Deaminase variants with altered sequence preference

Abstract

Provided herein are cytosine deaminase variants with enhanced preference for particular motifs, polynucleotides and vectors encoding the same, and methods of using the same.

Description

[0001] DEAMINASE VARIANTS WITH ALTERED SEQUENCE PREFERENCE

[0002] RELATED APPLICATIONS

[0003] This application claims benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 715,431, filed November 1, 2024, entitled “Deaminase Variants with Altered Sequence Preference”, the entire contents of which are incorporated herein by reference.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (W109470028WO00-SEQ-ARM.xml; Size: 133,289 bytes; and Date of Creation: October 30, 2025) are herein incorporated by reference in their entirety.

[0006] BACKGROUND

[0007] Cytosine deaminases are enzymes that catalyze the deamination of cytosine to produce uracil, which is ultimately converted to thymine during DNA synthesis. Cytosine deaminases possess the ability to edit cytosine bases without inducing double- stranded breaks.

[0008] SUMMARY

[0009] Cytosine deaminases, which deaminate cytosine (C) to produce uracil (U) — which is then converted to thymine (T) following DNA synthesis (e.g., during polymerase chain reaction, PCR) — have utility in base editing applications in particular when fused to RNA-guided nucleases or other sequence- specific DNA binding proteins. For example, apolipoprotein B mRNA editing enzyme 3A (APOBEC3A or A3 A) can direct locus- specific conversion of C’s to T’s when used in combination with a DNA-binding protein (e.g. Cas9). However, these base editors, even those that preferentially target certain xC motifs (where “x” represents a nucleobase), can deaminate all C’s within a 20 base pair sequence targeted by a guide sequence. Consequently, the use of cytosine deaminases can result in promiscuous deamination and off- target effects that may be detrimental and therefore unsuitable for gene editing.

[0010] Provided herein are cytosine deaminase variants that exhibit a preference to deaminate C’s in unique xC contexts (e.g., CC motifs and CG motifs), thereby reducing off-target genome editing both locally and globally. Such cytosine deaminase variants are useful for instances in which accurate and precise gene editing are important, such as in the context of developing therapeutics and / or treating disease.

[0011] #14540497v1 In some aspects, the disclosure provides a cytosine deaminase variant comprising one or more of: (i) an aspartic acid, isoleucine, leucine, or valine at a position corresponding to N24 of SEQ ID NO: 1; (ii) an aspartic acid, cysteine, phenylalanine, glutamic acid, histidine, methionine, asparagine, or threonine at a position corresponding to G27 of SEQ ID NO: 1; (iii) an isoleucine, methionine, or leucine at position corresponding to W98 of SEQ ID NO: 1; (iv) a glycine or asparagine at position corresponding to F102 of SEQ ID NO: 1; (v) a cysteine, isoleucine, asparagine, or valine at a position corresponding to S103 of SEQ ID NO: 1; (vi) an alanine, aspartic acid, methionine, glutamine, glycine, or asparagine at a position corresponding to R128 of SEQ ID NO: 1; (vii) an alanine, cysteine, glutamic acid, phenylalanine, glycine, methionine, tyrosine, histidine, isoleucine, leucine, threonine, valine, or tryptophan at a position corresponding to D 131 of SEQ ID NO: 1; (viii) a cysteine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, lysine, methionine, asparagine, glutamine, arginine, serine, threonine, or valine at a position corresponding to Y132 of SEQ ID NO: 1; (ix) an aspartic acid or glutamic acid at a position corresponding to L135 of SEQ ID NO: 1; (x) an alanine, cysteine, glutamic acid, glycine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, or valine at a position corresponding to Y136 of SEQ ID NO: 1; or (xi) an aspartic acid or glutamic acid at a position corresponding to LI 86.

[0012] In some embodiments, the cytosine deaminase variant comprises an aspartic acid, isoleucine, leucine, or valine at a position corresponding to N24 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an aspartic acid, cysteine, phenylalanine, glutamic acid, histidine, methionine, asparagine, or threonine at a position corresponding to G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a isoleucine, methionine, or leucine at a position corresponding to W98 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a glycine or asparagine at a position corresponding to F102 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a cysteine, isoleucine, asparagine, or valine at a position corresponding to SI 03 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an alanine, aspartic acid, methionine, glutamine, glycine, or asparagine at a position corresponding to R 128 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an alanine, cysteine, glutamic acid, phenylalanine, glycine, methionine, tyrosine, histidine, isoleucine, leucine, threonine, valine, or tryptophan at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a cysteine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, lysine, methionine, asparagine, glutamine, arginine, serine, threonine, or valine at a position corresponding to Y 132

[0013] #14540497v1 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an aspartic acid or glutamic acid at a position corresponding to L135 of SEQ ID NO: 1.

[0014] In some embodiments, the cytosine deaminase variant comprises an alanine, cysteine, glutamic acid, glycine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, or valine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an aspartic acid or glutamic acid at a position corresponding to LI 86.

[0015] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 1-10. In some embodiments, the cytosine deaminase variant comprises an amino acid sequencing having at least 95% identity to any one of SEQ ID NOs: 13-22, 41-49, 51-55, 58-86, 89-106, or 110-111. In some embodiments the cytosine deaminase variant comprises an amino acid sequence of any one of SEQ ID NOs: 13- 22, 41-49, 51-55, 58-86, 89-106, or 110-111. In some embodiments, the cytosine deaminase variant has increased cytosine-cytosine domain preference relative to a cytosine deaminase of SEQ ID NO: 1. In some embodiments, the cytosine deaminase is an apolipoprotein B mRNA editing enzyme, catalytic polypeptide (APOBEC).

[0016] In some aspects, this disclosure provides a cytosine deaminase variant comprising one or more of: (i) glycine, leucine, or glutamic acid at a position corresponding to N23 of SEQ ID NO: 1; (ii) tyrosine, phenylalanine, glycine, histidine, asparagine, or tryptophan at a position corresponding to R28 of SEQ ID NO: 1; (iii) asparagine, alanine, serine, proline, aspartic acid, glutamine, threonine, glycine, lysine, or cysteine at a position corresponding to H29 of SEQ ID NO: 1; (iv) valine at a position corresponding to K60 of SEQ ID NO: 1; (v) glutamic acid at a position corresponding to L62 of SEQ ID NO: 1; (vi) alanine at a position corresponding to S103 of SEQ ID NO: 1; (vii) tryptophan at a position corresponding to VI 10 of SEQ ID NO: 1; (viii) glycine or aspartic acid at a position corresponding to 1129 of SEQ ID NO: 1; (ix) valine at a position corresponding to D 131 of SEQ ID NO: 1; (x) proline at a position corresponding to Y132 of SEQ ID NO: 1; and (xi) lysine or arginine at a position corresponding to P134 of SEQ ID NO: 1.

[0017] In some embodiments, the cytosine deaminase variants comprises glycine, leucine, or glutamic acid at a position corresponding to N23 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variants comprises tyrosine, phenylalanine, glycine, histidine, asparagine, or tryptophan at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variants comprises asparagine, alanine, serine, proline, aspartic acid, glutamine, threonine, glycine, lysine, or cysteine at a position corresponding to H29 of SEQ ID

[0018] #14540497v1 NO: 1. In some embodiments, the cytosine deaminase variants comprises valine at a position corresponding to K60 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variants comprises glutamic acid at a position corresponding to L62 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variants comprises alanine at a position corresponding to SI 03 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variants comprises glycine or aspartic acid at a position corresponding to 1129 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variants comprises tryptophan at a position corresponding to VI 10 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variants comprises valine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variants comprises proline at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variants comprises lysine or arginine at a position corresponding to Pl 34 of SEQ ID NO: 1.

[0019] In some embodiments, the cytosine deaminase variants comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 1-10. In some embodiments, the cytosine deaminase variants comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 11-12, 23-40, 50, 56-67, or 87-88. In some embodiments, the cytosine deaminase variants comprises an amino acid sequence of any one of SEQ ID NOs: 11-12, 23-40, 50, 56-57, or 87-88. In some embodiments, the cytosine deaminase variants comprises increased cytosineguanosine domain preference relative to a cytosine deaminase of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variants comprises the cytosine deaminase is a helix-4 deaminase. In some embodiments, the cytosine deaminase variants comprises the cytosine deaminase is an apolipoprotein B mRNA editing enzyme, catalytic polypeptide (APOBEC). In some aspects, this disclosure provides a base editor comprising a DNA binding protein and the cytosine deaminase variant described herein. In some embodiments, the DNA binding protein is a CRISPR associated (Cas) protein. In some embodiments, the Cas protein is catalytically inactive. In some embodiments, the Cas protein is a catalytically inactive Cas9 protein.

[0020] In some embodiments, the cytosine deaminase variants comprises a peptide linker between the DNA binding domain and the cytosine deaminase. In some aspects, this disclosure provides a polynucleotide encoding a cytosine deaminase variant described herein or a base editor described herein. In some aspects, this disclosure provides a polynucleotide comprising: (a) a nucleic acid encoding a base editor comprising a deaminase variant and a polynucleotide binding protein; and (b) a base editor target site comprising a first polynucleotide binding protein sequence motif and a first deaminase sequence motif.

[0021] #14540497v1 In some aspects, this disclosure provides a polynucleotide comprising: (a) a nucleic acid encoding a base editor comprising a deaminase variant and a polynucleotide binding protein; and (b) a first base editor target site and a second base editor target site that flank the nucleotides encoding the deaminase variant of the nucleic acid; wherein the first base editor target site comprises a first polynucleotide binding protein sequence motif and a first deaminase sequence motif; and wherein the second base editor target site comprises a second polynucleotide binding protein sequence motif and a second deaminase sequence motif. In some embodiments, the polynucleotide binding protein is a catalytically inactive Cas protein. In some embodiments, the polynucleotide comprises a guide RNA comprising a homology region that is complementary to the first polynucleotide binding protein sequence motif and / or the second polynucleotide binding protein sequence motif. In some aspects, this disclosure provides a method of determining sequence preference of a plurality of deaminase variants, the method comprising: (i) transfecting a plurality of cells with a plurality of vectors, each vector of the plurality of vectors comprising: (a) a nucleic acid encoding a base editor comprising a deaminase variant and a polynucleotide binding protein; and (b) a first base editor target site; wherein the first base editor target site comprises a first polynucleotide binding protein sequence motif and a first deaminase sequence motif: (ii) amplifying vectors of the plurality of vectors that comprise an edited first deaminase sequence motif to produce amplicons that comprise a nucleic acid encoding the deaminase variant; and (iii) sequencing the amplicons to determine the sequence preference of the plurality of deaminase variants for the first deaminase sequence motif.

[0022] In some embodiments, sequencing comprises sequencing a contiguous polynucleotide comprising the nucleic acid encoding the deaminase variant and the first base editor target site using long-read sequencing. In some embodiments, the amplifying comprising contacting plurality of vectors with a first primer that is complementary to an edited first deaminase sequence motif. In some embodiments, this disclosure provides a method of determining sequence preference of a plurality of deaminase variants, the method comprising: (i) transfecting a plurality of cells with a plurality of vectors, each vector of the plurality of vectors comprising: (a) a nucleic acid encoding a base editor comprising a deaminase variant and a polynucleotide binding protein; and (b) a first base editor target site and a second base editor target site that flank the nucleotides encoding the deaminase variant of the nucleic acid; wherein the first base editor target site comprises a first polynucleotide binding protein sequence motif and a first deaminase sequence motif; wherein the second base editor target site comprises a second polynucleotide binding protein sequence motif and a second deaminase sequence motif; (ii) amplifying vectors of the plurality of vectors that comprise an edited first deaminase sequence

[0023] #14540497v1 motif and / or an edited second deaminase sequence motif to produce amplicons that comprise a nucleic acid encoding the deaminase variant, the amplifying comprising contacting the plurality of vectors with: (a) a first primer that is complementary to an edited first deaminase sequence motif and a second primer that is complementary to the vector; (b) a first primer that is complementary to an edited second deaminase sequence motif and a second primer that is complementary to the vector; or (c) a first primer that is complementary to an edited first deaminase sequence motif and a second primer that is complementary to an edited second deaminase sequence motif; and (iii) sequencing the amplicons to identify deaminase variants that edited the first deaminase sequence motif and / or the second deaminase sequence motif. In some embodiments, the first deaminase sequence motif and the second deaminase sequence motif are the same motif. In some embodiments, the first deaminase sequence motif and the second deaminase sequence motif are different motifs. In some embodiments, the first deaminase sequence motif and / or the second deaminase sequence motif comprise an AC motif, a CC motif, a GC motif or a TC motif. In some embodiments, amplifying comprises amplifying using RNase H-dependent PCR.

[0024] BRIEF DESCRIPTION OF DRAWINGS

[0025] FIG. 1 is an exemplary schematic of a polynucleotide encoding a single guide RNA (sgRNA) and an open reading frame (ORF) encoding an APOBEC3A variant flanked by xC sequence contexts and a catalytically dead Cas9 (dCas9) protein.

[0026] FIGs. 2A-2B relate to the sequence context preference of cytosine deaminase (APOBEC3A) variants with synonymous mutations (z.e., mutations in the coding sequence that do not change the corresponding amino acid; FIG. 2A) and nonsynonymous mutations (FIG. 2B). Some variants were identified as CC-specific variants (e.g., having a preference for CC motifs; FIG. 2B, y-axis), while others were identified as GC-specific variants (having a preference for GC motifs; FIG. 2B, x-axis).

[0027] FIG. 3 is an exemplary alignment of human APOBEC3A (top sequence, hs = Homo sapieri) with mouse APOBEC (second line, mm = Mus musculus), four other human APOBEC3s (lines 3-6), and AID (line 7). Arrows indicate positions at which mutations were observed to affect the sequence context preference (e.g., increase preference for GC and / or CC motifs). Asterisks indicate cytosine residues that are important for chelating zinc and well conserved across cytosine deaminases. The sequences of FIG. 3 should be considered contiguous across pages.

[0028] #14540497v1 FIG. 4 shows the effects of APOBEC3A substitution mutations on CC and GC preference. Negative values (redder) indicate higher GC preference. Positive values indicate CC preference.

[0029] FIG. 5 is a block plot showing the percentage of fragments mutated in constructs containing a synonymous variant (encoding a WT protein sequence) and in constructs encoding non-synonymous variants at positions 70, 72, 101, 106, and 130, which have been shown to impact deaminase activity.

[0030] FIGs. 6A-6B relate to activity level of APOBEC3A variants compared to WT APOBEC3A (heatmap, FIG. 6B) against six different target sequences (FIG. 6A).

[0031] DETAILED DESCRIPTION

[0032] Cytosine deaminases are enzymes that catalyze the deamination (removal of an amine group) of cytosine to produce uracil, which is then converted into thymine during DNA synthesis, such as by endogenous DNA synthesis or molecular biology techniques such as polymerase chain reaction (PCR). Cytosine deaminases are used for a number of applications in genome engineering and synthetic biology. One such application is the engineering of base editors, in which base-editing enzymes (e.g., a cytosine deaminase) is fused to a DNA-binding protein, such as CRISPR-associated protein, or Cas.

[0033] Currently used cytosine deaminases can promiscuously edit any cytosine within 20 base pairs of a target region (e.g., a region of interest for base editing). Additionally, cytosine deaminases can differ in their preferential sequence context (e.g., sequence motifs), with different deaminases exhibiting differential activity on cytosines in the context of xC, depending on the identity of the bases represented by x and y. Provided herein are cytosine deaminase variants that exhibit a preference for particular cytosine motifs, making them useful for precise and accurate cytosine base editing, thereby reducing off-target genome editing both locally and globally.

[0034] Cytosine deaminases can be broadly classified as being cytosine deaminases or cytidine deaminases. Naturally- occurring cytosine deaminases are primarily found in fungi and bacteria and convert cytosine into uracil. Cytidine deaminases are a group of enzymes that deaminate cytidine or deoxycytidine into uridine or deoxyuridine. Cytidine deaminases are involved in nucleoside metabolism and are present in a variety of organisms, including humans. As used herein, the term cytosine deaminase refers to a cytosine or cytidine deaminase.

[0035] #14540497v1 Subclasses of cytosine deaminases include prokaryotic cytosine deaminases (e.g., bacterial or fungal cytosine deaminases) and eukaryotic cytosine deaminases. Eukaryotic cytosine deaminases include activation-induced cytidine deaminase (AID) and apolipoprotein B mRNA editing enzyme, catalytic polypeptide (APOBEC). APOBEC subfamilies include APOBEC1, APOBEC2, APOBEC3, APOBEC4, and AID e.g., as described in Krishnan et al. PNAS 115.14 (2018): E3201-E3210. Within APOBEC subfamilies there are additional subgenera. For example, the APOBEC3 family comprises APOBEC3A WT (Human), APOB EC-3 isoform 3 WT (Mus musculus), APOBEC-3G WT (Human), APOBEC-3H WT (Human), APOBEC-3F WT (Human), APOBEC-3B WT (Human), and AICDA WT (Human).

[0036] Cytosine deaminases can also be grouped according to structure. One such group is known as the helix-4 deaminases, which can be distinguished from other deaminase groups based on the presence of a beta- strand between helix-4 and helix-5 of the protein structure. Cytosine deaminases belonging to the APOBEC family are considered helix-4 deaminases. In some embodiments, a helix-4 deaminase is ADAT2 (TadA-Tad2), ADAT3 (Tad3), Bd3614, ADAR (Tadl), RibD-like deaminase, XOO_2897 deaminase, OTT_1508 deaminase, AID (activation-induced cytidine deaminase, also called AICDA), APOBEC 1 (apolipoprotein B mRNA editing enzyme catalytic deaminase 1), APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D / E, APOBEC3F, APOBEC3G, APOBEC3H, or APOBEC4.

[0037] In some embodiments, this disclosure provides a cytosine deaminase variant comprising a mutation compared to a wild-type cytosine deaminase. In some embodiments, a wild-type cytosine deaminase is a helix-4 cytosine deaminase. In some embodiments, a wild-type cytosine deaminase is an APOBEC family cytosine deaminase. In some embodiments, a wild-type cytosine deaminase is an APOBEC 1 family cytosine deaminase. In some embodiments, a wildtype cytosine deaminase is an APOBEC2 family cytosine deaminase. In some embodiments, a wild-type cytosine deaminase is an APOBEC3 family cytosine deaminase. In some embodiments, a wild-type cytosine deaminase is an APOBEC4 family cytosine deaminase. In some embodiments, a wild-type cytosine deaminase is an AID family cytosine deaminase. In some embodiments, a wild-type cytosine deaminase is an APOBEC3A family cytosine deaminase. In some embodiments, a wild-type cytosine deaminases comprises the sequence of any one of SEQ ID NOs: 1-10. In some embodiments, a cytosine deaminase variant comprises a mutation compared to a wild-type cytosine deaminase comprising the sequence of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a mutation compared to a wild-type cytosine deaminase comprising the sequence of SEQ ID NO: 1

[0038] #14540497v1 In some embodiments, a cytosine deaminase variant comprises an amino acid sequence of at least 95% identity to SEQ ID NO: 1 and one or more mutations compared to SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises an amino acid sequence of at least 95% identity to any one of SEQ ID NOs: 1-10 and one or more mutations compared to SEQ ID NO: 1-10.

[0039] Some embodiments relate to amino acid sequences or nucleotide sequences having some percentage identity to a reference amino acid sequence or reference nucleotide sequence, respectively. As used herein, the term “identity” refers to the degree to which two or more sequences (e.g., amino acid sequences, nucleotide sequences) are related, as determined by the number of matches between each sequence. “Percent (%) identity” refers to the percentage of residues or nucleotides in a first amino acid or first nucleic acid sequence that are identical to the residues in a second amino acid sequence or second nucleic acid sequence. The skilled artisan will understand that determining percent identity may require a step of aligning a first and second sequence and / or introducing gaps. Several algorithms for aligning sequences and / or determining percent identity between sequences are known in the art. For example, EMBOSS NEEDLE (e.g., as described in Madeira F. et al., Nucleic Acids Research. 2024 Jul;52(Wl):W521-W525. PMID: 38597606) can be used to align sequences and determine percent identity between sequences.

[0040] In some embodiments, this disclosure provides a cytosine deaminase variant comprising a mutation e.g., a substitution, an insertion, or a deletion) at a position corresponding to a reference a cytosine deaminase. A cytosine deaminase variant (e.g., an APOBEC3A variant) having a “position corresponding to” a reference amino acid sequence (e.g., a reference APOBEC3A amino acid sequence like SEQ ID NO: 1) refers to a particular amino acid in the cytosine deaminase variant relative to the reference cytosine deaminase variant.

[0041] A cytosine deaminase variant (e.g., an APOBEC3A variant) may be identified relative to a reference sequence. In other words, the reference sequence can be used as a standard for mapping mutations between different cytosine deaminase variants (e.g., different APOBEC3A variants). This mapping may be used to compare cytosine deaminase variants comprising insertions or deletions that shift the location of a mutation relative to the reference sequence). In some embodiments, determining a position of a cytosine deaminase variant corresponding to a reference sequence comprises aligning the reference cytosine deaminase sequence (e.g., wildtype APOBEC3A) and the cytosine deaminase variant sequence (e.g., an APOBEC3A variant) (e.g., using Clustal Omega as described in Madeira et al., Nucleic Acids Res. 2024 Jul;52(Wl) W521-W525. PMID: 38597606). In some embodiments, this is determined using a structural

[0042] #14540497v1 alignment (e.g., as described in Bittrich et al., Bioinformatics, Volume 40, Issue 6, June 2024, doi.org / 10.1093 / bioinformatics / btae370).

[0043] Directly below are four examples of protein variants comprising a methionine at a position corresponding to Y38 of a reference sequence (Ref).

[0044] In a first example, the reference sequence (Ref) and the target sequence (Tar) align at every amino acid between positions 4 and 37 of the reference sequence. Position 38 of the target sequence corresponds to position 38 of the reference sequence. Position 38 of 112> sequence is methionine. Position 38 of the reference sequence is tyrosine.( SEQ ID N0.113)Re f 4 DTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPY 38

[0045] Tar 4 DTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPM 38

[0046] In a second example, the target sequence has a three amino acid deletion and a tyrosine to methionine substitution relative to the reference sequence. Because of the deletion, position 35 of the target sequence corresponds to position 38 of the reference sequence. Position 35 of (SEQ ID NO : 112 ) the target sequence is methionine. Position 38 of the reference sequence is tyrosine.

[0047] ( SEQ ID NO : 114 )

[0048] Re f 4 -DTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPY 38

[0049] Tar 4 -DT - TEDGKPVIRIFKKENGEFKIEYDRTFEPM 35

[0050] In a third example, the target sequence has a four amino acid insertion and a tyrosine to methionine substitution relative to the reference sequence. Because of the insertion, position 42 of the target sequence corresponds to position 38 of the reference sequence. Position 42 of the ( SEQ ID NO : 112 ) target sequence is methionine. Position 38 of the reference sequence is tyrosine.

[0051] ( SEQ ID NO : 115 ) Re f 4 DTDYITED - GKPVIRIFKKENGEFKIEYDRTFEPY 38

[0052] Tar 4 DTDYITEDQRQRGKPVIRIFKKENGEFKIEYDRTFEPM 42

[0053] In a fourth example, the target sequence differs in sequence at amino position 38, 42, 43, and 44. Position 38 of the target sequence corresponds to position 38 of the reference sequence. Position 38 of the target sequence is methionine. Position 38 of the reference seqii nb® N0 : 116> tyrosine.(SEQ ID N0.117 )

[0054] Re f 4 DTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPYGGYSTV 44

[0055] Tar 4 DTDYITEDGKPVIRIFKKENGEFKIEYDRTFEPMGGYRSE 44

[0056] In some instances, two different protein sequences may not correspond with one another.

[0057] For example, this may occur when the sequence identity or structural identity between two

[0058] #14540497v1 protein sequences is so low that an alignment cannot be made or the two different sequences do not align at a given position.

[0059] Cytosine deaminase variants described herein may have a preference for a specific polynucleotide sequence motif (e.g., a GC or CC motif). For example, a cytosine deaminase variant with a preference for a CC motif, in the presence of multiple CC motifs and multiple GC motifs, would deaminate CC motifs more frequently than it would deaminate GC motifs. In another example, a cytosine deaminase with a preference for a GC motif, in the presence of multiple GC motifs and multiple CC motifs, would deaminate GC motifs more frequently than it would deaminate CC motifs. In some embodiments, the preference of a cytosine deaminase is determined by comparing a first probability value (p-value) that the cytosine deaminase variant only deaminates a first motif (e.g., a CC) to a second p-value that the cytosine deaminase variant only deaminates a second motif (e.g., a GC motif) (p-values may be determined from experimental data e.g., as described in the Examples). In some embodiments, if the first p-value is less than the second p-value (e.g., at least 2 times less, at least 5 times less, at least 10 times less, at least 100 times less, at least 103times less, at least 104times less, at least 105times less or at least 106times less) then the deaminase has a preference for the first motif.

[0060] In some embodiments, this disclosure provides cytosine deaminase variants with increased preference for a CC sequence context (also called a CC-specific cytosine deaminase). Such cytosine deaminase variants have strong preference for specifically deaminating CC motifs and are therefore less prone to off-target deamination when used to deaminate cytosines in a CC sequence context. In some embodiments, a cytosine deaminase variant with increased preference for CC motifs comprises a mutation at a position corresponding to one or more of N24, G27, W98, F102, S103, R128, D131, Y132, L135, Y136, or L186 of SEQ ID NO: 1. In some embodiments the cytosine deaminase variant does not comprise a mutation at a position corresponding to 70, 72, 101, 106, and 130 of SEQ ID NO: 1.

[0061] N24 Cytosine Deaminase Variants

[0062] In some embodiments, a cytosine deaminase variant comprises a mutation at a position corresponding to N24 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a mutation at a position corresponding to N24 of SEQ ID NO: 1 and has at least 95% identity to any one of SEQ ID NOs: 1-7, 9-10.

[0063] In some embodiments, this disclosure provides a cytosine deaminase variant comprising an aspartic acid (also called aspartate), isoleucine, leucine, or valine at a position corresponding to N24 of SEQ ID NO: 1 (e.g., N24D, N24I, N24L, or N24V in a cytosine deaminase

[0064] #14540497v1 corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides an aspartic acid, isoleucine, leucine, or valine at a position corresponding to N24 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) relative to any one of SEQ ID NOs: 1-7, 9-10. In some embodiments, this disclosure provides an aspartic acid, isoleucine, leucine, or valine at position N24 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-7, 9-10 and an aspartic acid, isoleucine, leucine, or valine at a position corresponding to N24 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an aspartic acid, isoleucine, leucine, or valine at a position corresponding to N24 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an aspartic acid, isoleucine, leucine, or valine at position N24 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises an aspartic acid, isoleucine, leucine, or valine at position N24 of SEQ ID NO: 1.

[0065] In some embodiments, the cytosine deaminase variant comprises an aspartic acid at a position corresponding to N24 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an isoleucine at a position corresponding to N24 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a leucine at a position corresponding to N24 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a valine at a position corresponding to N24 of SEQ ID NO: 1.

[0066] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-7, 9-10 and an aspartic acid at a position corresponding to N24 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-7, 9-10 and an isoleucine at a position corresponding to N24 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-7, 9-10 and a leucine at a position corresponding to N24 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least

[0067] #14540497v1 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1- 7, 9-10 and a valine at a position corresponding to N24 of SEQ ID NO: 1.

[0068] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an aspartic acid at position N24 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an isoleucine at position N24 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a leucine at position N24 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a valine at position N24 of SEQ ID NO: 1.

[0069] In some embodiments, the cytosine deaminase variant comprises an aspartic acid at position N24 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an isoleucine at position N24 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a leucine at position N24 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a valine at position N24 of SEQ ID NO: 1.

[0070] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 13-16. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 13-16. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 13- 16. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 13-16. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 13-16. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence of any one of SEQ ID NOs: 13-16.

[0071] G27 Cytosine Deaminase Variants

[0072] In some embodiments, a cytosine deaminase variant comprises a mutation corresponding to position G27 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant

[0073] #14540497v1 comprises a mutation at a position corresponding to G27 of SEQ ID NO: 1 and has at least 95% identity to any one of SEQ ID NOs: 1-10.

[0074] In some embodiments, this disclosure provides a cytosine deaminase variant comprising a cysteine, phenylalanine, histidine, methionine, asparagine, or threonine at a position corresponding to G27 of SEQ ID NO: 1 (e.g., G27C, G27F, G27H, G27M, G27N, or G27T in a cytosine deaminase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides a cysteine, phenylalanine, histidine, methionine, asparagine, or threonine at a position corresponding to G27 of SEQ ID NO: 1 and one, two, three, or four additional mutations (e.g., amino acid substitutions) relative to any one of SEQ ID NOs: 1-10. In some embodiments, this disclosure provides a cytosine deaminase variant comprising a cysteine, phenylalanine, histidine, methionine, asparagine, or threonine at position G27 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1- 10 and a cysteine, phenylalanine, histidine, methionine, asparagine, or threonine at a position corresponding to G27 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising a cysteine, phenylalanine, histidine, methionine, asparagine, or threonine at a position corresponding to G27 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising a cysteine, phenylalanine, histidine, methionine, asparagine, or threonine at position G27 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a cysteine, phenylalanine, histidine, methionine, asparagine, or threonine at position G27 of SEQ ID NO: 1.

[0075] In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a phenylalanine at a position corresponding to G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a histidine at a position corresponding to G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a methionine at a position corresponding to G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a asparagine at a position corresponding to G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a threonine at a position corresponding to G27 of SEQ ID NO: 1.

[0076] #14540497v1 In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a cysteine at a position corresponding to G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a phenylalanine at a position corresponding to SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1- 10 and a histidine at a position corresponding to G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a methionine at a position corresponding to G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an asparagine at a position corresponding to G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a threonine at a position corresponding to G27 of SEQ ID NO: 1.

[0077] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cysteine at position G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a phenylalanine at position G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a histidine at position G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a methionine at position G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a asparagine at position G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a threonine at position G27 of SEQ ID NO: 1.

[0078] #14540497v1 In some embodiments, the cytosine deaminase variant comprises a cysteine at position G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a phenylalanine at position G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a histidine at position G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a methionine at position G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a asparagine at position G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a threonine at position G27 of SEQ ID NO: 1.

[0079] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 17-22. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 17-22. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 17- 22. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 17-22. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 17-22. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence of any one of SEQ ID NOs: 17-22.

[0080] W98 Cytosine Deaminase Variants

[0081] In some embodiments, a cytosine deaminase variant comprises a mutation corresponding to position W98 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a mutation at a position corresponding to W98 of SEQ ID NO: 1 and has at least 95% identity to any one of SEQ ID NOs: 1-10.

[0082] In some embodiments, this disclosure provides a cytosine deaminase variant comprising a isoleucine, methionine, or leucine at a position corresponding to W98 of SEQ ID NO: 1 (e.g., W98F, W98I, W98M, or W98L in a cytosine deaminase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides a isoleucine, methionine, or leucine at a position corresponding to W98 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) relative to any one of SEQ ID NOs: 1-10. In some embodiments, this disclosure provides a cytosine deaminase variant comprising a isoleucine, methionine, or leucine at position W98 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant

[0083] #14540497v1 comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a isoleucine, methionine, or leucine at a position corresponding to W98 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising a isoleucine, methionine, or leucine at a position corresponding to W98 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising a isoleucine, methionine, or leucine at position W98 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a isoleucine, methionine, or leucine at position W98 of SEQ ID NO: 1.

[0084] In some embodiments, the cytosine deaminase variant comprises a phenylalanine at a position corresponding to W98 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an isoleucine at a position corresponding to W98 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a methionine at a position corresponding to W98 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a leucine at a position corresponding to W98 of SEQ ID NO: 1.

[0085] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a phenylalanine at a position corresponding to W98 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a isoleucine at a position corresponding to W98 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1- 10 and a methionine at a position corresponding to W98 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1- 10 and a leucine at a position corresponding to W98 of SEQ ID NO: 1.

[0086] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a phenylalanine at position W98 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a isoleucine at position W98 of SEQ

[0087] #14540497v1 ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a methionine at position W98 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a leucine at position W98 of SEQ ID NO: 1.

[0088] In some embodiments, the cytosine deaminase variant comprises a phenylalanine at position W98 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an isoleucine at position W98 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a methionine at position W98 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a leucine at position W98 of SEQ ID NO: 1.

[0089] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 41-42 or 110-111. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 41-42 or 110-111. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 41-42 or 110-111. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 41- 42 or 110-111. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 41-42 or 110-111. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence of any one of SEQ ID NOs: 41-42 or 110-111.

[0090] F102 Cytosine Deaminase Variants

[0091] In some embodiments, a cytosine deaminase variant comprises a mutation corresponding to position F102 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a mutation at a position corresponding to F102 of SEQ ID NO: 1 and has at least 95% identity to any one of SEQ ID NOs: 1-10.

[0092] In some embodiments, this disclosure provides a cytosine deaminase variant comprising a glycine or asparagine at a position corresponding to F102 of SEQ ID NO: 1 (e.g., F102G or F102N in a cytosine deaminase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides a glycine or asparagine at a position corresponding to F102 of SEQ ID NO:

[0093] #14540497v1 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) relative to any one of SEQ ID NOs: 1-10. In some embodiments, this disclosure provides a cytosine deaminase variant comprising a glycine or asparagine at position F102 of SEQ ID NO: 1 and one, two, three or four additional mutations e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1- 10 and a glycine or asparagine at a position corresponding to F102 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising a glycine or asparagine at a position corresponding to F102 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising a glycine or asparagine at position Fl 02 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a glycine or asparagine at position F102 of SEQ ID NO: 1.

[0094] In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to F102 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an asparagine at a position corresponding to F102 of SEQ ID NO: 1.

[0095] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a glycine at a position corresponding to Fl 02 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an asparagine at a position corresponding to F102 of SEQ ID NO: 1.

[0096] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glycine at position F102 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an asparagine at position F102 of SEQ ID NO: 1.

[0097] In some embodiments, the cytosine deaminase variant comprises a glycine at position F102 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an asparagine at position Fl 02 of SEQ ID NO: 1.

[0098] #14540497v1 In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 43-44. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 43-44. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 43- 44. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 43-44. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 43-44. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence of any one of SEQ ID NOs: 43-44.

[0099] S103 Cytosine Deaminase Variants

[0100] In some embodiments, a cytosine deaminase variant comprises a mutation corresponding to position SI 03 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a mutation at a position corresponding to S103 of SEQ ID NO: 1 and has at least 95% identity to SEQ ID NO: 1 or 6.

[0101] In some embodiments, this disclosure provides a cytosine deaminase variant comprising alanine, asparagine, cysteine, isoleucine, or valine at a position corresponding to S103 of SEQ ID NO: 1 (e.g., S103A, S103N, S103C, S 1031, or S103V in a cytosine deaminase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides an alanine, asparagine, cysteine, isoleucine, or valine at a position corresponding to S103 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) relative to SEQ ID NO: 1 or 6. In some embodiments, this disclosure provides a cytosine deaminase variant comprising alanine, asparagine, cysteine, isoleucine, or valine at position S103 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity of SEQ ID NO: 1 or 6 and an alanine, asparagine, cysteine, isoleucine, or valine at a position corresponding to S103 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising alanine, asparagine, cysteine, isoleucine, or valine at a position corresponding to SI 03 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least

[0102] #14540497v1 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising alanine, asparagine, cysteine, isoleucine, or valine at position SI 03 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises alanine, asparagine, cysteine, isoleucine, or valine at position SI 03 of SEQ ID NO: 1.

[0103] In some embodiments, the cytosine deaminase variant comprises alanine at a position corresponding to SI 03 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises asparagine at a position corresponding to S103 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises cysteine at a position corresponding to SI 03 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises isoleucine at a position corresponding to SI 03 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises valine at a position corresponding to S103 of SEQ ID NO: 1.

[0104] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 or 6 and an alanine at a position corresponding to SI 03 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 or 6 and an asparagine at a position corresponding to SI 03 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 or 6 and a cysteine at a position corresponding to SI 03 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 or 6 and an isoleucine at a position corresponding to SI 03 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 or 6 and a valine at a position corresponding to S103 of SEQ ID NO: 1.

[0105] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine at position SI 03 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine at position S103 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and

[0106] #14540497v1 cysteine at position SI 03 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and isoleucine eat position S103 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and valine at position SI 03 of SEQ ID NO: 1.

[0107] In some embodiments, the cytosine deaminase variant comprises alanine at position SI 03 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises alanine at position SI 03 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises cystine at position SI 03 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises isoleucine at position SI 03 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises valine at position S103 of SEQ ID NO: 1.

[0108] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 45-49. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 45-49. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 45- 49. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 45-49. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 45-49. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence of any one of SEQ ID NOs: 45-49.

[0109] R128 Cytosine Deaminase Variants

[0110] In some embodiments, a cytosine deaminase variant comprises a mutation corresponding to position R128 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a mutation at a position corresponding to R128 of SEQ ID NO: 1 and has at least 95% identity to any one of SEQ ID NOs: 1-10.

[0111] In some embodiments, this disclosure provides a cytosine deaminase variant comprising alanine, aspartic acid, glycine, methionine, or glutamine at a position corresponding to R 128 of SEQ ID NO: 1 (e.g., R128A, R128D, R128G, R128M, or R128Q in a cytosine deaminase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides an alanine,

[0112] #14540497v1 aspartic acid, glycine, methionine, or glutamine at a position corresponding to R128 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) relative to any one of SEQ ID NOs: 1-10. In some embodiments, this disclosure provides a cytosine deaminase variant comprising alanine, aspartic acid, glycine, methionine, or glutamine at position R128 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an alanine, aspartic acid, glycine, methionine, or glutamine at a position corresponding to R128 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine, aspartic acid, glycine, methionine, or glutamine at a position corresponding to R128 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine, aspartic acid, glycine, methionine, or glutamine at position R128 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises alanine, aspartic acid, glycine, methionine, or glutamine at position R128 of SEQ ID NO: 1.

[0113] In some embodiments, the cytosine deaminase variant comprises alanine at a position corresponding to R 128 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises aspartic acid at a position corresponding to R 128 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises glycine at a position corresponding to R 128 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises methionine at a position corresponding to R128 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises glutamine at a position corresponding to R 128 of SEQ ID NO: 1.

[0114] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an alanine at a position corresponding to R 128 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an aspartic acid at a position corresponding to R128 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1- 10 and a glycine at a position corresponding to R128 of SEQ ID NO: 1. In some embodiments,

[0115] #14540497v1 the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a methionine at a position corresponding to R128 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a glutamine at a position corresponding to R128 of SEQ ID NO: 1.

[0116] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine at position R 128 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and aspartic acid at position R128 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glycine at position R 128 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and methionine at position R128 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamine at position R128 of SEQ ID NO: 1.

[0117] In some embodiments, the cytosine deaminase variant comprises alanine at position R 128 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises aspartic acid at position R 128 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises glycine at position R 128 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises methionine at position R 128 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises glutamine at position R128 of SEQ ID NO: 1.

[0118] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 51-55. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 51-55. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 51- 55. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 51-55. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99.5% identity to

[0119] #14540497v1 any one of SEQ ID NOs: 51-55. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence of any one of SEQ ID NOs: 51-55.

[0120] D131 Cytosine Deaminase Variants

[0121] In some embodiments, a cytosine deaminase variant comprises a mutation corresponding to position D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a mutation at a position corresponding to D 131 of SEQ ID NO: 1 and has at least 95% identity to any one of SEQ ID NOs: 1-10.

[0122] In some embodiments, this disclosure provides a cytosine deaminase variant comprising valine, histidine, isoleucine, leucine, tryptophan, alanine, cysteine, glutamic acid, phenylalanine, glycine, methionine, lysine, or threonine at a position corresponding to D131 of SEQ ID NO: 1 (e.g., D131V, D131H, D131I, D131L, D131W, D131A, D131C, D131E, D131F, D131G, D131M, D131K, or D131T in a cytosine deaminase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides a valine, histidine, isoleucine, leucine, tryptophan, alanine, cysteine, glutamic acid, phenylalanine, glycine, methionine, lysine, or threonine at a position corresponding to D 131 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) relative to any one of SEQ ID NOs: 1-10. In some embodiments, this disclosure provides a cytosine deaminase variant comprising valine, histidine, isoleucine, leucine, tryptophan, alanine, cysteine, glutamic acid, phenylalanine, glycine, methionine, lysine, or threonine at position D131 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a valine, histidine, isoleucine, leucine, tryptophan, alanine, cysteine, glutamic acid, phenylalanine, glycine, methionine, lysine, or threonine at a position corresponding to D131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising valine, histidine, isoleucine, leucine, tryptophan, alanine, cysteine, glutamic acid, phenylalanine, glycine, methionine, lysine, or threonine at a position corresponding to D 131 of SEQ ID NO: 1.

[0123] In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising valine at position D131 of SEQ ID NO: 1. In

[0124] #14540497v1 some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising histidine at position D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising isoleucine at position D131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising leucine at position D131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising tryptophan at position D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising alanine at position D131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising cysteine at position D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising glutamic acid at position D131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising phenylalanine at position D131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising glycine at position D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising methionine at position D131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising lysine at position D131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least

[0125] #14540497v1 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising threonine at position D 131 of SEQ ID NO: 1.

[0126] In some embodiments, a cytosine deaminase variant comprises valine at position D131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises histidine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises isoleucine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises leucine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises tryptophan at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises alanine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises cysteine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises glutamic acid at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises phenylalanine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises glycine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises methionine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises lysine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises threonine at a position corresponding to D 131 of SEQ ID NO: 1.

[0127] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a valine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1- 10 and a histidine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an isoleucine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a leucine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a tryptophan at a

[0128] #14540497v1 position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an alanine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a cysteine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a glutamic acid at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a phenylalanine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a glycine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a methionine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a lysine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a threonine at a position corresponding to D 131 of SEQ ID NO: 1.

[0129] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and valine at position D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and histidine at position D131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and isoleucine at position D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%,

[0130] #14540497v1 or at least 99%) identity with SEQ ID NO: 1 and leucine at position D131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tryptophan at position D131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine at position D131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and cysteine at position D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamic acid at position D131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and phenylalanine at position D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glycine at position D131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and methionine at position D131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and lysine at position D131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and threonine at position D 131 of SEQ ID NO: 1.

[0131] In some embodiments, a cytosine deaminase variant comprises valine at position D131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises histidine at position D131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises isoleucine at position D131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises leucine at position D131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises tryptophan at position D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises alanine at position D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises cysteine at position D131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises glutamic acid at position D131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant

[0132] #14540497v1 comprises phenylalanine at position D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises glycine at position D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises methionine at position D 131 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises lysine at position D131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an threonine at position D131 of SEQ ID NO: 1.

[0133] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 58-70. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 58-70. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 58- 70. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 58-70. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 58-70. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence of any one of SEQ ID NOs: 58-70.

[0134] Y132 Cytosine Deaminase Variants

[0135] In some embodiments, a cytosine deaminase variant comprises a mutation corresponding to position Y132 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a mutation at a position corresponding to Y132 of SEQ ID NO: 1 and has at least 95% identity to any one of SEQ ID NOs: 1-10.

[0136] In some embodiments, this disclosure provides a cytosine deaminase variant comprising cysteine, glycine, histidine, lysine, asparagine, glutamine, arginine, serine, proline, aspartic acid, glutamic acid, methionine, valine, isoleucine, or threonine at a position corresponding to Y132 of SEQ ID NO: 1 (e.g., Y132A, Y132C, Y132G, Y132H, Y132K, Y132N, Y132Q, Y132R, Y132S, Y132P, Y132D, Y132E, Y132M, Y132V, Y132I, or Y132T in a cytosine deaminase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides an cysteine, glycine, histidine, lysine, asparagine, glutamine, arginine, serine, proline, aspartic acid, glutamic acid, methionine, valine, isoleucine, or threonine at a position corresponding to Y132 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) relative to any one of SEQ ID NOs: 1-10. In some embodiments, this disclosure provides a cytosine deaminase variant comprising cysteine, glycine, histidine, lysine, asparagine, glutamine,

[0137] #14540497v1 arginine, serine, proline, aspartic acid, glutamic acid, methionine, valine, isoleucine, or threonine at position Y132 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a cysteine, glycine, histidine, lysine, asparagine, glutamine, arginine, serine, proline, aspartic acid, glutamic acid, methionine, valine, isoleucine, or threonine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising cysteine, glycine, histidine, lysine, asparagine, glutamine, arginine, serine, proline, aspartic acid, glutamic acid, methionine, valine, isoleucine, or threonine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising cysteine, glycine, histidine, lysine, asparagine, glutamine, arginine, serine, proline, aspartic acid, glutamic acid, methionine, valine, isoleucine, or threonine at position Y132 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises cysteine, glycine, histidine, lysine, asparagine, glutamine, arginine, serine, proline, aspartic acid, glutamic acid, methionine, valine, isoleucine, or threonine at position Y132 of SEQ ID NO: 1.

[0138] In some embodiments, the cytosine deaminase variant comprises alanine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises cysteine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises glycine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises histidine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises lysine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises asparagine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises glutamine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises arginine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises serine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises proline at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises

[0139] #14540497v1 aspartic acid at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises glutamic acid at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises methionine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises valine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises isoleucine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises threonine at a position corresponding to Y132 of SEQ ID NO: 1.

[0140] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an alanine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a cysteine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1- 10 and a glycine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a histidine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a lysine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an asparagine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a glutamine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an arginine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a serine at a position

[0141] #14540497v1 corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a proline at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an aspartic acid at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a glutamic acid at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a methionine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a valine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an isoleucine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a threonine at a position corresponding to Y 132 of SEQ ID NO: 1.

[0142] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and alanine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and cysteine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glycine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and histidine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with

[0143] #14540497v1 SEQ ID NO: 1 and lysine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and asparagine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and arginine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and serine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and proline at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and aspartic acid at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamic acid at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and methionine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and valine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and isoleucine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and threonine at position Y132 of SEQ ID NO: 1.

[0144] In some embodiments, the cytosine deaminase variant comprises alanine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises cysteine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises glycine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises histidine at position Y132 of SEQ ID NO: 1. In some

[0145] #14540497v1 embodiments, the cytosine deaminase variant comprises lysine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises asparagine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises glutamine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises arginine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises serine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises proline at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises aspartic acid at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises glutamic acid at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises methionine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises valine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises isoleucine at position Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises threonine at position Y132 of SEQ ID NO: 1.

[0146] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 71-86. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 71-86. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 71- 86. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 71-86. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 71-86. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence of any one of SEQ ID NOs: 71-86.

[0147] L135 Cytosine Deaminase Variants

[0148] In some embodiments, a cytosine deaminase variant comprises a mutation corresponding to position LI 35 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a mutation at a position corresponding to L135 of SEQ ID NO: 1 and has at least 95% identity to any one of SEQ ID NOs: 1-10.

[0149] In some embodiments, this disclosure provides a cytosine deaminase variant comprising aspartic acid or glutamic acid at a position corresponding to L135 of SEQ ID NO: 1 (e.g.,

[0150] #14540497v1 L135D or L135E in a cytosine deaminase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides an aspartic acid or glutamic acid at a position corresponding to L135 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) relative to any one of SEQ ID NOs: 1-10. In some embodiments, this disclosure provides a cytosine deaminase variant comprising aspartic acid or glutamic acid at position L135 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an aspartic acid glutamic acid at a position corresponding to LI 35 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising aspartic acid or glutamic acid at a position corresponding to LI 35 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising aspartic acid or glutamic acid at position L135 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises aspartic acid or glutamic acid at position L135 of SEQ ID NO: 1.

[0151] In some embodiments, the cytosine deaminase variant comprises aspartic acid at a position corresponding to LI 35 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises glutamic acid at a position corresponding to L135 of SEQ ID NO: 1.

[0152] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an aspartic acid at a position corresponding to L135 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a glutamic acid at a position corresponding to L135 of SEQ ID NO: 1.

[0153] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and aspartic acid at position LI 35 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glutamic acid at position L135 of SEQ ID NO: 1.

[0154] #14540497v1 In some embodiments, the cytosine deaminase variant comprises aspartic acid at position L135 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises glutamic acid at position LI 35 of SEQ ID NO: 1.

[0155] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 89-90. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 89-90. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 89- 90. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 89-90. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 89-90. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence of any one of SEQ ID NOs: 89-90.

[0156] Y136 Cytosine Deaminase Variants

[0157] In some embodiments, a cytosine deaminase variant comprises a mutation corresponding to position Y136 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a mutation at a position corresponding to Y136 of SEQ ID NO: 1 and has at least 95% identity to any one of SEQ ID NOs: 1-10.

[0158] In some embodiments, this disclosure provides a cytosine deaminase variant comprising an alanine, cysteine, glutamic acid (also called glutamate), glycine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, or valine at a position corresponding to Y136 of SEQ ID NO: 1 (e.g., Y136A, Y136C, Y136E, Y136G, Y136I, Y136K, Y136L, Y136M, Y136N, Y136Q, Y136R, Y136S, Y136T, or Y136V in a cytosine deaminase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides an alanine, cysteine, glutamic acid, glycine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, or valine at a position corresponding to Y 136 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) relative to any one of SEQ ID NOs: 1-10. In some embodiments, this disclosure provides a cytosine deaminase variant comprising an alanine, cysteine, glutamic acid, glycine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, or valine at position Y136 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises:

[0159] #14540497v1 an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an alanine, cysteine, glutamic acid, glycine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, or valine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising an alanine, cysteine, glutamic acid, glycine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, or valine at a position corresponding to Y 136 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising an alanine, cysteine, glutamic acid, glycine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, or valine at position Y136 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises an alanine, cysteine, glutamic acid, glycine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, or valine at position Y136 of SEQ ID NO: 1.

[0160] In some embodiments, the cytosine deaminase variant comprises an alanine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a glutamic acid at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a isoleucine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a lysine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a leucine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a methionine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an asparagine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an glutamine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an arginine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a serine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a

[0161] #14540497v1 serine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a threonine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a valine at a position corresponding to Y136 of SEQ ID NO: 1.

[0162] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an alanine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a cysteine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1- 10 and a glutamic acid at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1- 10 and a glycine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an isoleucine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a lysine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a methionine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an asparagine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a glutamine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an arginine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine

[0163] #14540497v1 deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a serine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a threonine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a valine at a position corresponding to Y 136 of SEQ ID NO: 1.

[0164] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an alanine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cysteine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamic acid at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glycine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a isoleucine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a lysine at position Y 136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a leucine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an methionine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an asparagine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO:

[0165] #14540497v1 1 and a glutamine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an arginine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a serine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a threonine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a valine at position Y136 of SEQ ID NO: 1.

[0166] In some embodiments, the cytosine deaminase variant comprises an alanine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a cysteine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a glutamic acid at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a glycine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a lysine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a leucine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a methionine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an asparagine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a glutamine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an arginine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a serine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a threonine at position Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a valine at position Y136 of SEQ ID NO: 1.

[0167] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 91-104. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 91-104. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 91- 104. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 91-104. In some embodiments, the

[0168] #14540497v1 cytosine deaminase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 91-104. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence of any one of SEQ ID NOs: 91-104.

[0169] L186 Cytosine Deaminase Variants

[0170] In some embodiments, a cytosine deaminase variant comprises a mutation corresponding to position LI 86 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a mutation at a position corresponding to LI 86 of SEQ ID NO: 1 and has at least 95% identity to any one of SEQ ID NOs: 1-10.

[0171] In some embodiments, this disclosure provides a cytosine deaminase variant comprising an aspartic acid or glutamic acid at a position corresponding to L186 of SEQ ID NO: 1 (e.g., L186D or L186E in a cytosine deaminase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides an aspartic acid, isoleucine, leucine, or valine at a position corresponding to LI 86 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) relative to any one of SEQ ID NOs: 1-10. In some embodiments, this disclosure provides a cytosine deaminase variant comprising an aspartic acid or glutamic acid at position LI 86 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an aspartic acid or glutamic acid at a position corresponding to LI 86 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising an aspartic acid or glutamic acid at a position corresponding to LI 86 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising an aspartic acid or glutamic acid at position LI 86 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises an aspartic acid or glutamic acid at position LI 86 of SEQ ID NO: 1.

[0172] In some embodiments, the cytosine deaminase variant comprises an aspartic acid at a position corresponding to LI 86 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a glutamic acid at a position corresponding to LI 86 of SEQ ID NO: 1.

[0173] #14540497v1 In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an aspartic acid at a position corresponding to L186 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a glutamic acid at a position corresponding to L186 of SEQ ID NO: 1.

[0174] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an aspartic acid at position LI 86 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamic acid at position L186 of SEQ ID NO: 1.

[0175] In some embodiments, the cytosine deaminase variant comprises an aspartic acid at position LI 86 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a glutamic acid at position LI 86 of SEQ ID NO: 1.

[0176] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 105-106. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 105-106. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 105- 106. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 105-106. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 105-106. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence of any one of SEQ ID NOs: 105-106.

[0177] In some embodiments, this disclosure provides cytosine deaminase variants with increased preference for a GC sequence context (also called a GC-specific cytosine deaminase). Such cytosine deaminase variants have strong preference for specifically deaminating GC motifs and are therefore less prone to off-target deamination when used to deaminate cytosines in a GC sequence context. In some embodiments, a cytosine deaminase variant with increased preference for GC motifs comprises a mutation at a position corresponding to N23, R28, H29, K60, L62, VI 10, 1129, or P134 of SEQ ID NO: 1.

[0178] #14540497v1 N23 Cytosine Deaminase Variants

[0179] In some embodiments, a cytosine deaminase variant comprises a mutation corresponding to position N23 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a mutation at a position corresponding to N23 of SEQ ID NO: 1 and has at least 95% identity to any one of SEQ ID NOs: 1-7, 9-10.

[0180] In some embodiments, this disclosure provides a cytosine deaminase variant comprising a glycine or leucine at a position corresponding to N23 of SEQ ID NO: 1 (e.g., N23G or N23L in a cytosine deaminase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides a cytosine deaminase comprising a glycine or leucine at a position corresponding to N23 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) relative to any one of SEQ ID NOs: 1-7, 9-10. In some embodiments, this disclosure provides a cytosine deaminase variant comprising a glycine or leucine at position N23 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-7, 9-10 and a glycine or leucine at a position corresponding to N23 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising a glycine or leucine at a position corresponding to N23 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising a glycine or leucine at position N23 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a glycine or leucine at position N23 of SEQ ID NO: 1.

[0181] In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to N23 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a leucine at a position corresponding to N23 of SEQ ID NO: 1.

[0182] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-7, 9-10 and a glycine at a position corresponding to N23 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-7, 9-10 and a leucine at a position corresponding to N23 of SEQ ID NO: 1.

[0183] #14540497v1 In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glycine at position N23 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a leucine at position N23 of SEQ ID NO: 1.

[0184] In some embodiments, the cytosine deaminase variant comprises a glycine at position N23 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a leucine at position N23 of SEQ ID NO: 1.

[0185] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 11-12. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 11-12. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 11- 12. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 11-12. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 11-12. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence of any one of SEQ ID NOs: 11-12.

[0186] R28 Cytosine Deaminase Variants

[0187] In some embodiments, a cytosine deaminase variant comprises a mutation corresponding to position R28 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a mutation at a position corresponding to R28 of SEQ ID NO: 1 and has at least 95% identity to any one of SEQ ID NOs: 1-10.

[0188] In some embodiments, this disclosure provides a cytosine deaminase variant comprising a phenylalanine, glycine, histidine, asparagine, tryptophan, or tyrosine at a position corresponding to R28 of SEQ ID NO: 1 (e.g., R28F, R28G, R28H, R28N, R28W, or R28Y in a cytosine deaminase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides a cytosine deaminase variant comprising a phenylalanine, glycine, histidine, asparagine, tryptophan, or tyrosine at a position corresponding to R28 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) relative to any one of SEQ ID NOs: 1-7, 9-10. In some embodiments, this disclosure provides a cytosine deaminase

[0189] #14540497v1 variant comprising a phenylalanine, glycine, histidine, asparagine, tryptophan, or tyrosine at position R28 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a phenylalanine, glycine, histidine, asparagine, tryptophan, or tyrosine at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising a phenylalanine, glycine, histidine, asparagine, tryptophan, or tyrosine at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising a phenylalanine, glycine, histidine, asparagine, tryptophan, or tyrosine at position R28 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a phenylalanine, glycine, histidine, asparagine, tryptophan, or tyrosine at position R28 of SEQ ID NO: 1.

[0190] In some embodiments, the cytosine deaminase variant comprises a phenylalanine at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a histidine at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an asparagine at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a tryptophan at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a tyrosine at a position corresponding to R28 of SEQ ID NO: 1.

[0191] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a phenylalanine at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a glycine at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1- 10 and a histidine at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments,

[0192] #14540497v1 the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an asparagine at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a tryptophan at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a tyrosine at a position corresponding to R28 of SEQ ID NO: 1.

[0193] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a phenylalanine at position R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glycine at position R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a histidine at position R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an asparagine at position R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tryptophan at position R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a tyrosine at position R28 of SEQ ID NO: 1.

[0194] In some embodiments, the cytosine deaminase variant comprises a phenylalanine at position R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a glycine at position R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a histidine at position R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an asparagine at position R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a tryptophan at position R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a tyrosine at position R28 of SEQ ID NO: 1.

[0195] #14540497v1 In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 23-28. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 23-28. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 23- 28. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 23-28. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 23-28. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence of any one of SEQ ID NOs: 23-28.

[0196] H29 Cytosine Deaminase Variants

[0197] In some embodiments, a cytosine deaminase variant comprises a mutation corresponding to position H29 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a mutation at a position corresponding to H29 of SEQ ID NO: 1 and has at least 95% identity to any one of SEQ ID NOs: 1-10.

[0198] In some embodiments, this disclosure provides a cytosine deaminase variant comprising an alanine, cysteine, aspartic acid, glycine, lysine, asparagine, proline, glutamine, serine, or threonine at a position corresponding to H29 of SEQ ID NO: 1 (e.g., H29A, H29C, H29D, H29G, H29K, H29N, H29P, H29Q, H29S, or H29T in a cytosine deaminase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides a cytosine deaminase variant comprising an alanine, cysteine, aspartic acid, glycine, lysine, asparagine, proline, glutamine, serine, or threonine at a position corresponding to H29 of SEQ ID NO: 1 and one, two, three or four additional mutations e.g., amino acid substitutions) relative to any one of SEQ ID NOs: 1- 10. In some embodiments, this disclosure provides a cytosine deaminase variant comprising an alanine, cysteine, aspartic acid, glycine, lysine, asparagine, proline, glutamine, serine, or threonine at position H29 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an alanine, cysteine, aspartic acid, glycine, lysine, asparagine, proline, glutamine, serine, or threonine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at

[0199] #14540497v1 least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising an alanine, cysteine, aspartic acid, glycine, lysine, asparagine, proline, glutamine, serine, or threonine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising an alanine, cysteine, aspartic acid, glycine, lysine, asparagine, proline, glutamine, serine, or threonine at position H29 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises an alanine, cysteine, aspartic acid, glycine, lysine, asparagine, proline, glutamine, serine, or threonine at position H29 of SEQ ID NO: 1.

[0200] In some embodiments, the cytosine deaminase variant comprises an alanine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an aspartic acid at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a lysine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an asparagine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a proline at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a glutamine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a serine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a threonine at a position corresponding to H29 of SEQ ID NO: 1.

[0201] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an alanine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a cysteine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1- 10 and an aspartic acid at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least

[0202] #14540497v1 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1- 10 and a glycine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a lysine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an asparagine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a proline at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a glutamine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a serine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a threonine at a position corresponding to H29 of SEQ ID NO: 1.

[0203] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an alanine at position H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cysteine at position H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an aspartic acid at position H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glycine at position H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a lysine at position H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least

[0204] #14540497v1 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an asparagine at position H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a proline at position H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamine at position H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a serine at position H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a threonine at position H29 of SEQ ID NO: 1.

[0205] In some embodiments, the cytosine deaminase variant comprises an alanine at position H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a cysteine at position H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an aspartic acid at position H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a glycine at position H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a lysine at position H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an asparagine at position H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a proline at position H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a glutamine at position H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a serine at position H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a threonine at position H29 of SEQ ID NO: 1.

[0206] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 29-38. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 29-38. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 29- 38. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 29-38. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99.5% identity to

[0207] #14540497v1 any one of SEQ ID NOs: 29-38. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence of any one of SEQ ID NOs: 29-38.

[0208] K60 Cytosine Deaminase Variants

[0209] In some embodiments, a cytosine deaminase variant comprises a mutation corresponding to position K60 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a mutation at a position corresponding to K60 of SEQ ID NO: 1 and has at least 95% identity to any one of SEQ ID NOs: 1, 3, or 5-6.

[0210] In some embodiments, this disclosure provides a cytosine deaminase variant comprising a valine at a position corresponding to K60 of SEQ ID NO: 1 (e.g., K60V in a cytosine deaminase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides a valine at a position corresponding to K60 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) relative to any one of SEQ ID NOs: 1, 3, or 5-6. In some embodiments, this disclosure provides a cytosine deaminase variant comprising a valine at position K60 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1, 3, or 5-6 and a valine at a position corresponding to K60 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising a valine at a position corresponding to K60 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising a valine at position K60 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a valine at position K60 of SEQ ID NO: 1.

[0211] In some embodiments, the cytosine deaminase variant comprises a valine at a position corresponding to K60 of SEQ ID NO: 1.

[0212] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1, 3, or 5-6 and a valine at a position corresponding to K60 of SEQ ID NO: 1.

[0213] #14540497v1 In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a valine at position K60 of SEQ ID NO: 1.

[0214] In some embodiments, the cytosine deaminase variant comprises a valine at position K60 of SEQ ID NO: 1.

[0215] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 39. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 39. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 39. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 39. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 39In some embodiments, the cytosine deaminase variant comprises an amino acid sequence of SEQ ID NO: 39.

[0216] L62 Cytosine Deaminase Variants

[0217] In some embodiments, a cytosine deaminase variant comprises a mutation corresponding to position L62 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a mutation at a position corresponding to L62 of SEQ ID NO: 1 and has at least 95% identity to any one of SEQ ID NOs: 1, 3, or 6.

[0218] In some embodiments, this disclosure provides a cytosine deaminase variant comprising a glutamic acid at a position corresponding to L62 of SEQ ID NO: 1 e.g., L62E in a cytosine deaminase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides a glutamic acid at a position corresponding to L62 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) relative to any one of SEQ ID NOs: 1, 3, or 6. In some embodiments, this disclosure provides a cytosine deaminase variant comprising a glutamic acid at position L62 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1, 3, or 6 and a glutamic acid at a position corresponding to L62 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising a

[0219] #14540497v1 glutamic acid at a position corresponding to L62 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising a glutamic acid at position L62 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a glutamic acid at position L62 of SEQ ID NO: 1.

[0220] In some embodiments, the cytosine deaminase variant comprises a glutamic acid at a position corresponding to L62 of SEQ ID NO: 1.

[0221] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1, 3, or 6 and a glutamic acid at a position corresponding to L62 of SEQ ID NO: 1.

[0222] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glutamic acid at position L62 of SEQ ID NO: 1.

[0223] In some embodiments, the cytosine deaminase variant comprises a glutamic acid at position L62 of SEQ ID NO: 1.

[0224] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 40. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 40. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 40. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 40. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99.5% identity to SEQ ID NO: 40. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence of SEQ ID NO: 40.

[0225] VI 10 Cytosine Deaminase Variants

[0226] In some embodiments, a cytosine deaminase variant comprises a mutation corresponding to position VI 10 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a mutation at a position corresponding to VI 10 of SEQ ID NO: 1 and has at least 95% identity to any one of SEQ ID NOs: 1-10.

[0227] In some embodiments, this disclosure provides a cytosine deaminase variant comprising a tryptophan at a position corresponding to VI 10 of SEQ ID NO: 1 (e.g., V 110W in a cytosine

[0228] #14540497v1 deaminase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides a tryptophan at a position corresponding to VI 10 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) relative to any one of SEQ ID NOs: 1-10. In some embodiments, this disclosure provides a cytosine deaminase variant comprising tryptophan at position VI 10 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a tryptophan at a position corresponding to VI 10 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising tryptophan at a position corresponding to VI 10 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises: an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a cytosine deaminase variant comprising tryptophan at position VI 10 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises tryptophan at position VI 10 of SEQ ID NO: 1.

[0229] In some embodiments, the cytosine deaminase variant comprises tryptophan at a position corresponding to VI 10 of SEQ ID NO: 1.

[0230] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a tryptophan at a position corresponding to VI 10 of SEQ ID NO: 1.

[0231] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and tryptophan at position VI 10 of SEQ ID NO: 1.

[0232] In some embodiments, the cytosine deaminase variant comprises tryptophan at position VI 10 of SEQ ID NO: 1.

[0233] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 50. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 50. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 50. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 50. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least

[0234] #14540497v1 99.5% identity to SEQ ID NO: 50. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence of SEQ ID NO: 50.

[0235] 1129 Cytosine Deaminase Variants

[0236] In some embodiments, a cytosine deaminase variant comprises a mutation corresponding to position 1129 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a mutation at a position corresponding to 1129 of SEQ ID NO: 1 and has at least 95% identity to any one of SEQ ID NOs: 1-10.

[0237] In some embodiments, this disclosure provides a cytosine deaminase variant comprising glycine or aspartic acid at a position corresponding to 1129 of SEQ ID NO: 1 (e.g., I129G or I129D in a cytosine deaminase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides a cytosine deaminase variant comprising a glycine or an aspartic acid at a position corresponding to 1129 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) relative to any one of SEQ ID NOs: 1-10. In some embodiments, this disclosure provides a cytosine deaminase variant comprising glycine or aspartic acid at position 1129 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a glycine or aspartic acid at a position corresponding to 1129 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glycine or aspartic acid at a position corresponding to 1129 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a glycine or aspartic acid at position 1129 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises glycine or aspartic acid at position 1129 of SEQ ID NO: 1.

[0238] In some embodiments, the cytosine deaminase variant comprises glycine at a position corresponding to 1129 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises aspartic acid at a position corresponding to 1129 of SEQ ID NO: 1.

[0239] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a glycine at a position corresponding to 1129 of SEQ ID NO: 1. In some

[0240] #14540497v1 embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1- 10 and an aspartic acid at a position corresponding to 1129 of SEQ ID NO: 1.

[0241] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and glycine at position 1129 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and aspartic acid at position 1129 of SEQ ID NO: 1.

[0242] In some embodiments, the cytosine deaminase variant comprises glycine at position 1129 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises aspartic acid at position 1129 of SEQ ID NO: 1.

[0243] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 56-57. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 56-57. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 56- 57. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 56-57. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 56-57. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence of any one of any one of SEQ ID NOs: 56-57.

[0244] P134 Cytosine Deaminase Variants

[0245] In some embodiments, a cytosine deaminase variant comprises a mutation corresponding to position Pl 34 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a mutation at a position corresponding to P134 of SEQ ID NO: 1 and has at least 95% identity to any one of SEQ ID NOs: 1-10.

[0246] In some embodiments, this disclosure provides a cytosine deaminase variant comprising a lysine or arginine at a position corresponding to P134 of SEQ ID NO: 1 (e.g., P134K or P134R in a cytosine deaminase corresponding to SEQ ID NO: 1). In some embodiments, this disclosure provides a cytosine deaminase variant comprising a lysine or asparagine at a position corresponding to P134 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) relative to any one of SEQ ID NOs: 1-10. In some embodiments, this

[0247] #14540497v1 disclosure provides a cytosine deaminase variant comprising a lysine or arginine at position P134 of SEQ ID NO: 1 and one, two, three or four additional mutations (e.g., amino acid substitutions) in SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a lysine or arginine at a position corresponding to P134 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a lysine or arginine at a position corresponding to P134 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a lysine or arginine at position Pl 34 of SEQ ID NO: 1. In some embodiments, a cytosine deaminase variant comprises a lysine or arginine at position Pl 34 of SEQ ID NO: 1.

[0248] In some embodiments, the cytosine deaminase variant comprises a lysine at a position corresponding to Pl 34 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an arginine at a position corresponding to P134 of SEQ ID NO: 1.

[0249] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and a glycine at a position corresponding to Pl 34 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with any one of SEQ ID NOs: 1-10 and an aspartic acid at a position corresponding to P134 of SEQ ID NO: 1.

[0250] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and a lysine at position Pl 34 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% (e.g., at least 95%, at least 98%, or at least 99%) identity with SEQ ID NO: 1 and an arginine at position P134 of SEQ ID NO: 1.

[0251] In some embodiments, the cytosine deaminase variant comprises a lysine at position Pl 34 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises an arginine at position Pl 34 of SEQ ID NO: 1.

[0252] In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 87-88. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 95% identity to

[0253] #14540497v1 any one of SEQ ID NOs: 87-88. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 98% identity to any one of SEQ ID NOs: 87- 88. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs: 87-88. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence having at least 99.5% identity to any one of SEQ ID NOs: 87-88. In some embodiments, the cytosine deaminase variant comprises an amino acid sequence of any one of SEQ ID NOs: 87-88.

[0254] Base Editors

[0255] Some embodiments of this disclosure relate to base editors comprising a cytosine deaminase variant described herein. Generally speaking, base editors are composed of a DNA binding domain and a nucleobase-modifying domain (e.g., a cytosine deaminase). The design of the base editor can facilitate modification of nucleobases within a particular region, such as a locus in a genome. Base editors are typically engineered to change individual bases in DNA sequences without inducing double- stranded DNA breaks. Nucleobase modification occurs via chemical modification by the base editor, such as the removal of an amino group (e.g., via catalyzing the hydrolytic deamination of cytosine in the case of a base editor comprising a cytosine deaminase variant) to convert one base to another. Base editors that target cytosine can be created via the fusion of a DNA-binding protein (e.g., a catalytically inactive Cas9) with a cytosine deaminase, e.g., a cytosine deaminase variant provided herein, and a guide RNA (gRNA) that is complementary to a target DNA sequence or a portion thereof. The gRNA directs the DNA-binding protein to the target DNA sequence by hybridizing to the target sequence. Upon guiding the DNA-binding protein to the target DNA sequence, the DNA- binding protein binds to a protospacer adjacent motif (PAM) site immediately adjacent to the target DNA sequence. After binding of the target DNA sequence by the DNA binding protein, the base-editing enzyme (e.g., a cytosine deaminase variant described herein) edits the base for which it exhibits affinity. Following editing of a nucleobase by a base editor, endogenous DNA repair machinery can replicate the edited DNA sequence and incorporate the edit into the cellular genome, e.g. by converting uracil to thymine following deamination of a cytosine (e.g., by a cytosine deaminase variant described herein). Molecular biology assays, such as polymerase chain reaction (PCR) can also be used following editing of a nucleobase by a base editor to convert the edited base (e.g., uracil) to a different base (e.g., thymine). Different types of base editors are known in the art, e.g., as described in Anzalon et al., Nature Biotechnology

[0256] #14540497v1 38.7 (2020): 824-844. In some embodiments, a DNA-binding protein is a nickase Cas (z.e., a Cas protein in which one of two endonuclease domains is inactivated).

[0257] In some embodiments, this disclosure provides a polynucleotide encoding a cytosine deaminase or a base editor provided herein. The term “polynucleotides”, used interchangeably with the term “nucleic acids”, refers to polymers of nucleotides (e.g., deoxyribonucleic acids, modified nucleotides, etc.).

[0258] In some embodiments, this disclosure provides a base editor comprising a DNA binding protein and a cytosine deaminase variant provided herein (e.g. a cytosine deaminase variant comprising the sequence of any one of SEQ ID NOs: 11-106). In some embodiments, a DNA binding protein is an Argonaute protein. In some embodiments, an Argonaute protein is catalytically inactive. In some embodiments, a DNA binding protein is a CRISPR associated (Cas) protein. In some embodiments, a Cas protein is catalytically inactive. In some embodiments, a Cas protein is a catalytically inactive Cas9 protein. In some embodiments, a base editor provided herein comprises a peptide linker between the DNA binding domain and the cytosine deaminase. In some embodiments, a peptide linker is a glycine serine linker.

[0259] Some embodiments of the disclosure relate to a polynucleotide encoding a base editor and / or a base editor target site. Such polynucleotides can be introduced into cells (e.g., E. coli cells), for example, to harness endogenous cellular engineering to edit a base within the genome of the cell or to identify sequence preference of a base editor. In some embodiments, this disclosure provides a polynucleotide comprising (a) a nucleic acid encoding a base editor comprising a deaminase variant and a polynucleotide binding protein; and (b) a base editor target site comprising a first polynucleotide binding protein sequence motif and a first deaminase sequence motif. In some embodiments, this disclosure provides a polynucleotide comprising: (a) a nucleic acid encoding a base editor comprising a deaminase variant and a polynucleotide binding protein; and (b) a first base editor target site and a second base editor target site that flank the nucleotides encoding the deaminase variant of the nucleic acid; wherein the first base editor target site comprises a first polynucleotide binding protein sequence motif and a first deaminase sequence motif; and wherein the second base editor target site comprises a second polynucleotide binding protein sequence motif and a second deaminase sequence motif. In general, a base editor target site refers to a nucleotide sequence (e.g., a DNA sequence) or region that the base editor is designed to target to induce a nucleobase modification (e.g., a cytosine for deamination). In some embodiments, the polynucleotide binding protein (e.g., DNA-binding protein) is a catalytically inactive Cas protein. In some embodiments, a polynucleotide provided herein comprises a guide RNA (gRNA) comprising a homology region

[0260] #14540497v1 that is complementary to a first polynucleotide binding protein sequence motif and / or a second polynucleotide binding protein sequence motif.

[0261] In some embodiments, a polynucleotide encoding a base editor comprises a promoter. In some embodiments, a promoter is a T5 promoter. In some embodiments, a promoter is an arabinose promoter. In some embodiments, a polynucleotide encoding a base editor comprises a T5 promoter and an arabinose promoter. In some embodiments, a polynucleotide encoding a base editor comprises a His tag. In some embodiments, a polynucleotide encoding a base editor comprises a region encoding a uracil glycosylase inhibitor (UGI).

[0262] In some embodiments, a polynucleotide comprises, from 5’ to 3’, a first promoter, a sequence encoding a guide RNA, a second promoter, a first base-editor target site, a sequence encoding a cytosine deaminase variant (e.g., a cytosine deaminase variant provided herein), a second base-editor target site, and a sequence encoding a DNA-binding protein (e.g., a catalytically inactive Cas9). In some embodiments, a polynucleotide comprises, from 5’ to 3’, a first promoter, a first base-editor site, a sequence encoding a cytosine deaminase variant (e.g., a cytosine deaminase variant provided herein), a second base-editor target site, and a sequence encoding a guide RNA.

[0263] A “polynucleotide binding protein sequence motif’ to a sequence to which the DNA- binding protein (e.g., Cas9) of the base editor binds. In some embodiments, a polynucleotide binding protein sequence motif (e.g., a first polynucleotide binding protein sequence motif) is a sequence that is complementary to a guide RNA (gRNA).

[0264] A “deaminase sequence motif’ refers to a sequence that is recognized by a cytosine deaminase and comprises a target cytosine to be deaminated. In some embodiments, a deaminase sequence motif comprises a GC motif. In some embodiments, a deaminase sequence motif comprises a CC motif. In some embodiments, a deaminase sequence motif comprises an AC motif. In some embodiments, a deaminase sequence motif comprises a TC motif. In some embodiments, a deaminase sequence motif (e.g., a first deaminase sequence motif) is complementary to a gRNA or a portion thereof. In some embodiments, a polynucleotide binding protein sequence motif comprises a deaminase sequence motif.

[0265] In some embodiments, a polynucleotide comprises at least one base editor target site. In some embodiments, a polynucleotide comprises two base editor target sites. In some embodiments, a polynucleotide comprises more than two base editor target sites. In some embodiments, a base editor target site comprises or is adjacent to a CC motif. In some embodiments, a base editor target site comprises or is adjacent to a GC motif. In some

[0266] #14540497v1 embodiments, a base editor target site comprises or is adjacent to an AC motif. In some embodiments, a base editor target site comprises or is adjacent to a TC motif.

[0267] A guide RNA (gRNA) refers to an RNA polymer that (1) comprises a targeting region which is complementary to a target (e.g., a sequence comprising a cytosine to deaminate) and (2) that is capable of facilitating binding of a ribonucleoprotein complex comprising the gRNA to the target. In some embodiments, the gRNA is a CRISPR associated protein (Cas) gRNA. In some embodiments, a Cas gRNA polynucleotide refers to a two polynucleotide system comprising a tracrRNA and a crRNA e.g., as described in Karvelis et al., RNA Biol. 2013 May;10(5):841-51. PMID: 23535272. The tracrRNA comprises a sequence encoding a stem loop structure that associates with the Cas protein (e.g., dead Cas9, dCas9). The crRNA comprises a homology region that is complementary to the target (e.g., a sequence comprising a cytosine to deaminate) and a region that is complementary to the tracrRNA. The crRNA and the tracrRNA may form a complex with the Cas protein, which in turn binds to a target DNA or RNA polynucleotide (depending on the Cas type). In some embodiments, a Cas gRNA polynucleotide refers to a single guide RNA (sgRNA) polynucleotide e.g., as described in Jinek et al., Science. 2012 Aug 17;337(6096):816-21. doi: 10.1126 / science.1225829. A sgRNA comprises a sequence encoding a stem loop structure that associates with the Cas protein and a polynucleotide binding protein sequence motif. Many Cas proteins bind to polynucleotides (e.g., double stranded DNA) in a position that is adjacent to a protospacer adjacent motif (PAM) site. In some embodiments, a DNA binding protein is an RNA-guided nuclease. In some embodiments, the polynucleotide binding protein sequence motif is complementary to a target (e.g., a portion of a sequence comprising a cytosine to deaminate) that is adjacent to a PAM site (e.g., sufficiently adjacent such that Cas binding to the target can take place). Methods of making and using gRNAs are known in the art, e.g., as described in Mohr, et al., FEBS J. 2016 Sep;283(17):3232-8. PMCID: PMC5014588. In some embodiments, a base editor provided herein comprises a gRNA comprising an RNA aptamer, a DNA binding protein, and a cytosine deaminase variant provided herein. In some embodiments, the cytosine deaminase variant is fused to an RNA aptamer binding moiety. The term “RNA aptamer binding moiety” refers to a structural region or functional group within an RNA aptamer (single- stranded RNA that binds target molecules by folding into a three-dimensional shape) that interacts with or binds to a target (e.g., a target DNA sequence).

[0268] Methods of Determining Sequence Preference

[0269] #14540497v1 This disclosure also describes methods of determining sequence preference of a cytosine deaminase variant provided herein.

[0270] Deaminase preference can be determined by combining polynucleotides (e.g., DNA polynucleotides) of known sequence with a deaminase (e.g., cytosine deaminase variants or adenosine deaminase variants) in conditions that facilitate the deaminating activity of the deaminase variant, amplifying the deaminated polynucleotides, sequencing the amplified deaminated polynucleotides, and comparing the sequence of the deaminated polynucleotides to the original sequence of the polynucleotides to identify nucleotides that were deaminated to determine common motifs where deamination occurred. Any suitable polynucleotide of known sequence (e.g., a polynucleotide encoding a cytosine deaminase or a cytosine deaminase variant) can be used to determine sequence preference of a deaminase variant. In some aspects, this disclosure provides a method of determining sequence preference of a plurality of deaminase variants, the method comprising: (i) transfecting a plurality of cells with a plurality of vectors, each vector of the plurality of vectors comprising: (a) a nucleic acid encoding a base editor comprising a deaminase variant (e.g., a cytosine deaminase variant or an adenosine deaminase variant) and a polynucleotide binding protein; and (b) a first base editor target site; wherein the first base editor target site comprises a first polynucleotide binding protein sequence motif and a first deaminase sequence motif; (ii) amplifying vectors of the plurality of vectors that comprise an edited first deaminase sequence motif to produce amplicons that comprise a nucleic acid encoding the deaminase variants; and (iii) sequencing the amplicons to determine the sequence preference of the plurality of deaminase variants for the first deaminase sequence motif (e.g., in a manner which preserves the linkage of the phenotype (edited sequence motif) and genotype (deaminase variant)).

[0271] Adenosine base editors typically comprise an adenosine deaminase and a DNA binding protein (e.g., a dCas), e.g., as discussed in Gaudelli et. al., Nature 551, 464-471 (2017). Adenosine deaminases deaminate adenosine into Inosine, which is read as guanosine by DNA polymerases. Thus, an adenosine deaminases may covert a T:A base pair into a C:G base pair.

[0272] A “plurality” refers to at least 2. In some embodiments, a plurality refers to at least 5, at least 10, at least 50, at least 100, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, or more. In some embodiments, a plurality refers to 2-20,000. In some embodiments, a plurality refers to 100-1,000. In some embodiments, a plurality refers to 500- 5,000. In some embodiments, a plurality refers to 1,000-10,000.

[0273] A “plurality of deaminase variants” refers to at least 2 deaminase variants. In some embodiments, a plurality refers to at least 5, at least 10, at least 50, at least 100, at least 500, at

[0274] #14540497v1 least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, or more deaminase variants. In some embodiments, a plurality refers to 2-20,000 deaminase variants. In some embodiments, a plurality refers to 100-1,000 deaminase variants. In some embodiments, a plurality refers to 500-5,000 deaminase variants. In some embodiments, a plurality refers to 1,000-10,000 deaminase variants.

[0275] A “plurality of cells” refers to at least 2 cells. In some embodiments, a plurality refers to at least 5, at least 10, at least 50, at least 100, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, or more cells. In some embodiments, a plurality refers to 2-20,000 cells. In some embodiments, a plurality refers to 100-1,000 cells. In some embodiments, a plurality refers to 500-5,000 cells. In some embodiments, a plurality refers to 1,000-10,000 cells.

[0276] A “plurality of vectors” refers to at least 2 vectors. In some embodiments, a plurality refers to at least 5, at least 10, at least 50, at least 100, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, or more vectors. In some embodiments, a plurality refers to 2-20,000 vectors. In some embodiments, a plurality refers to 100-1,000 vectors. In some embodiments, a plurality refers to 500-5,000 vectors. In some embodiments, a plurality refers to 1,000-10,000 vectors. In some embodiments, a plurality of vectors comprises a vector that does not encode a base editor. In some embodiments, a plurality of vectors comprises a vector that does not encode a base editor target site. In some embodiments, a plurality of vectors comprises a vector that does not encode a base editor, a base editor target site, a gRNA, and / or a DNA-binding protein.

[0277] “Transfecting” a cell refers to introducing a polynucleotide (e.g., a polynucleotide encoding a base editor and a base editor target site) into the cell. In some embodiments, transfecting comprises using a viral vector. In some embodiments, transfecting comprises using a non- viral vector. In some embodiments, transfecting comprises electroporation. In some embodiments, an object of transfecting a plurality of cells is to transfect no more than nucleotide encoding a deaminase variant (e.g., vector) per cell. In some embodiments, transfection comprising transfecting with a multiplicity of infection (MOI) that is less than or equal to 1 (e.g., less than 0.75, less than 0.5 or less than 0.25).

[0278] In some embodiments, cells of the plurality of cells are bacterial cells (e.g., E.coli strains DH5a, DH10B, BL21, BL21-DE3). In some embodiments, the plurality of cells are mammalian, yeast or insect cells. In some embodiments, the plurality of cells are human cells.

[0279] In some embodiments, a vector encodes, from 5’ to 3’, a first promoter, a guide RNA, a second promoter, a first base-editor target site, a deaminase variant (e.g., a cytosine deaminase

[0280] #14540497v1 variant or an adenosine deaminase variant), a second base-editor target site, and a DNA-binding protein (e.g., a catalytically inactive Cas9). In some embodiments, a vector encodes, from 5’ to 3’, a first promoter, a first base-editor site, a cytosine deaminase variant (e.g., a cytosine deaminase variant provided herein), a second base-editor target site, and a guide RNA. In some embodiments, a vector encodes, from 5’ to 3’, a first promoter, a guide RNA, a second promoter, a first base-editor target site, a deaminase variant (e.g., a cytosine deaminase variant or adenosine deaminase variant), a second base-editor target site, a DNA-binding protein (e.g., a catalytically inactive Cas9), a UGI, and a His tag. In some embodiments, a vector encodes, from 5’ to 3’, a first promoter, a first base-editor target site, a cytosine deaminase variant (e.g., a cytosine deaminase variant provided herein), a second base-editor target site, a guide RNA, a UGI, and a His tag.

[0281] In some embodiments, amplifying comprises contacting a plurality of vectors with a first primer that is complementary to an edited (e.g., deaminated) first deaminase sequence motif. In some embodiments, amplifying comprises amplifying using polymerase chain reaction (PCR). In some embodiments, amplifying comprises amplifying using RNase H-dependent PCR (rhPCR). rhPCR uses blocked primers and RNase H2 enzyme to increase the accuracy of amplification (e.g., reduce the introduction of errors) and reduce the formation of primer dimers (see Dobosy et al. BMC Biotech, 2011. 11(80); PMID: 21831278).

[0282] In some embodiments, sequencing comprises sequencing a contiguous polynucleotide comprising a nucleic acid encoding a deaminase variant and a first base editor target site using long-read sequencing. When using long-read sequencing, primers specific to target DNA sequences that have been edited (e.g., deaminated by a cytosine deaminase variant provided herein) are not required to amplify the target DNA sequences prior to long-read sequencing. Long-read sequencing allows for the sequencing of entire DNA molecules (e.g., long sequences), rather than small portions of DNA, allowing for complete coverage of long amplicons. In other words, long read sequencing allows identification of modifications to the target site and the cytosine deaminase variant from a single read. In some embodiments, sequencing comprises next-generation sequencing (NGS). In some embodiments, sequencing does not comprise long-read sequencing. When sequencing with techniques that sequence shorter regions of DNA, primers specific for the edited sequences can be used to amplify DNA regions of interest (e.g., target DNA sequences). Additionally, RNAse H-dependent PCR (rhPCR) can be used to improve the preference of amplification of DNA regions of interest prior to sequencing using techniques that sequence shorter regions of DNA. In some embodiments,

[0283] #14540497v1 amplifying comprises contacting a plurality of vectors with a first primer that is complementary to an edited first deaminase sequence motif.

[0284] The cytosine deaminase variants provided herein can be useful for specifically deaminating multiple cytosines in particular motifs. In some aspects, the disclosure provides a method of determining sequence preference of a plurality of deaminase variants, the method comprising: (i) transfecting a plurality of cells with a plurality of vectors, each vector of the plurality of vectors comprising: (a) a nucleic acid encoding a base editor comprising a deaminase variant and a polynucleotide binding protein; and (b) a first base editor target site and a second base editor target site that flank the nucleotides encoding the deaminase variant of the nucleic acid; wherein the first base editor target site comprises a first polynucleotide binding protein sequence motif and a first deaminase sequence motif; wherein the second base editor target site comprises a second polynucleotide binding protein sequence motif and a second deaminase sequence motif; (ii) amplifying vectors of the plurality of vectors that comprise an edited first deaminase sequence motif and / or an edited second deaminase sequence motif to produce amplicons that comprise a nucleic acid encoding the deaminase variant, the amplifying comprising contacting the plurality of vectors with: (a) a first primer that is complementary to an edited first deaminase sequence motif and a second primer that is complementary to the vector; (b) a first primer that is complementary to an edited second deaminase sequence motif and a second primer that is complementary to the vector; or (c) a first primer that is complementary to an edited first deaminase sequence motif and a second primer that is complementary to an edited second deaminase sequence motif; and (iii) sequencing the amplicons to identify deaminase variants that edited the first deaminase sequence motif and / or the second deaminase sequence motif.

[0285] In some embodiments, a first deaminase sequence motif and a second deaminase sequence motif (e.g., an additional deaminase sequence motif) are the same motif. In some embodiments, a first deaminase sequence motif and a second deaminase sequence motif are different motifs. In some embodiments, a first deaminase sequence motif and / or a second deaminase sequence motif comprise an AC motif, a CC motif, a GC motif, or a TC motif. In some embodiments, a first deaminase sequence motif comprises an AC motif and a second deaminase sequence motif comprises an AC motif. In some embodiments, a first deaminase sequence motif comprises an AC motif and a second deaminase sequence motif comprises a CC motif. In some embodiments, a first deaminase sequence motif comprises an AC motif and a second deaminase sequence motif comprises a GC motif. In some embodiments, a first deaminase sequence motif comprises an AC motif and a second deaminase sequence motif

[0286] #14540497v1 comprises a TC motif. In some embodiments, a first deaminase sequence motif comprises a CC motif and a second deaminase sequence motif comprises an AC motif. In some embodiments, a first deaminase sequence motif comprises a CC motif and a second deaminase sequence motif comprises a CC motif. In some embodiments, a first deaminase sequence motif comprises a CC motif and a second deaminase sequence motif comprises a GC motif. In some embodiments, a first deaminase sequence motif comprises a CC motif and a second deaminase sequence motif comprises a TC motif. In some embodiments, a first deaminase sequence motif comprises a GC motif and a second deaminase sequence motif comprises an AC motif. In some embodiments, a first deaminase sequence motif comprises a GC motif and a second deaminase sequence motif comprises a CC motif. In some embodiments, a first deaminase sequence motif comprises a GC motif and a second deaminase sequence motif comprises a GC motif. In some embodiments, a first deaminase sequence motif comprises a GC motif and a second deaminase sequence motif comprises a TC motif. In some embodiments, a first deaminase sequence motif comprises a TC motif and a second deaminase sequence motif comprises an AC motif. In some embodiments, a first deaminase sequence motif comprises a TC motif and a second deaminase sequence motif comprises a CC motif. In some embodiments, a first deaminase sequence motif comprises a TC motif and a second deaminase sequence motif comprises a GC motif. In some embodiments, a first deaminase sequence motif comprises a TC motif and a second deaminase sequence motif comprises a TC motif.

[0287] In some embodiments, a vector is a viral vector (e.g., an adeno-associated vector, a lentiviral vector, or an adenoviral vector). In some embodiments, a plurality of vectors collectively comprises at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) of the plurality of deaminase variants. In some embodiments, a plurality of vectors collectively comprises 100% of the plurality of deaminase variants.

[0288] In some embodiments, a plurality of cells comprises bacterial cells (e.g., E. coli strains DH5a, DH10B, BL21, or BL21-DE3). In some embodiments, a plurality of cells comprises mammalian, yeast, or insect cells. In some embodiments, a plurality of cells comprises human cells. In some embodiments, a plurality of cells collectively comprises at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) of the plurality of deaminase variants. In some embodiments, a plurality of cells collectively comprises 100% of the plurality of deaminase variants.

[0289] Method of Editing a Specific Sequence Context

[0290] #14540497v1 The cytosine deaminase variants provided herein are useful for selectively editing cytosines present in a specific sequence (e.g., editing a specific sequence context). In some embodiments, this disclosure provides a method of specifically editing a GC sequence context using a cytosine deaminase variant provided herein. In some embodiments, a method of specifically editing a GC sequence context comprising using a cytosine deaminase variant comprising a sequence of any one of SEQ ID NOs: 11-106. In some embodiments, this disclosure provides a method of specifically editing a CC sequence context using a cytosine deaminase variant provided herein. In some embodiments, a method of specifically editing a CC sequence context comprises using a cytosine deaminase variant comprising a sequence of any one of SEQ ID NOs: 11-106.

[0291] In some embodiments, this disclosure provides a method of removing an amine using a cytosine deaminase variant provided herein (e.g., a cytosine deaminase variant comprising the sequence of any one of SEQ ID NOs: 11-106), wherein the amine is adjacent to or comprises an AC motif, a CC motif, a GC motif, or a TC motif. In some embodiments, an amine is adjacent to or comprises an AC motif. In some embodiments, an amine is adjacent to or comprises a CC motif. In some embodiments, an amine is adjacent to or comprises a GC motif. In some embodiments, an amine is adjacent to or comprises a TC motif.

[0292] In some embodiments, this disclosure provides a method of selectively deaminating a cytosine, the method comprising using a cytosine deaminase variant provided herein (e.g., a cytosine deaminase variant comprising the sequence of any one of SEQ ID NOs: 11-106), wherein the cytosine is adjacent to or comprises an AC motif, a CC motif, a GC motif, or a TC motif. In some embodiments, the cytosine is adjacent to or comprises an AC motif. In some embodiments, the cytosine is adjacent to or comprises a CC motif. In some embodiments, the cytosine is adjacent to or comprises a GC motif. In some embodiments, the cytosine is adjacent to or comprises a TC motif.

[0293] As described herein (e.g., see below), some cytosine deaminase variants comprise increased activity and / or specificity for a cytosine in a particular three nucleotide motif. In some embodiments, a method comprises deaminating a cytosine of a particular three nucleotide motif using a particular cytosine deaminase variant (e.g., using a base editor comprising the particular cytosine deaminase variant) that has increased activity and / or specificity for the particular three nucleotide motif.

[0294] In some embodiments, a cytosine deaminase variant comprises increased activity for a cytosine positioned between a T and an A (e.g., a “TCA” motif). In some embodiments, the cytosine deaminase variant comprises a histidine at a position corresponding to Y132 of SEQ ID

[0295] #14540497v1 NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 76.

[0296] In some embodiments, a cytosine deaminase variant comprises increased activity for a cytosine positioned between a T and a C (e.g., a “TCC” motif). In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to F102 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the cytosine deaminase variant comprises an alanine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 71. In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the cytosine deaminase variant comprises an arginine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 83.

[0297] In some embodiments, a cytosine deaminase variant comprises increased activity for a cytosine positioned between a T and a G. In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to F102 of SEQ ID NO: 1 (e.g., a “TCG” motif). In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the cytosine deaminase variant comprises an arginine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 83.

[0298] In some embodiments, a cytosine deaminase variant comprises increased activity for a cytosine positioned between a T and a T (e.g., a “TCT” motif). In some embodiments, the cytosine deaminase variant comprises a histidine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 76.

[0299] In some embodiments, a cytosine deaminase variant comprises increased activity for a cytosine positioned between a G and an A (e.g., a “GCA” motif). In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the cytosine deaminase variant comprises an arginine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 83.

[0300] #14540497v1 In some embodiments, a cytosine deaminase variant comprises increased activity for a cytosine positioned between a G and a C (e.g., a “GCC” motif). In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to F102 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the cytosine deaminase variant comprises an alanine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the cytosine deaminase variant comprises a proline at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the cytosine deaminase variant comprises a serine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the cytosine deaminase variant comprises an glycine at a position corresponding to N23 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the cytosine deaminase variant comprises an asparagine at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the cytosine deaminase variant comprises an alanine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 71. In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the cytosine deaminase variant comprises a histidine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, the cytosine deaminase variant comprises an arginine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the cytosine deaminase variant comprises a serine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the cytosine deaminase variant comprises a threonine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the cytosine deaminase variant comprises a valine at a position

[0301] #14540497v1 corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 104.

[0302] In some embodiments, a cytosine deaminase variant comprises increased activity for a cytosine positioned between a G and a T (e.g., a “GCT” motif). In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 72.

[0303] In some embodiments, a cytosine deaminase variant comprises increased activity for a cytosine positioned between a C and an A (e.g., a “CCA” motif). In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to F102 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the cytosine deaminase variant comprises an arginine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 83.

[0304] In some embodiments, a cytosine deaminase variant comprises increased activity for a cytosine positioned between a C and a C (e.g., a “CCC” motif). In some embodiments, the cytosine deaminase variant comprises a glutamate at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 60. In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to Fl 02 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the cytosine deaminase variant comprises an asparagine at a position corresponding to F102 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the cytosine deaminase variant comprises a threonine at a position corresponding to G27 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the cytosine deaminase variant comprises an alanine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 30. In some

[0305] #14540497v1 embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the cytosine deaminase variant comprises a lysine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the cytosine deaminase variant comprises an asparagine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the cytosine deaminase variant comprises a proline at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the cytosine deaminase variant comprises a serine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to N23 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the cytosine deaminase variant comprises a histidine at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the cytosine deaminase variant comprises an asparagine at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the cytosine deaminase variant comprises an alanine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the cytosine deaminase variant comprises a histidine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, the cytosine deaminase variant comprises a lysine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the cytosine deaminase variant comprises an asparagine at a position corresponding to Y132 of SEQ ID NO: 1. In some

[0306] #14540497v1 embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 80. In some embodiments, the cytosine deaminase variant comprises an arginine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the cytosine deaminase variant comprises a serine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the cytosine deaminase variant comprises a threonine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the cytosine deaminase variant comprises an alanine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 92. In some embodiments, the cytosine deaminase variant comprises an isoleucine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 95. In some embodiments, the cytosine deaminase variant comprises a methionine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 98. In some embodiments, the cytosine deaminase variant comprises an asparagine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 99. In some embodiments, the cytosine deaminase variant comprises a serine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 102. In some embodiments, the cytosine deaminase variant comprises a threonine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 103. In some embodiments, the cytosine deaminase variant comprises a valine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 104.

[0307] In some embodiments, a cytosine deaminase variant comprises increased activity for a cytosine positioned between a C and a G (e.g., a “CCG” motif). In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to N23 of SEQ ID

[0308] #14540497v1 NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the cytosine deaminase variant comprises an arginine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 92. In some embodiments, the cytosine deaminase variant comprises a threonine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 103.

[0309] In some embodiments, a cytosine deaminase variant comprises increased activity for a cytosine positioned between a C and a T (e.g., a “CCT” motif). In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to F102 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to N23 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the cytosine deaminase variant comprises an alanine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises a histidine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 71. In some embodiments, the cytosine deaminase variant comprises a lysine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the cytosine deaminase variant comprises an asparagine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 80. In some embodiments, the cytosine deaminase variant comprises an arginine at a position corresponding to Y 132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the cytosine deaminase variant comprises a serine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine

[0310] #14540497v1 deaminase variant comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the cytosine deaminase variant comprises a threonine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the cytosine deaminase variant comprises an alanine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 92. In some embodiments, the cytosine deaminase variant comprises an isoleucine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 95. In some embodiments, the cytosine deaminase variant comprises an asparagine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 101. In some embodiments, the cytosine deaminase variant comprises a serine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 102. In some embodiments, the cytosine deaminase variant comprises a threonine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 103. In some embodiments, the cytosine deaminase variant comprises a valine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 104.

[0311] In some embodiments, a cytosine deaminase variant comprises increased activity for a cytosine positioned between an A and an A (e.g., a “ACA” motif). In some embodiments, the cytosine deaminase variant comprises an alanine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 58. In some embodiments, the cytosine deaminase variant comprises a glutamate at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 60. In some embodiments, the cytosine deaminase variant comprises a threonine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 67. In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to Fl 02 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID

[0312] #14540497v1 NO: 43. In some embodiments, the cytosine deaminase variant comprises a serine at a position corresponding to H29 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to N23 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the cytosine deaminase variant comprises a histidine at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the cytosine deaminase variant comprises an asparagine at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the cytosine deaminase variant comprises an alanine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 71. In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the cytosine deaminase variant comprises a histidine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, the cytosine deaminase variant comprises a lysine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the cytosine deaminase variant comprises an asparagine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 80. In some embodiments, the cytosine deaminase variant comprises a proline at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, the cytosine deaminase variant comprises an arginine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the cytosine deaminase variant comprises a serine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the cytosine deaminase variant comprises a threonine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 85. In some

[0313] #14540497v1 embodiments, the cytosine deaminase variant comprises an alanine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 92. In some embodiments, the cytosine deaminase variant comprises an isoleucine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 95. In some embodiments, the cytosine deaminase variant comprises a threonine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 103. In some embodiments, the cytosine deaminase variant comprises a valine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 104.

[0314] In some embodiments, a cytosine deaminase variant comprises increased activity for a cytosine positioned between an A and a C (e.g., a “ACC” motif). In some embodiments, the cytosine deaminase variant comprises an alanine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 58. In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 59. In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 62. In some embodiments, the cytosine deaminase variant comprises a valine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 68. In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to N23 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the cytosine deaminase variant comprises an isoleucine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO:95.

[0315] In some embodiments, a cytosine deaminase variant comprises increased activity for a cytosine positioned between an A and a G (e.g., a “ACG” motif). In some embodiments, the

[0316] #14540497v1 cytosine deaminase variant comprises a glycine at a position corresponding to F102 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to N23 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the cytosine deaminase variant comprises a histidine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, the cytosine deaminase variant comprises a serine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the cytosine deaminase variant comprises a threonine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 92. In some embodiments, the cytosine deaminase variant comprises an isoleucine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 95. In some embodiments, the cytosine deaminase variant comprises a valine at a position corresponding to Y136 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 104.

[0317] In some embodiments, a cytosine deaminase variant comprises increased activity for a cytosine positioned between an A and a T (e.g., a “ACT” motif). In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to N23 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to Y132 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 72.

[0318] In some embodiments, a cytosine deaminase variant comprises increased specificity (e.g., relative to a wildtype cytosine deaminase) for a cytosine positioned between an A and a C (e.g., a “ACC” motif) compared to other three nucleotide motifs (e.g., at least 10%, at least 25%, at least 50%, at least 75%, or at least 100% increased activity for the ACC motif compared to other three nucleotide motifs). In some embodiments, the cytosine deaminase variant comprises an alanine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the

[0319] #14540497v1 cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 58. In some embodiments, the cytosine deaminase variant comprises a cysteine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 59. In some embodiments, the cytosine deaminase variant comprises a glycine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 62. In some embodiments, the cytosine deaminase variant comprises a valine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 68.

[0320] In some embodiments, a cytosine deaminase variant comprises increased specificity (e.g., relative to a wildtype cytosine deaminase) for a cytosine positioned between an A and an A (e.g., a “ACA” motif) compared to other three nucleotide motifs (e.g., at least 10%, at least 25%, at least 50%, at least 75%, or at least 100% increased activity for the ACA motif compared to other three nucleotide motifs). In some embodiments, the cytosine deaminase variant comprises a valine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 68. In some embodiments, the cytosine deaminase variant comprises a tyrosine at a position corresponding to D 131 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 70. In some embodiments, the cytosine deaminase variant comprises a asparagine at a position corresponding to R28 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 26.

[0321] In some embodiments, a cytosine deaminase variant comprises increased specificity (e.g., relative to a wildtype cytosine deaminase) for a cytosine positioned between an T and an A (e.g., a “TCA” motif) compared to other three nucleotide motifs (e.g., at least 10%, at least 25%, at least 50%, at least 75%, or at least 100% increased activity for the TCA motif compared to other three nucleotide motifs). In some embodiments, the cytosine deaminase variant comprises a valine at a position corresponding to K60 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the cytosine deaminase variant comprises an aspartic acid at a position corresponding to N24 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the cytosine deaminase variant comprises an alanine at a position corresponding to R 128 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of

[0322] #14540497v1 SEQ ID NO: 51. In some embodiments, the cytosine deaminase variant comprises an glutamine at a position corresponding to R128 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 55.

[0323] In some embodiments, a cytosine deaminase variant comprises increased specificity (e.g., relative to a wildtype cytosine deaminase) for a cytosine positioned between a C and a C (e.g., a “CCC” motif) compared to other three nucleotide motifs (e.g., at least 10%, at least 25%, at least 50%, at least 75%, or at least 100% increased activity for the CCC motif compared to other three nucleotide motifs). In some embodiments, the cytosine deaminase variant comprises an isoleucine at a position corresponding to N24 of SEQ ID NO: 1. In some embodiments, the cytosine deaminase variant comprises the amino acid sequence of SEQ ID NO: 14.

[0324] EXAMPLES

[0325] Example 1. Screening of APOBEC3A Variants

[0326] Cytosine deaminases are enzymes that deaminate (remove an amine) cytosine bases to produce uracil, which is converted to thymine following DNA synthesis (e.g., polymerase chain reaction, PCR). Cytosine deaminases can be fused to DNA binding protein (like catalytically dead Cas9); such enzymes are useful for genome editing, but can promiscuously deaminate any cytosine within 20 base pairs of a particular target motif, leading to local and global off-target effects when used for genome editing. Local off-target effects can be a result of a cytosine deaminases that can deaminate multiple different sequence motifs with similar a preference. Global off-target effects may be a result of off-target binding of the DNA binding protein combined with local off-target effects of the deaminase. To generate cytosine deaminases with higher sequence context preference, and thus reduced off-target effects, thousands of cytosine deaminase variants having mutations compared to a wild-type human APOB EC 3A were produced and tested for sequence context (e.g., sequence motif) preference.

[0327] An E. coli vector was developed in which DNA encoding an APOBEC3A variant could be inserted upstream of dCas9 and downstream of the AraC promoter, allowing for induction of a APOBEC3A-dCas9 fusion gene using arabinose. The plasmid also encoded a single guide RNA (sgRNA) under control of an IPTG-inducible promoter. The APOBEC3A variant was flanked by target sites for the sgRNA. When bacteria containing this plasmid were grown in media with arabinose and IPTG, the APOBEC3A-dCas9 fusion and the sgRNA were both expressed. The dCas9 subsequently brought the APOBEC3A to the target sites flanking APOBEC3A coding sequence. The dCas9-gRNA complex opened the DNA at the target site forming a DNA-RNA hybrid with one strand and leaving the other strand single- stranded. This

[0328] #14540497v1 gave the AP0BEC3A deaminase variant the opportunity to edit two C’s in the single- stranded portion. One of the C’s was preceded by a G and the other by a C, giving the variant the opportunity to mutate C’s in two contexts.

[0329] A single variant site APOBEC3A library with thousands of APOBEC3A variants was synthesized and ligated into this construct. The ligation reaction was transformed into E. coli. The transformation reaction was grown for 2.5 hours in minimal media without arabinose and IPTG, but with 2% glucose to minimize leaky expression from the construct. Next the cells were transferred to minimal media containing arabinose and IPTG and grown for 1 to 4 hours. The cells were then lysed and the plasmid population from the culture was purified using a plasmid miniprep.

[0330] Each plasmid copy contained two pairs of C’s on either side of the APOBEC3A coding sequence and each C could be deaminated or not, resulting in four possible mutation states for each pair of C’s. After induction, the C’s could become “CC” (unmutated), “T(orU)C” (only GC context mutated), “CT(orU)” (only CC context mutated), and “TT(or UU)” (both contexts mutated).

[0331] To determine which variants deaminated each of the C’s, two methods of amplifying and sequencing the target DNA were used.

[0332] In the first method, PCR was performed using primers specific to one of the four mutation states. To ensure that the PCR was specific to just one mutation state, RNase Il- dependent PCR (rhPCR) was used. Experiments using synthetic rhPCR targets and the rhPCR primers specific for each state indicated that in each case, a target with a given mutation state could be specifically amplified while only minimally amplifying other states. To get a complete picture of the mutations caused by each variant, four rhPCRs were performed along with a PCR using primers which flank the mutation target sites therefore amplified the entire pool of variants. Each of these PCR products was fragmented and converted into a sequencing library then sequenced on an Illumina NextSeq. The frequency of each variant in each pool was counted and then normalized to the read count of the synonymous variants in the reaction, and then normalized again to the frequency of that variant in the PCR with the flanking primers. This provided a relative enrichment level for each variant in each mutation context. A higher relative enrichment level indicated that the variant had an increased tendency to mutate that C or pair of C’s relative to the WT enzyme.

[0333] In the second method, PCR was performed using primers that flank the deaminase target sites to generate amplicons that contained the target sites and the entire coding sequence of APOBEC3A variants. An Oxford Nanopore sequencing library (long-read sequencing library)

[0334] #14540497v1 was generated from the amplicons, and the library was sequenced using the Minion sequencer, which sequenced entire amplicons. Long-reads generated using the Minion sequencer allowed for the direct linking of each encoded APOBEC3A variant to the deamination that the respective variant induced at its target sites. Results indicated that certain substitutions at positions 23, 24, 27-30, 60, 62, 98, 102, 103, 126, 129, 131, 132, 134-136 and 186 impact APOBEC3A CC or GC preference (FIG. 4 and Table 1). Additionally, control data from Oxford Nanopore sequencing illustrated that variants having non-synonymous mutations as positions previously shown to be critical for deaminase activity have much lower activity compared to variants having synonymous mutations at those same positions, verifying the data identifying novel variants with increased deaminase activity (FIG. 5). Variants were identified as having a preference for GC motifs (“GC-specific variants”) where the ratio of the GC p-value over the CC p-value was less than 1 x 10’5. Variants were identified as having a preference for CC motifs (“CC-specific variants”) where the ratio of the CC p-value over the GC p-value was less than 1 x 10’5. See Table 1 for a list of variants that have a CC preference or a GC preference. The distribution of GC- and CC-specific variants compared to variants that edited either GC or CC motifs or neither GC nor CC motifs is shown in FIG. 2 (control variants with synonymous mutations shown in FIG. 2A; experimental variants shown in FIG. 2B) (see Methods for more details). Of the APOBEC3A positions described to influence deaminase preference

[0335] FIG. 3 shows an alignment of APOBEC3A with mouse APOBEC, four additional human APOBEC3s, and AID (all helix-4 deaminases). Homologous mutations at positions identified in APOBEC3A (indicated by arrows) as conferring sequence context preference may confer similar preference in other helix-4 deaminases.

[0336] Further experiments were performed to identify APOBEC3A variants with markedly higher or lower activity compared to wild-type APOBEC3A at specific xCx (where “x” is a nucleobase; also called NCN) motifs. Briefly, the APOBEC3A library was ligated into base editor constructs with target sites containing at least one example of all 16 possible NCN sequences (FIG. 6A). Expression of the base editor and the guide RNA was then induced and cells were incubated for 1-4 hours, and plasmids from the cells were harvested. Target sites and variants were then sequenced using Oxford Nanopore sequencing. For each target basedeaminase variant combination, the frequency of a base that was a C (unmutated) was compared to how often it was a T (mutated). The mutation was therefore calculated as: (mutated counts) / (mutated counts + unmutated counts)

[0337] The variant library contained numerous synonymous variants. The average mutation rate for each target base in the presence of a synonymous variant was calculated to get a baseline

[0338] #14540497v1 reading for the behavior of wild-type AP0BEC3A. For every non- synonymous variant, an “Activity Level Relative to WT” was calculated as:

[0339] (mutation rate in variant) / (average mutation rate among synonymous variants

[0340] An “Activity Level Relative to WT” value of 1 indicated that the variant mutated the target base with a similar rate to synonymous variants. A value of less than 1 indicated it is less active and a value of more than 1 indicated that it is more active (FIG. 6B).

[0341] Taken together, the data demonstrate that the cytosine deaminase variants identified herein are highly specific for particular motifs (e.g., CC motifs and / or GC motifs) and are thus useful in gene editing applications to target cytosine bases for editing while reducing local and global off-target editing.

[0342] Methods

[0343] Variants with preference differences for the “GC” or “CC” contexts were defined using a statistical test:

[0344] 1. The variant contained in each sequencing read was determined and each target site in each sequencing read was scored as having: a. Both C’s mutated. b. Only the C in the “CC” context mutated. c. Only the C in the “GC” context mutated. d. Neither C mutated.

[0345] 2. For each variant the percentage of scored targets sites from each of those four categories was calculated.

[0346] 3. Distributions of the “CC”-only mutated (category b) and “GC”-only mutated (category c) percentages for the synonymous variants were calculated.

[0347] 4. A “CC”-only p-value and “GC”-only p-value was calculated for each variant in the panel using the distribution calculated for the synonymous variants as a null distribution.

[0348] 5. Variants where the p-value for the “CC”-only category was 105-times lower than the p- value for the “GC”-only category were categorized as having a “preference for CC context over GC”.

[0349] 6. Variants where the p-value for the “GC”-only category was 105-times lower than the p- value for the “CC”-only category were categorized as having a “preference for GC context over CC”.

[0350] #14540497v1 Table 1. Sequences of exemplary cytosine deaminase variants.

[0351] #14540497v1

[0352] #14540497v1

[0353] #14540497v1

[0354] #14540497v1

[0355] #14540497v1

[0356] #14540497v1

[0357] #14540497v1

[0358] #14540497v1

[0359] #14540497v1

[0360] #14540497v1

[0361] #14540497v1

Claims

CLAIMSWhat is claimed is:

1. A cytosine deaminase variant comprising one or more of:(i) an aspartic acid, isoleucine, leucine, or valine at a position corresponding to N24 of SEQ ID NO: 1;(ii) an aspartic acid, cysteine, phenylalanine, glutamic acid, histidine, methionine, asparagine, or threonine at a position corresponding to G27 of SEQ ID NO: 1;(iii) an isoleucine, methionine, or leucine at position corresponding to W98 of SEQ ID NO: 1;(iv) a glycine or asparagine at position corresponding to F102 of SEQ ID NO: 1;(v) a cysteine, isoleucine, asparagine, or valine at a position corresponding to S103 of SEQ ID NO: 1;(vi) an alanine, aspartic acid, methionine, glutamine, glycine, or asparagine at a position corresponding to R 128 of SEQ ID NO: 1;(vii) an alanine, cysteine, glutamic acid, phenylalanine, glycine, methionine, tyrosine, histidine, isoleucine, leucine, threonine, valine, or tryptophan at a position corresponding to D131 of SEQ ID NO: 1;(viii) a cysteine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, lysine, methionine, asparagine, glutamine, arginine, serine, threonine, or valine at a position corresponding to Y 132 of SEQ ID NO: 1;(ix) an aspartic acid or glutamic acid at a position corresponding to L135 of SEQ ID NO: 1;(x) an alanine, cysteine, glutamic acid, glycine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, or valine at a position corresponding to Y 136 of SEQ ID NO: 1; or(xi) an aspartic acid or glutamic acid at a position corresponding to LI 86.

2. The cytosine deaminase variant of claim 1, comprising an aspartic acid, isoleucine, leucine, or valine at a position corresponding to N24 of SEQ ID NO: 1.

3. The cytosine deaminase variant of claim 1 or claim 2, comprising an aspartic acid, cysteine, phenylalanine, glutamic acid, histidine, methionine, asparagine, or threonine at a position corresponding to G27 of SEQ ID NO: 1.#14540497v14. The cytosine deaminase variant of any one of claims 1-3, comprising an isoleucine, methionine, or leucine at a position corresponding to W98 of SEQ ID NO: 1.

5. The cytosine deaminase variant of any one of claims 1-4, comprising a glycine or asparagine at a position corresponding to F102 of SEQ ID NO: 1.

6. The cytosine deaminase variant of any one of claims 1-5, comprising a cysteine, isoleucine, asparagine, or valine at a position corresponding to SI 03 of SEQ ID NO: 1.

7. The cytosine deaminase variant of any one of claims 1-6, comprising an alanine, aspartic acid, methionine, glutamine, glycine, or asparagine at a position corresponding to R128 of SEQ ID NO: 1.

8. The cytosine deaminase variant of any one of claims 1-7, comprising an alanine, cysteine, glutamic acid, phenylalanine, glycine, methionine, tyrosine, histidine, isoleucine, leucine, threonine, valine, or tryptophan at a position corresponding to D 131 of SEQ ID NO: 1.

9. The cytosine deaminase variant of any one of claims 1-8, comprising a cysteine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, lysine, methionine, asparagine, glutamine, arginine, serine, threonine, or valine at a position corresponding to Y132 of SEQ ID NO: 1.

10. The cytosine deaminase variant of any one of claims 1-9, comprising an aspartic acid or glutamic acid at a position corresponding to LI 35 of SEQ ID NO: 1.

11. The cytosine deaminase variant of any one of claims 1-10, comprising an alanine, cysteine, glutamic acid, glycine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, threonine, or valine at a position corresponding to Y136 of SEQ ID NO: 1.

12. The cytosine deaminase variant of any one of claims 1-11, comprising an aspartic acid or glutamic acid at a position corresponding to LI 86.#14540497v113. The cytosine deaminase variant of claim 1 or claim 2, comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 1-10.

14. The cytosine deaminase variant of claim 1, comprising an amino acid sequencing having at least 95% identity to any one of SEQ ID NOs: 13-22, 41-49, 51-55, 58-86, 89-106, or 110- 111.

15. The cytosine deaminase variant of claim 14, comprising an amino acid sequence of any one of SEQ ID NOs: 13-22, 41-49, 51-55, 58-86, 89-106, or 110-111.

16. The cytosine deaminase variant of any one of claims 1-15, wherein the cytosine deaminase variant has increased cytosine-cytosine domain preference relative to a cytosine deaminase of SEQ ID NO: 1.

17. The cytosine deaminase variant of any one of claims 1-16, wherein the cytosine deaminase is an apolipoprotein B mRNA editing enzyme, catalytic polypeptide (APOBEC).

18. A cytosine deaminase variant comprising one or more of:(i) glycine, leucine, or glutamic acid at a position corresponding to N23 of SEQ ID NO: 1;(ii) tyrosine, phenylalanine, glycine, histidine, asparagine, or tryptophan at a position corresponding to R28 of SEQ ID NO: 1;(iii) asparagine, alanine, serine, proline, aspartic acid, glutamine, threonine, glycine, lysine, or cysteine at a position corresponding to H29 of SEQ ID NO: 1;(iv) valine at a position corresponding to K60 of SEQ ID NO: 1;(v) glutamic acid at a position corresponding to L62 of SEQ ID NO: 1;(vi) alanine at a position corresponding to S103 of SEQ ID NO: 1;(vii) tryptophan at a position corresponding to VI 10 of SEQ ID NO: 1;(viii) glycine or aspartic acid at a position corresponding to 1129 of SEQ ID NO: 1;(ix) valine at a position corresponding to D 131 of SEQ ID NO: 1;(x) proline at a position corresponding to Y132 of SEQ ID NO: 1; and(xi) lysine or arginine at a position corresponding to Pl 34 of SEQ ID NO: 1.#14540497v119. The cytosine deaminase variant of claim 18, comprising glycine, leucine, or glutamic acid at a position corresponding to N23 of SEQ ID NO: 1.

20. The cytosine deaminase variant of claim 18, comprising tyrosine, phenylalanine, glycine, histidine, asparagine, or tryptophan at a position corresponding to R28 of SEQ ID NO: 1.

21. The cytosine deaminase variant of claim 18, comprising asparagine, alanine, serine, proline, aspartic acid, glutamine, threonine, glycine, lysine, or cysteine at a position corresponding to H29 of SEQ ID NO: 1.

22. The cytosine deaminase variant of claim 18, comprising valine at a position corresponding to K60 of SEQ ID NO: 1.

23. The cytosine deaminase variant of claim 18, comprising glutamic acid at a position corresponding to L62 of SEQ ID NO: 1.

24. The cytosine deaminase variant of claim 18, comprising alanine at a position corresponding to SI 03 of SEQ ID NO: 1.

25. The cytosine deaminase variant of claim 18, comprising glycine or aspartic acid at a position corresponding to 1129 of SEQ ID NO: 1.

26. The cytosine deaminase variant of claim 18, comprising tryptophan at a position corresponding to VI 10 of SEQ ID NO: 1.

27. The cytosine deaminase variant of claim 18, comprising valine at a position corresponding to D 131 of SEQ ID NO:

128. The cytosine deaminase variant of claim 18, comprising proline at a position corresponding to Y 132 of SEQ ID NO: 1.

29. The cytosine deaminase variant of claim 18, comprising lysine or arginine at a position corresponding to Pl 34 of SEQ ID NO: 1.#14540497v130. The cytosine deaminase variant of any one of claims 18-29, comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 1-10.

31. The cytosine deaminase variant of claim 18, comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 11-12, 23-40, 50, 56-67, or 87-88.

32. The cytosine deaminase variant of claim 14, comprising an amino acid sequence of any one of SEQ ID NOs: 11-12, 23-40, 50, 56-57, or 87-88.

33. The cytosine deaminase variant of any one of claims 18-32, comprising increased cyto sine-guano sine domain preference relative to a cytosine deaminase of SEQ ID NO: 1.

34. The cytosine deaminase variant of any one of claims 18-33, wherein the cytosine deaminase is a helix-4 deaminase.

35. The cytosine deaminase variant of any one of claims 18-32 or 34, wherein the cytosine deaminase is an apolipoprotein B mRNA editing enzyme, catalytic polypeptide (APOBEC).

36. A base editor comprising a DNA binding protein and the cytosine deaminase variant of any one of claims 1-35.

37. The base editor of claim 36, wherein the DNA binding protein is a CRISPR associated (Cas) protein.

38. The base editor of claim 37, wherein the Cas protein is catalytically inactive.

39. The base editor of claim 38, wherein the Cas protein is a catalytically inactive Cas9 protein.

40. The base editor of claim 39, comprising a peptide linker between the DNA binding domain and the cytosine deaminase.#14540497v141. A polynucleotide encoding the cytosine deaminase variant of any one of claims 1-35 or the base editor of any one of claims 36-40.

42. A polynucleotide comprising:(a) a nucleic acid encoding a base editor comprising a deaminase variant and a polynucleotide binding protein; and(b) a base editor target site comprising a first polynucleotide binding protein sequence motif and a first deaminase sequence motif.

43. A polynucleotide comprising:(a) a nucleic acid encoding a base editor comprising a deaminase variant and a polynucleotide binding protein; and(b) a first base editor target site and a second base editor target site that flank the nucleotides encoding the deaminase variant of the nucleic acid; wherein the first base editor target site comprises a first polynucleotide binding protein sequence motif and a first deaminase sequence motif; and wherein the second base editor target site comprises a second polynucleotide binding protein sequence motif and a second deaminase sequence motif.

44. The polynucleotide of claim 43, wherein the polynucleotide binding protein is a catalytically inactive Cas protein.

45. The polynucleotide of claim 44, comprising a guide RNA comprising a homology region that is complementary to the first polynucleotide binding protein sequence motif and / or the second polynucleotide binding protein sequence motif.

46. A method of determining sequence preference of a plurality of deaminase variants, the method comprising:(i) transfecting a plurality of cells with a plurality of vectors, each vector of the plurality of vectors comprising:(a) a nucleic acid encoding a base editor comprising a deaminase variant and a polynucleotide binding protein; and(b) a first base editor target site;#14540497v1wherein the first base editor target site comprises a first polynucleotide binding protein sequence motif and a first deaminase sequence motif;(ii) amplifying vectors of the plurality of vectors that comprise an edited first deaminase sequence motif to produce amplicons that comprise a nucleic acid encoding the deaminase variant; and(iii) sequencing the amplicons to determine the sequence preference of the plurality of deaminase variants for the first deaminase sequence motif.

47. The method of claim 46, wherein sequencing comprises sequencing a contiguous polynucleotide comprising the nucleic acid encoding the deaminase variant and the first base editor target site using long-read sequencing.

48. The method of claim 46 or claim 47, wherein the amplifying comprising contacting plurality of vectors with a first primer that is complementary to an edited first deaminase sequence motif.

49. A method of determining sequence preference of a plurality of deaminase variants, the method comprising:(i) transfecting a plurality of cells with a plurality of vectors, each vector of the plurality of vectors comprising:(a) a nucleic acid encoding a base editor comprising a deaminase variant and a polynucleotide binding protein; and(b) a first base editor target site and a second base editor target site that flank the nucleotides encoding the deaminase variant of the nucleic acid; wherein the first base editor target site comprises a first polynucleotide binding protein sequence motif and a first deaminase sequence motif; wherein the second base editor target site comprises a second polynucleotide binding protein sequence motif and a second deaminase sequence motif;(ii) amplifying vectors of the plurality of vectors that comprise an edited first deaminase sequence motif and / or an edited second deaminase sequence motif to produce amplicons that comprise a nucleic acid encoding the deaminase variant, the amplifying comprising contacting the plurality of vectors with:#14540497v1(a) a first primer that is complementary to an edited first deaminase sequence motif and a second primer that is complementary to the vector;(b) a first primer that is complementary to an edited second deaminase sequence motif and a second primer that is complementary to the vector; or(c) a first primer that is complementary to an edited first deaminase sequence motif and a second primer that is complementary to an edited second deaminase sequence motif; and(iii) sequencing the amplicons to identify deaminase variants that edited the first deaminase sequence motif and / or the second deaminase sequence motif.

50. The method of claim 49, wherein the first deaminase sequence motif and the second deaminase sequence motif are the same motif.

51. The method of claim 49, wherein the first deaminase sequence motif and the second deaminase sequence motif are different motifs.

52. The method of any one of claims 49-51, wherein the first deaminase sequence motif and / or the second deaminase sequence motif comprise an AC motif, a CC motif, a GC motif or a TC motif.

53. The method of any one of claims 46-52, wherein amplifying comprises amplifying using RNase H-dependent PCR.#14540497v1

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