% for ABEmaxAW and to 0.13% for the Cas9(D10A) nickase-only control (FIG. 21). These findings confirm that ABEmaxAW maintains strong DNA base editing activity while exhibiting much lower transcriptome-wide

RNA editing compared to ABEmax.

[0399] In order to determine the potential biological significance of the A-to-I edits observed with ABEmax and ABEmaxAW, the ENSEMBLE Variant Effector Predictor was used to determine where the edits were located within mRNA transcripts in the transcriptome-wide sequencing data (FIG. 3A). The RNA editing associated with ABEmax was spread across the transcriptome and not localized to particular regions (FIG. 12). Only 4.2% of the A-to-I edits were in a protein coding region; of these, 69% lead to coding changes (FIG. 3B). Next, SIFT was used to predict the impact of these coding changes on protein function, revealing that 58% of the coding A-to-I mutations are predicted to have a deleterious impact on protein function (FIG. 3C). In total ABEmax induced 1,138 A-to-I mutations predicted to be deleterious to protein function, compared to 535 for Cas9(D10A) alone. This was reduced to 727 for ABEmaxAW (FIG. 3C). Mutations in the 3' or 5' UTR can also be deleterious to protein function, but the effects of such mutations are not readily prcdictablc(JO). Finally, it is noted that the biological consequence even of mutations that genuinely impair protein function are likely to be minimized by the very low average A-to-I RNA editing frequency of 0.21% for ABEmax, and 0.14% for ABEmaxAW, compared to 0.13% for the Cas9(D10A) nickase-only control.

Materials and Methods for Examples 1-5

Plasmid construction

[0400] All mammalian cell expression plasmids were constructed by USER cloning from gBlock gene fragments (Integrated DNA Technologies), as previously described (32).

Phusion U Green Multiplex PCR Master Mix (ThermoFisher) was used for amplification of DNA. sgRNA plasmids were constructed by blunt end ligation of a linear PCR product generated by encoding the 20-nt variable protospacer sequence onto the 5' end of an amplification primer and treating the resulting piece to KLD Enzyme Mix (New England Biolabs) according to the manufacturers’ instruction. Machl chemically competent E. coli (ThermoFisher) cells were used for plasmid construction. Mammalian cell culture

[0401] All cells were cultured and maintained at 37 °C with 5 % C02. Antibiotics were not used for cell culture. HEK293T cells (ATCC CRL-3216) and HeLa cells (ATCC CCL-2) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) plus GlutaMax

(ThermoFisher) supplemented with 10 % (v/v) fetal bovine serum (FBS). K562 cells (ATCC CCL-243) were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium plus GlutaMax (ThermoFisher) supplemented with 10 % (v/v) fetal bovine serum (FBS). U20S cells (ATCC HTB-96) were cultured in MyCoy’s 5A Medium plus GlutaMax

(ThermoFisher) supplemented with 10 % (v/v) fetal bovine serum (FBS).

Preparation of plasmids for mammalian cell transfection

[0402] To obtain endotoxin-free plasmids for transfection, 45 mF of Machl cells

(ThermoFisher) expressing freshly-transformed plasmid were pelleted by centrifugation (6000 g, 5 minutes, 4 °C) and purified using ZymoPURE II Plasmid Midi Prep Kits (Zymo Research), according to the manufacturer’s instructions with the inclusion of the optional step of passing the plasmid across the EndoZero Spin column (Zymo Research). Plasmid yield was quantified using a Nanodrop and by electrophoresis on a 1% agarose Tris/Borate/EDTA gel supplemented with ethidium bromide.

Mammalian cell lipofection and genomic DNA isolation

[0403] HEK293T cells were seeded on 48-well poly-D-lysine coated plates (Coming) 18-20 hours before lipofection. Fipofection was performed at a cell density of 65%. Unless otherwise stated, cells were transfected with 462 ng of nuclease- or base-editor expression plasmid DNA, 138 ng of sgRNA expression plasmid DNA, and 100 ng of TadA-dimer expression plasmid if this was included for“in trans” analysis of RNA editing. 1.4 pF Fipofectamine 2000 (ThermoFisher) was used per well. Cells were harvested 48 hours or 5 days, as indicated, after transfection.

[0404] HeFa cells were seeded in 250 pF of media on 48-well collagen coated plates (Coming) at a density of 70,000-80,000 cells per mF 20-24 hours before lipofection so cells were approximately 85% confluent at the time of transfection. A total of 200 ng of plasmid was used per well, consisting of a mixture of 154 ng of base editor or Cas9 nickase plasmid and 46 ng of sgRNA expression vector plasmid. 1 pF of HeFafect (OZ Biosciences) was used per well according to the manufacturer’s instmctions. DNA extraction was performed exactly as described above for HEK293T cells. Genomic DNA isolation

[0405] Media was removed and cells were washed once with IX DPBS (ThermoFisher). Genomic DNA extraction was performed by addition of 100 m L freshly prepared lysis buffer (10 mM Tris-HCl, pH 7.0, 0.05% SDS, 25 m g/ml Proteinase K (Sigma Aldrich)) directly into the 48-well culture well. The extraction solution was incubated at 37 °C for 60 minutes and then 80 °C for 20 minutes.

Mammalian cell nucleofection

[0406] 560 ng Cas9(D10A)- or base-editor expression plasmid was combined with 240 ng sgRNA-expression plasmid in a volume that did not exceed 1.5 pL. Detailed plasmid maps for plasmids ABEmax and ABEmaxAW are depicted in FIGs. 13A-13B. This combined plasmid mixture was nucleofected in a final volume of 20 pL per sample in a 16-well Nucleocuvette strip (Lonza). K562 cells were nucleofected using the SF Cell Line 4D- Nucleofector X Kit (Lonza) with 5x10 ^s cells per sample (program FF-120), according to the manufacturer’s protocol. U20S cells were nucleofected using the Nucleofector X Kit with 3- 4xl0 ⁵ cells per sample (program DN-100), according to the manufacturer’s protocol. RNA and DNA were isolated 48 hours post-nucleofection. U20S cells were trypsinized and resuspended in PBS, and K562 cells were directly resuspended in PBS before being spun down by centrifugation (800 * g, 2 minutes) to isolate cell pellets. Cell pellets were resuspended in PBS (20 pL) and 3 pL was placed in 50 pL DNA lysis buffer (10 mM Tris- HCl, pH 7.0, 0.05% SDS, 25 p g/ml Proteinase K (Sigma Aldrich)), which was incubated on a heat block at 37 °C for 60 minutes and then 80 °C for 20 minutes. The remaining 17 pL of cells suspended in PBS was pelleted again by centrifugation (800 * g, 2 minutes) and RNA extraction was begun on these pellets with the addition of RLT Plus Lysis Buffer (Qiagen) to the cell pellet. RNA isolation proceeded with the RNEasy PLUS Mini Kit (Qiagen), as described below.

RNA isolation from mammalian cells

[0407] Cells were transfected with the indicated construct, and unless otherwise stated, an sgRNA for the LDLR target site. In the case of HEK293T cells, at the same time as genomic DNA was harvested from one set of wells that had been transfected with editor in

combination with LDLR sgRNA, a second set of wells that had undergone identical treatment were lysed for RNA harvest. RNA isolation was performed with the RNeasy PLUS Mini Kit (Qiagen) according to the manufacturer’s instructions. In short, RNA isolation began with removal of the culture media and washing of the cells with IX DBPS (ThermoFisher). 350 mE RLT Plus buffer (Qiagen) was added to each well, cells were homogenized by pipetting and transferred into a DNA eliminator column and the subsequent binding and washing steps for RNA isolation using the RNEasy columns were performed as recommended by the manufacturer. Upon elution of RNA from the RNEasy column with 45 pL of RNAse free water (Qiagen), 2 pL of RNAseOUT inhibitor (ThermoFisher) was added to prevent RNA degradation and RNA was stored at -80 °C. cDNA generation for targeted RNA amplicon sequencing

[0408] cDNA generation was performed with Superscript IV (ThermoFisher) according to the manufacturer’s instructions. A poly-T primer was used to selectively amplify mRNAs in the cDNA synthesis step. The optional step of RNAse degradation prior to amplification of cDNAs was included to improve the efficiency of PCR. It is noted that this step was particularly important for RSL1D1 PCR.

Preparation of genomic DNA and RNA amplicons for high-throughput sequencing

( HTS )

[0409] A two-step PCR protocol was performed as previously reported (1). In brief, 1 pL of isolated genomic DNA was input into the first round of PCR (PCR1). Phusion U Multiplex Master Mix (Thermo Fisher) was used for both PCR steps. PCR1 was performed with the primers listed in Table 2 for the appropriate sgRNA treatment for 30 cycles with an annealing temperature of 61 °C and an extension time at 72 °C for 15 seconds. Upon verification that PCR1 was successful by running the products on a 2% agarose gel, the barcoding PCR (PCR2) was set up using primers to incorporate barcodes for Illumina sequencing. All primers were ordered from Integrated DNA Technologies (IDT). After PCR2, up to 240 samples with different barcode combinations were combined and purified by gel extraction using the QIAquick Gel Extraction Kit (Qiagen). A second column was used for full removal of agarose and ethidium bromide before the product was quantified using the QBit ssDNA HS Assay Kit (ThermoFisher) and sequenced using an Illumina MiSeq with 220-260-bp single-end reads.

[0410] For RNA, primers were used as listed in Table 2 to amplify the targeted region of cDNA. qPCR was used for all experiments to avoid over- amplification of the cDNA.

RSF1D1 required more PCR cycles (34) than IP90 and CTNNB1 (32 each) using the cycling conditions of 98 °C for 1 minute 30 seconds, then cycles of (98 °C for 10 seconds, 60 °C for 15 seconds, and 72 °C for 15 seconds) followed by a final extension of 2 minutes at 72 °C. No-RT controls and no-input controls were also processed by qPCR and carried forward onto the MiSeq for each experiment. In no instances did either control exceed 2.5% of the number of aligned reads for the particular experiment when compared to the corresponding RNA samples.

[0411] For assessing the number of adenosines within an amplicon that showed greater than 0.1% editing, the % G for each adenosine position was measured and counted in Microsoft Excel using the formula =COUNTIF(C85:HS85, ">0.001"), where C85:HS85 represent the range of cells containing the frequency of bases called as a guanosine when the interrogated nucleoside is an adeosnine (for non-adenosine positions the value within the C85:HS85 range is set to zero).

Analysis ofHTS data for DNA sequencing and targeted amplicon sequencing

[0412] Batch analysis with Crispresso2 (33) was used for targeted amplicon and DNA sequencing analysis(33 ). For DNA analysis, a 30-bp window was used to quantify indels around the DNA nick site. Otherwise, the default parameters were used for analysis. The output file“Reference.NUCLEOTIDE_PERCENTAGE_SUMMARY.txt” was imported into Microsoft Excel for quantification of editing frequencies, and

“CRISPRessoBatch_quantification_of_editing_frequency.txt� � for quantification of indel frequencies.

[0413] For analysis of RNA amplicon editing, no sgRNA flag was used. Instead, the output file“Reference.NUCLEOTIDE_PERCENTAGE_SUMMARY.txt” was imported into Microsoft Excel for analysis of A-to-G editing rates associated with each sample (inosine in RNA is read as a guanosine by polymerases).

[0414] Prism (GraphPad) was used to generate dot plots and bar plots of these data. For instances in the text where means have been calculated across multiple genomic or transcriptomic loci, the standard deviations reported represent the standard deviation of the mean for all biological replicates.

Preparation of RNA libraries for RNA-Seq

[0415] Total RNA was applied to 01igo-dT(25) Dynabeads (Thermofisher) to enrich for polyadenylated transcripts. Stranded RNA-seq libraries were generated from these samples using the PrepX mRNA 48 kit (Takara) on the Apollo 324 followed by barcoding and amplification (12 cycles). Following PCR and bead cleanup with AmpureXP beads

(Beckman Coulter), libraries were visualized on a 2200 TapeStation (Agilent) and quantified using a Library Quantification Kit (KAPA Biosystems) for multiplexing libraries were sequenced on a NextSeq high-throughput flowcell (Illumina) as 150 bp paired-end reads. RNA-Seq data analysis

[0416] Analysis of the transcriptome-wide editing RNA sequencing data was performed as follows. Prior to the analyses described below, Fastq files were generated using Bcl2Fastq2, then trimmed using Trimmomatic version 0.32 to remove adaptor sequences, unpaired sequences, and low-quality bases. Sam alignments were created using HISAT2 to align paired reads from each of three biological replicates to the hg38 human reference genome (UCSC). Precomputed HISAT2 indexes where obtained from

ccb.jhu.edu/software/hisat2/index.shtml. The resulting sam files were sorted and indexed using the samtools software package. Sorted bam alignments from three biological replicates were combined using samtools in order to increase coverage and provide high-quality variant calls. Combined bams were randomly down- sampled to 120 million aligned reads for each condition using a random number generator. The standard error of the mean was found by repeated random down-sampling (from the total number of aligned reads to 120 million aligned reads) and measuring the spread in the variant calling results, which arise from different random sampling events.

[0417] Variant calling was performed using the freebayes software package version 1.2.0 (github.com/ekg/freebayes), an inherently probabilistic measure which accounts for error.

The resulting VCF files were filtered with vcftools to retain only A-to-G variants, common variants, and variant calls with a call quality greater than or equal to 20, thus removing sites with less than a 0.99 probability of corresponding to a position where a real A-to-I edit has occurred. Thus the variant calling performed here considers read depth at a specific adenosine, number of edited reads at that position, mapping quality, and base call quality, and using all of these indicators, returns the probability that there is bona fide RNA editing at that given adenosine.

Effect prediction of the A-to-I variants identified by RNA-Seq

[0418] The Variant Effect Predictor (ENSEMBLE) was used to determine the location within a transcript of each A-to-I edit found in the sample treated with either ABEmax,

Cas9(D10A), or ABEmaxAW, and whether the mutation was synonymous or non- synonymous. The category“downstream gene variant” includes mutations found within a region 5 kb downstream of the start of a gene and the category“upstream gene variant” includes mutations found in the region 5 kb upstream of a protein-coding region.“Intergenic regions” includes A-to-I mutations occurring in non-coding regions more than 5 kb away from the beginning or end of a coding region. SIFT (sift.bii.a-star.edu. sg) was used to predict the outcome of non- synonymous mutations on protein function. High and low confidence calls were made using standard SIFT parameters.

Calculation of the average frequency of A-to-I editing across the transcriptome

[0419] To calculate the average frequency of A-to-I RNA editing among adenosines sequenced in transcriptome-wide sequencing analysis, REDItools were used to quantify the % A-to-I editing in each sample (github.com/tflati/reditools2.0). All nucleotides were removed except adenosines from the analysis, and then removed all adenosines with a read coverage less than 20 to avoid errors due to low sampling. Next, the number of adenosines converted to an inosine in each sample were calculated and this number was divided by the total number of adenosines in the dataset after filtering to obtain a percentage of adenosines edited to inosine in the transcriptome. Calculation of s.e.m. was performed as described in the variant calling section.

Analysis of the transcriptome-wide position ofA-to-I edits

[0420] The transcriptome-wide RNA sequencing data was demultiplexed and aligned as described above. Bins 1,000,0000 nucleotides wide were created along the human genome using bedtools makewindows. The high confidence A-to-I edits were counted per bin using bedtools coverage. Finally, the data was plotted in R using plot_ly and Ideoviz, to show SNP density per bin.

Table 1. Guide RNA sequences. PAM sequences are in italics. For sgRNA LDLR, a 5' G was included in the sgRNA expression cassette to enable efficient expression of the sgRNA from the U6 promoter. This 5' G is indicated as [G].

Table 2. Primers used for amplification of genomic DNA or cDNA for HTS. NO: 27)

Table 3. List of amplicon sequences used for alignment and analysis of HTS reads. Table 4. List of primers used to amplify genomic off-target loci. These primers have been published previously (1, 2) but are listed here for completeness.

Table 5. List of interrogated off-target genomic loci (31 ), with guide RNA sequences and amplicons used for alignment.

Table 6. List of plasmid accession numbers from Addgene.

pCMV-ABEmax(TadA, TadA*E59A) 125662

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EQUIVALENTS AND SCOPE

[0421] In the 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 one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0422] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the disclosure, or embodiments of the disclosure, is/are referred to as comprising particular elements and/or features, certain embodiments of the disclosure or embodiments of the disclosure consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms“comprising” and“containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included.

Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0423] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the present disclosure, the specification shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0424] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.