Adenosine deaminase variants and uses thereof

Abstract

The present invention provides adenosine deaminase variants capable of deaminating adenine and / or cytosine in a target polynucleotide (e.g., DNA). The present disclosure also provides multimolecular complexes, fusion proteins, base editors, and base editing systems comprising adenosine deaminase variants having adenine and cytosine deaminase activity and / or cytosine deaminase specificity, and methods of use thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 229,057, filed August 3, 2021, and U.S. Provisional Application No. 63 / 166,778, filed March 26, 2021, the entire contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The above ASCII copy, created on March 25, 2022, is designated 180802-048002PCT_SL and is 2,073,042 bytes in size. [Background technology]

[0003] Deaminases, such as adenosine deaminase and cytidine deaminase, have been used in systems and compositions useful for targeted editing of nucleic acid sequences. For example, targeted cleavage and / or modification of genomic DNA is a highly promising approach for studying gene function and may even provide novel therapeutics for human genetic diseases. Currently available base editors and base editing systems utilize cytidine deaminase, which converts targeted C·G base pairs to T·A, and / or adenosine deaminase, which converts A·T base pairs to G·C. Exemplary base editors previously described include cytidine base editors (e.g., BE4), adenine base editors (e.g., ABE7.10), and base editors containing both adenine deaminase and cytidine deaminase. There is a need in the art for improved base editors that can induce modifications within target sequences with greater diversity, specificity, and efficiency. Summary of the Invention

[0004] As described below, the present disclosure features adenosine deaminase variants capable of deaminating adenine and / or cytosine in a target polynucleotide (e.g., DNA), and adenosine deaminase variants capable of predominantly deaminating cytosine in a target polynucleotide. For example, aspects of the disclosure provide adenosine deaminase variants that have increased cytosine deaminase activity and / or cytosine deaminase specificity (e.g., about 10-fold, 30-fold, 50-fold, 70-fold, or more) compared to a reference adenosine deaminase (e.g., TadA*8.20 or TadA*8.19). In embodiments, the adenosine deaminase variant maintains the level of adenosine deaminase activity (e.g., at least about 30%, 40%, 50%, 60%, 70%, or more of the activity) of a reference adenosine deaminase (e.g., TadA*8.20, TadA*8.19, or any of the variants listed in Tables 1A-1F (e.g., 1.2, 1.4, 1.6, 1.12, 1.14, 1.17, or 1.19)).

[0005] Aspects of the present invention relate to novel engineered deaminases, as well as methods and compositions comprising the same, generated through multiple iterations of protein engineering (e.g., directed evolution, structure-based combinatorial screening, and mutation-based combinatorial screening). For example, an adenosine deaminase (e.g., ABE8 TadA*) capable of deaminating adenine in DNA was engineered from an ssDNA-acting deaminase of adenine to a deaminase capable of deaminating both adenine and cytosine (e.g., in nucleic acid molecules). Further engineering efforts have succeeded in altering the specificity of the adenosine deaminase so that it deaminates cytosine (e.g., in DNA) without significant adenosine deaminase activity. Successive rounds of such directed evolution, crystal structure-based combinatorial screening, and mutation-based combinatorial screening have yielded at least two novel classes of base editors derived from TadA*: (1) cytosine and adenine base editors (CABEs) that perform both adenine-to-guanine (A-to-G) and cytosine-to-thymine (C-to-T) base editing within the same editing window, and (2) TadA*-derived cytosine base editors (CBE-Ts) that possess predominantly cytidine deaminase activity (e.g., with little or no detectable adenosine deaminase activity).

[0006] The engineered deaminases provided herein have advantages over existing deaminases. For example, when used in conjunction with base editors, such deaminases may result in improved off-target properties (CBE-T / TadC has lower guide-independent off-targets compared to BE4 rAPOBEC) and may have a more precise editing window (CBE-T / TadC has a more focused editing window compared to BE4 rAPOBEC, and allele distribution data supports this). Furthermore, with respect to on-target editing, the data provided herein indicate that CBE-T may be at least as active as BE4.

[0007] In one aspect, the disclosed invention features an adenosine deaminase variant that has increased cytidine deaminase activity and / or increased cytidine deaminase specificity relative to a reference adenosine deaminase, wherein the adenosine deaminase variant includes two or more amino acid modifications relative to the reference adenosine deaminase.

[0008] In another aspect, the disclosed invention features adenosine deaminase variants having increased cytidine deaminase activity and / or increased cytidine deaminase specificity relative to a reference adenosine deaminase, wherein the adenosine deaminase variants include modifications relative to the reference adenosine deaminase in one or more of region A comprising amino acid residues 82-84, region B comprising amino acid residues 27-30 and 47-49, region C comprising residues 107-115, or the C-terminal helix comprising residues 139-167: 10 20 30 40 50 MSEVEFSHEY WMRHALTLAK RARDEREVPV GAVLVLNNRV IGEGWNRAIG 60 70 80 90 100 LHDPTAHAEI MALRQGGLVM QNYRLYDATL YSTFEPCVMC AGAMIHSRIG 110 120 130 140 150 RVVFGVRNAK TGAAGSLMDV LHHPGMNHRV EITEGILADE CAALLCRFFR 160 MPRRVFNAQK KAQSSTD (SEQ ID NO: 1).

[0009] In another aspect, the invention of the present disclosure provides a method for the production of a medicament comprising the following sequence: 10 20 30 40 50 MSEVEFSHEY WMRHALTLAK RARDEREVPV GAVLVLNNRV IGEGWNRAIG 60 70 80 90 100 LHDPTAHAEI MALRQGGLVM QNYRLYDATL YSTFEPCVMC AGAMIHSRIG 110 120 130 140 150 RVVFGVRNAK TGAAGSLMDV LHHPGMNHRV EITEGILADE CAALLCRFFR 160 MPRRVFNAQK KAQSSTD (SEQ ID NO: 1) The present invention features adenosine deaminase variants that contain one or more modifications in an amino acid sequence having at least about 70% or greater identity to SEQ ID NO: 1. The adenosine deaminase variants have increased cytidine deaminase activity and / or increased cytidine deaminase specificity compared to a reference adenosine deaminase. The one or more modifications do not include an R amino acid at position 48 of SEQ ID NO: 1, or a corresponding modification in another adenosine deaminase.

[0010] In another aspect, the invention of the present disclosure provides a method for the production of a medicament comprising the following sequence: 10 20 30 40 50 MSEVEFSHEY WMRHALTLAK RARDEREVPV GAVLVLNNRV IGEGWNRAIG 60 70 80 90 100 LHDPTAHAEI MALRQGGLVM QNYRLYDATL YSTFEPCVMC AGAMIHSRIG 110 120 130 140 150 RVVFGVRNAK TGAAGSLMDV LHHPGMNHRV EITEGILADE CAALLCRFFR 160 MPRRVFNAQK KAQSSTD (SEQ ID NO: 1) and adenosine deaminase variants comprising one or more modifications at amino acid positions selected from one or more of 2, 4, 6, 13, 27, 29, 100, 112, 114, 115, 162, and 165 of an amino acid sequence having at least about 70% or greater identity to, or corresponding positions in another adenosine deaminase.

[0011] In another aspect, the invention of the present disclosure provides a method for the production of a medicament comprising the following sequence: 10 20 30 40 50 MSEVEFSHEY WMRHALTLAK RARDEREVPV GAVLVLNNRV IGEGWNRAIG 60 70 80 90 100 LHDPTAHAEI MALRQGGLVM QNYRLYDATL YSTFEPCVMC AGAMIHSRIG 110 120 130 140 150 RVVFGVRNAK TGAAGSLMDV LHHPGMNHRV EITEGILADE CAALLCRFFR 160 MPRRVFNAQK KAQSSTD (SEQ ID NO: 1) S2H, V4K, V4S, V4T, V4Y, F6G, F6H, F6Y, H8Q, R13G, T17A, T17W, R23Q, E27C, E27G, E27H, E27K, E27Q, E27S, E27G, P29A, P29G, P29K, V30F, V30I, R47G, R47S, A48G, I49K, I49M, I49N, I49Q, I49T, G67W, I76H, I76R, I76W, Y76H, Y76R, Y7 6W, F84A, F84M, H96N, G100A, G100K, T111H, G112H, A114C, G115M, M118L, H122G, H122R, H122T, N127I, N127K, N127P, A142E, R147H, A158V, Q159S, A162C, A162N, A162Q, and S165P, or a corresponding modification in another deaminase.

[0012] In another aspect, the invention of the present disclosure provides a method for the production of a medicament comprising the following sequence: 10 20 30 40 50 MSEVEFSHEY WMRHALTLAK RARDEREVPV GAVLVLNNRV IGEGWNRAIG 60 70 80 90 100 LHDPTAHAEI MALRQGGLVM QNYRLYDATL YSTFEPCVMC AGAMIHSRIG 110 120 130 140 150 RVVFGVRNAK TGAAGSLMDV LHHPGMNHRV EITEGILADE CAALLCRFFR 160 MPRRVFNAQK KAQSSTD (SEQ ID NO: 1) E27H, Y76I, and F84M; E27H, I49K, and Y76I; E27S, I49K, Y76I, and A162N; E27K and D119N; E27H and Y76I; E27S, I49K, and G67W; E27S, I49K, and Y76I; I49T, G67W, and H96N; E27C, Y76I, and D119N; R13G, E27Q, and N127K; T17A, E27H, I49M, Y76I, and M118L; I49Q, Y76I, and G115M; S2H, I49K, Y76I, and G112H; R47S and R107C; H8Q, I49Q, and Y76I; T17A, A48G, S82T, and A142E; E27G and I49N; E27G, D77G, and S165P; E27S, I49K, and S82T; E27S, I49K, S82T, and G115M; E27S, V30I, I49K, and S82T; E27S, V30F, I49K, S82T, F84A, R107C, and A142E; E27S, V30F, I49K, S82T, F84A, G112H, and A142E; E27S, V30F, I49K, S82T, F84A, G115M, and A142E; E27S, I49K, S82T, F84L, and R107C; E27S, I49K, S82T, F84L, and G112H; E27S, I49K, S82T, F84L, and G115M; E27S, I49K, S82T, F84L, R107C, and G112H; E27S, I49K, S82T, F84L, R107C, and G115M; E27S, I49K, S82T, F84L, R107C, and A142E; E27S, I49K, S82T, F84L, G112H, and A142E; E27S, I49K, S82T, F84L, G115M, and A142E; E27S, I49K, S82T, F84L, R107C, G112H, G115M, and A142E; E27S, V30I, I49K, S82T, and F84L; E27S, P29G, I49K, and S82T; E27S, P29G, I49K, S82T, and G115M; E27S, P29G, I49K, S82T, and A142E; P29G, I49K, and S82T;E27G, I49K, and S82T; E27G, I49K, S82T, R107C, and A142E; V4K, E27H, I49K, Y76I, and A114C; V4K, E27H, I49K, Y76I, and D77G; F6Y, E27H, I49K, Y76I, G100A, and H122R; V4T, E27H, I49K, Y76R, and H122G; F6Y, E27H, I49K, and Y76W; F6Y, E27H, I49K, Y76I, and D119N; F6Y, E27H, I49K, Y76I, and A114C; F6Y, E27H, I49K, and Y76I; V4K, E27H, I49K, Y76W, and H122T; F6G, E27H, I49K, Y76R, and G100K; F6H, E27H, I49K, Y76I, and H122N; E27H, I49K, Y76I, and A114C; F6Y, E27H, I49K, Y76H, H122R, and T166I; E27H, I49K, Y76I, and N127P; R23Q, E27H, I49K, and Y76R; E27H, I49K, Y76H, H122R, and A158V; F6Y, E27H, I49K, Y76I, and T111H; E27H, I49K, Y76I, and R147H; E27H, I49K, Y76I, and A143E; F6Y, E27H, I49K, and Y76R; T17W, E27H, I49K, Y76H, H122G, and A158V; V4S, E27H, I49K, A143E, and Q159S; E27H, I49K, Y76I, N127I, and A162Q; T17A, E27H, and A48G; T17A, E27K, and A48G; T17A, E27S, and A48G; T17A, E27S, A48G, and I49K; T17A, E27G, and A48G; T17A, A48G, and I49N; T17A, E27G, A48G, and I49N; T17A, E27Q, and A48G; E27S, I49K, S82T, and R107C; E27S, I49K, S82T, and G112H; E27S, I49K, S82T, and A142E; E27S, I49K, S82T, R107C, and G112H; E27S, I49K, S82T, R107C, and G115M; E27S, I49K, S82T, R107C, and A142E;E27S, I49K, S82T, G112H, and A142E; E27S, I49K, S82T, G115M, and A142E; E27S, I49K, S82T, R107C, G112H, G115M, and A142E; E27S, V30I, I49K, S82T, and R107C; E27S, V30I, I49K, S82T, and G112H; E27S, V30I, I49K, S82T, and G115M; E27S, V30I, I49K, S82T, and A142E; E27S, V30I, I49K, S82T, R107C, and G112H; E27S, V30I, I49K, S82T, R107C, and G115M; E27S, V30I, I49K, S82T, R107C, and A142E; E27S, V30I, I49K, S82T, G112H, and A142E; E27S, V30I, I49K, S82T, G115M, and A142E; E27S, V30I, I49K, S82T, R107C, G112H, G115M, and A142E; E27S, V30L, I49K, and S82T; E27S, V30L, I49K, S82T, and R107C; E27S, V30L, I49K, S82T, and G112H; E27S, V30L, I49K, S82T, and G115M; E27S, V30L, I49K, S82T, and A142E; E27S, V30L, I49K, S82T, R107C, and G112H; E27S, V30L, I49K, S82T, R107C, and G115M; E27S, V30L, I49K, S82T, R107C, and A142E; E27S, V30L, I49K, S82T, G112H, and A142E; E27S, V30L, I49K, S82T, G115M, and A142E; E27S, V30L, I49K, S82T, R107C, G112H, G115M, and A142E; E27S, V30F, I49K, S82T, and F84A; E27S, V30F, I49K, S82T, F84A, and R107C; E27S, V30F, I49K, S82T, F84A, and G112H; E27S, V30F, I49K, S82T, F84A, and G115M; E27S, V30F, I49K, S82T, F84A, and A142E;E27S, V30F, I49K, S82T, F84A, R107C, and G112H; E27S, V30F, I49K, S82T, F84A, R107C, and G115M; E27S, V30F, I49K, S82T, F84A, R107C, G112H, G115M, and A142E; E27S, I49K, S82T, and F84L; E27S, I49K, S82T, F84L, and A142E; E27S, V30I, I49K, S82T, F84L, and R107C; E27S, V30I, I49K, S82T, F84L, and G112H; E27S, V30I, I49K, S82T, F84L, and G115M; E27S, V30I, I49K, S82T, F84L, and A142E; E27S, V30I, I49K, S82T, F84L, R107C, and G112H; E27S, V30I, I49K, S82T, F84L, R107C, and G115M; E27S, V30I, I49K, S82T, F84L, R107C, and A142E; E27S, V30I, I49K, S82T, F84L, G112H, and A142E; E27S, V30I, I49K, S82T, F84L, G115M, and A142E; E27S, V30I, I49K, S82T, F84L, R107C, G112H, G115M, and A142E; E27S, P29G, I49K, S82T, and R107C; E27S, P29G, I49K, S82T, and G112H; E27S, P29G, I49K, S82T, R107C, and G112H; E27S, P29G, I49K, S82T, R107C, and G115M; E27S, P29G, I49K, S82T, R107C, and A142E; E27S, P29G, I49K, S82T, G112H, and A142E; E27S, P29G, I49K, S82T, G115M, and A142E; E27S, P29G, I49K, S82T, R107C, G112H, G115M, and A142E; P29G, I49K, S82T, and R107C; P29G, I49K, S82T, and G112H; P29G, I49K, S82T, and G115M; P29G, I49K, S82T, and A142E; P29G, I49K, S82T, R107C, and G112H;P29G, I49K, S82T, R107C, and G115M; P29G, I49K, S82T, R107C, and A142E; P29G, I49K, S82T, G112H, and A142E; P29G, I49K, S82T, G115M, and A142E; P29G, I49K, S82T, R107C, G112H, G115M, and A142E; P29K, I49K, and S82T; P29K, I49K, S82T, and R107C; P29K, I49K, S82T, and G112H; P29K, I49K, S82T, and G115M; P29K, I49K, S82T, and A142E; P29K, I49K, S82T, R107C, and G112H; P29K, I49K, S82T, R107C, and G115M; P29K, I49K, S82T, R107C, and A142E; P29K, I49K, S82T, G112H, and A142E; P29K, I49K, S82T, G115M, and A142E; P29K, I49K, S82T, R107C, G112H, G115M, and A142E; P29K, V30I, I49K, and S82T; P29K, V30I, I49K, S82T, and R107C; P29K, V30I, I49K, S82T, and G112H; P29K, V30I, I49K, S82T, and G115M; P29K, V30I, I49K, S82T, and A142E; P29K, V30I, I49K, S82T, R107C, and G112H; P29K, V30I, I49K, S82T, R107C, and G115M; P29K, V30I, I49K, S82T, R107C, and A142E; P29K, V30I, I49K, S82T, G112H, and A142E; P29K, V30I, I49K, S82T, G115M, and A142E; P29K, V30I, I49K, S82T, R107C, G112H, G115M, and A142E; P29K, I49K, S82T, and F84L; P29K, I49K, S82T, F84L, and R107C; P29K, I49K, S82T, F84L, and G112H; P29K, I49K, S82T, F84L, and G115M; P29K, I49K, S82T, F84L, and A142E;P29K, I49K, S82T, F84L, R107C, and G112H; P29K, I49K, S82T, F84L, R107C, and G115M; P29K, I49K, S82T, F84L, R107C, and A142E; P29K, I49K, S82T, F84L, G112H, and A142E; P29K, I49K, S82T, F84L, R107C, G112H, G115M, and A142E; P29K, I49K, S82T, F84L, R107C, G112H, G115M, and A142E; P29K, V30I, I49K, S82T, and F84L; P29K, V30I, I49K, S82T, F84L, and R107C; P29K, V30I, I49K, S82T, F84L, and G112H; P29K, V30I, I49K, S82T, F84L, and G115M; P29K, V30I, I49K, S82T, F84L, and A142E; P29K, V30I, I49K, S82T, F84L, R107C, and G112H; P29K, V30I, I49K, S82T, F84L, R107C, and G115M; P29K, V30I, I49K, S82T, F84L, R107C, and A142E; P29K, V30I, I49K, S82T, F84L, G112H, and A142E; P29K, V30I, I49K, S82T, F84L, G115M, and A142E; P29K, V30I, I49K, S82T, F84L, R107C, G112H, G115M, and A142E; E27G, I49K, S82T, and R107C; E27G, I49K, S82T, and G112H; E27G, I49K, S82T, and G115M; E27G, I49K, S82T, and A142E; E27G, I49K, S82T, R107C, and G112H; E27G, I49K, S82T, R107C, and G115M; E27G, I49K, S82T, G112H, and A142E; E27G, I49K, S82T, G115M, and A142E; E27G, I49K, S82T, R107C, G112H, G115M, and A142E; E27H, I49K, and S82T; E27H, I49K, S82T, and R107C; E27H, I49K, S82T, and G112H; E27H, I49K, S82T, and G115M;E27H, I49K, S82T, and A142E; E27H, I49K, S82T, R107C, and G112H; E27H, I49K, S82T, R107C, and G115M; E27H, I49K, S82T, R107C, and A142E; E27H, I49K, S82T, G112H, and A142E; E27H, I49K, S82T, G115M, and A142E; E27H, I49K, S82T, R107C, G112H, G115M, and A142E; E27S, and S82T; E27S, S82T, and R107C; E27S, S82T, and G112H; E27S, S82T, and G115M; E27S, S82T, and A142E; E27S, S82T, R107C, and G112H; E27S, S82T, R107C, and G115M; E27S, S82T, R107C, and A142E; E27S, S82T, G112H, and A142E; E27S, S82T, G115M, and A142E; E27S, S82T, R107C, G112H, G115M, and A142E; P29A and S82T; P29A, S82T, and R107C; P29A, S82T, and G112H; P29A, S82T, and G115M; P29A, S82T, and A142E; P29A, S82T, R107C, and G112H; P29A, S82T, R107C, and G115M; P29A, S82T, R107C, and A142E; P29A, S82T, G112H, and A142E; P29A, S82T, G115M, and A142E; P29A, S82T, R107C, G112H, G115M, and A142E; E27S, V30I, and S82T; E27S, V30I, S82T, and R107C; E27S, V30I, S82T, and G112H; E27S, V30I, S82T, and G115M; E27S, V30I, S82T, and A142E; E27S, V30I, S82T, R107C, and G112H; E27S, V30I, S82T, R107C, and G115M; E27S, V30I, S82T, R107C, and A142E; E27S, V30I, S82T, G112H, and A142E; E27S, V30I, S82T, G115M, and A142E;E27S, V30I, S82T, R107C, G112H, G115M, and A142E; P29A, V30I, S82T, and F84L; P29A, V30I, S82T, F84L, and R107C; P29A, V30I, S82T, F84L, and G112H; P29A, V30I, S82T, F84L, and G115M; P29A, V30I, S82T, F84L, and A142E; P29A, V30I, S82T, F84L, R107C, and G112H; P29A, V30I, S82T, F84L, R107C, and G115M; P29A, V30I, S82T, F84L, R107C, and A142E; P29A, V30I, S82T, F84L, G112H, and A142E; P29A, V30I, S82T, F84L, G115M, and A142E; P29A, V30I, S82T, F84L, R107C, G112H, G115M, and A142E; E27S, P29A, V30L, I49K, S82T, F84L, R107C, G112H, G115M, and A142E; V4K and A114C; V4K and D77G; F6Y, G100A, and H122R; V4T, I76R, and H122G; F6Y and I76W; F6Y and D119N; F6Y and A114C; V4K, I76W, and H122T; F6G, I76R, and G100K; F6H and H122N; F6Y, I76H, H122R, and T166I; R23Q and I76R; I76H, H122R, and A158V; F6Y and T111H; T111H, H122G, and A162C; F6Y and I76R; T17W, I76H, H122G, and A158V; V4S, I76Y, A143E, and Q159S; N127I and A162Q; E27H, Y76I, F84M, and F149Y; E27H, I49K, Y76I, and F149Y; T17A, E27H, I49M, Y76I, M118L, and F149Y; T17A, A48G, S82T, A142E, and F149Y; E27G, and F149Y; E27G, I49N, and F149Y; E27H, Y76I, F84M, Y147D, F149Y, T166I, and D167N;E27H, I49K, Y76I, Y147D, F149Y, T166I, D167N; T17A, E27H, I49M, Y76I, M118L, Y147D, F149Y, T166I, and D167N; T17A, A48G, S82T, A142E, Y147D, F149Y, T166I, and D167N; E27G, Y147D, F149Y, T166I, and D167N; E27G, I49N, Y147D, F149Y, T166I, and D167N; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, and A142E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, D119N, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, H122G, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, N127P, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, A142E, and A143E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, and A143E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, D119N, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, H122G, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, N127P, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, A142E, and A143E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, and A143E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, A114C, G115M, and A142E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, D119N, and A142E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, H122G, and A142E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, N127P, and A142E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, A142E, and A143E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, and A143E; F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, D119N, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, H122G, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, N127P, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, D119N, H122G, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, D119N, N127P, and A142E;F6Y, E27H, I49K, S82T, R107C, G112H, G115M, H122G, N127P, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, D119N, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, H122G, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, N127P, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, A142E, and A143E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, and A143E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, H122G, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, N127P, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, A142E, and A143E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, and A143E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, D119N, H122G, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, D119N, N127P, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, H122G, N127P, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, H122G, and A142E;F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, N127P, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, H122G, N127P, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, H122G, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, N127P, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, A142E, and A143E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, and A143E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, H122G, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, N127P, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, H122G, N127P, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, A142E, and A143E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, and A143E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, A142E, and A143E;and a combination of amino acid modifications selected from one or more of F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, and A143E, or a combination of corresponding modifications in another adenosine deaminase;

[0013] In another aspect, the disclosed invention features a fusion protein including a polynucleotide-programmable DNA-binding domain and an adenosine deaminase variant of any of the above aspects or embodiments thereof.

[0014] In another aspect, the disclosed invention features a multimolecular complex including a polynucleotide-programmable DNA-binding protein, an adenosine deaminase variant of any of the above aspects or embodiments thereof, and a guide RNA.

[0015] In another aspect, the disclosed invention features a base editing system including an adenosine deaminase variant of any of the above aspects or embodiments thereof, a polynucleotide-programmable DNA-binding protein, and one or more guide polynucleotides, wherein the base editing system performs A to G and C to T edits of a target polynucleotide.

[0016] In another aspect, the disclosed invention features a base editing system including the fusion protein of any of the above aspects or embodiments thereof and one or more guide polynucleotides, wherein the base editing system performs A to G and C to T editing of a target polynucleotide.

[0017] In another aspect, the disclosed invention features a polynucleotide encoding the adenosine deaminase variant of any of the above aspects or embodiments thereof, a fusion protein of any of the above aspects or embodiments thereof, a multimolecular complex of any of the above aspects or embodiments thereof, or a base editing system of any of the above aspects or embodiments thereof.

[0018] In another aspect, the disclosed invention features a cell containing the vector of any of the above aspects or embodiments thereof.

[0019] In another aspect, the disclosed invention features a vector including a polynucleotide of any of the above aspects or embodiments thereof.

[0020] In another aspect, the disclosed invention features a composition comprising the adenosine deaminase variant of any of the above aspects or embodiments thereof, the fusion protein of any of the above aspects or embodiments thereof, the multimolecular complex of any of the above aspects or embodiments thereof, the base editing system of any one of the above aspects or embodiments thereof, the polynucleotide of any of the above aspects or embodiments thereof, the vector of any one of the above aspects or embodiments thereof, or the cell of any of the above aspects or embodiments thereof.

[0021] In another aspect, the disclosed invention features a method of editing the genome of a cell, the method including contacting a target polynucleotide sequence in the cell with a fusion protein of any of the above aspects or embodiments thereof and one or more guide polynucleotides to effect one or more modifications in the genome of the cell, thereby editing the genome of the cell.

[0022] In another aspect, the disclosed invention features a method for editing the genome of a cell, the method including contacting a target polynucleotide sequence in a cell of an organism with a multimolecular complex of any of the above aspects or embodiments thereof, or a base editing system of any of the above aspects or embodiments thereof, to produce one or more modifications in the genome of the cell, thereby editing the genome of the cell.

[0023] In another aspect, the disclosed invention features a method of treating a genetic disease or disorder in a subject, the method including contacting a target polynucleotide in a cell of the subject with a fusion protein of any of the above aspects or embodiments thereof and one or more guide polynucleotides, to effect one or more modifications in the genome of the cell, thereby treating the genetic disease or disorder in the subject.

[0024] In another aspect, the disclosed invention features a method of treating a genetic disease or disorder in a subject, the method comprising administering to a cell of the subject a multimolecular complex of any of the above aspects or embodiments thereof, a base editing system of any one of the above aspects or embodiments thereof, a vector of any of the above aspects or embodiments thereof, a cell of any of the above aspects or embodiments thereof, or a composition of any of the above aspects or embodiments thereof, to produce one or more modifications in the genome of the cell, thereby treating the genetic disease or disorder in the subject.

[0025] In another aspect, the disclosed invention features a method for C to T editing in a target polynucleotide, the method including contacting the target polynucleotide with a fusion protein of any one of the above aspects or embodiments thereof and one or more guide polynucleotides, thereby editing the target polynucleotide.

[0026] In another aspect, the disclosed invention features a method for editing a C to T in a target polynucleotide, the method including contacting a target polynucleotide sequence with a multimolecular complex of any of the above aspects or embodiments thereof, or a base editing system of any of the above aspects or embodiments thereof, thereby editing the target polynucleotide.

[0027] In another aspect, the disclosed invention features a method for introducing an A to G edit and / or a C to T edit in the genome of a cell. The method includes introducing into the cell a fusion protein of any of the above aspects or embodiments thereof and a guide polynucleotide that creates an A to G edit, a C to T edit, or a combination thereof, in the genome of the cell.

[0028] In another aspect, the disclosed invention features a method for introducing an A to G edit and / or a C to T edit in the genome of a cell. The method includes introducing a multimolecular complex of any of the above aspects or embodiments thereof, or a base editing system of any of the above aspects or embodiments thereof, into a cell to effect an A to G edit, a C to T edit, or a combination thereof, in the genome of the cell.

[0029] In another aspect, the disclosed invention features a method for correcting a single nucleotide polymorphism (SNP) in a polynucleotide, the method comprising contacting a target polynucleotide with a fusion protein of any of the above aspects or embodiments thereof and one or more guide polynucleotides, thereby editing the SNP by deaminating the SNP or its complementary nucleobase.

[0030] In another aspect, the disclosed invention features a method for correcting a single nucleotide polymorphism (SNP) in a polynucleotide, the method comprising contacting a target polynucleotide sequence with the multimolecular complex of any of the above aspects or embodiments thereof, or the base editing system of any of the above aspects or embodiments thereof, thereby editing the SNP by deaminating the SNP or its complementary nucleobase.

[0031] In another aspect, the disclosed invention features a method for editing a regulatory sequence present in the genome of a cell, the method comprising contacting the regulatory sequence with a fusion protein of any of the above aspects or embodiments thereof and one or more guide polynucleotides, thereby editing the regulatory sequence.

[0032] In another aspect, the disclosed invention features a method for editing a regulatory sequence present in the genome of a cell, the method comprising contacting the regulatory sequence with a multimolecular complex of any of the above aspects or embodiments thereof, or a base editing system of any of the above aspects or embodiments thereof, thereby editing the regulatory sequence.

[0033] In another aspect, the disclosed invention features a method for producing an adenosine deaminase variant with increased cytidine deaminase activity and / or cytidine deaminase specificity, the method comprising cloning an adenosine deaminase variant having the following sequence: 10 20 30 40 50 MSEVEFSHEY WMRHALTLAK RARDEREVPV GAVLVLNNRV IGEGWNRAIG 60 70 80 90 100 LHDPTAHAEI MALRQGGLVM QNYRLYDATL YSTFEPCVMC AGAMIHSRIG 110 120 130 140 150 RVVFGVRNAK TGAAGSLMDV LHHPGMNHRV EITEGILADE CAALLCRFFR 160 MPRRVFNAQK KAQSSTD (SEQ ID NO: 1) The one or more modifications may include generating one or more modifications in an amino acid sequence having at least 70% amino acid identity to S2H, V4K, V4S, V4T, V4Y, F6G, F6H, F6Y, H8Q, R13G, T17A, T17W, R23Q, E27C, E27G, E27H, E27K, E27Q, E27S, E27G, P29A, P29G, P29K, V30F, V30I, R47G, R47S, A48G, I49K, I49M, I49N, I49Q, I49T, G67W, I76H, I76R, I76W, Y76H, Y76R, Y76W, F84A, F84M, H96N, G100A, G100K, T111H, G112H, A114C, G115M, M118L, H122G, H122R, H122T, N127I, N127K, N127P, A142E, R147H, A158V, Q159S, A162C, A162N, A162Q, and S165P, or the corresponding amino acid positions in another adenosine deaminase.

[0034] In another aspect, the disclosed invention features an adenosine deaminase variant produced by the method of any of the above aspects or embodiments thereof.

[0035] In another aspect, the invention of the present disclosure features a kit comprising the fusion protein of any of the above aspects or embodiments thereof, the multimolecular complex of any of the above aspects or embodiments thereof, the base editing system of any of the above aspects or embodiments thereof, the polynucleotide of any of the above aspects or embodiments thereof, the vector of any of the above aspects or embodiments thereof, the cell of any of the above aspects or embodiments thereof, or the composition of any of the above aspects or embodiments thereof, and instructions for its use in base editing.

[0036] In any of the above aspects or embodiments thereof, the adenosine deaminase variant comprising the modification has the following amino acid sequence: 10 20 30 40 50 MSEVEFSHEY WMRHALTLAK RARDEREVPV GAVLVLNNRV IGEGWNRAIG 60 70 80 90 100 LHDPTAHAEI MALRQGGLVM QNYRLYDATL YSTFEPCVMC AGAMIHSRIG 110 120 130 140 150 RVVFGVRNAK TGAAGSLMDV LHHPGMNHRV EITEGILADE CAALLCRFFR 160 MPRRVFNAQK KAQSSTD (SEQ ID NO: 1) It has at least about 70% or more amino acid sequence identity to

[0037] In any of the above aspects or embodiments thereof, the modification is present at amino acid positions selected from one or more of 2, 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167 of an amino acid sequence having at least about 70% or more amino acid sequence identity to SEQ ID NO:1, or a corresponding amino acid position in another adenosine deaminase. In any of the above aspects or embodiments thereof, the two or more modifications are selected from the group consisting of S2X, V4X, F6X, H8X, R13X, T17X, ​​R23X, E27X, P29X, V30X, R47X, A48X, I49X, G67X, Y76X, D77X, S82X, F84X, H96X, G1X, and / or ... and D167X, or the corresponding amino acid positions in another adenosine deaminase. In any of the above aspects or embodiments thereof, the two or more modifications are a first modification at amino acid position 2 and a second modification at amino acid position 3 selected from one or more of: 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167. a first modification at amino acid position 4 and one or more further modifications at amino acid positions selected from one or more of: 2, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167;a first modification at amino acid position 6 and one or more further modifications at amino acid positions selected from one or more of 2, 4, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167; a first modification at amino acid position 13 and one or more further modifications at amino acid positions selected from one or more of 2, 4, 6, 8, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167; one or more further modifications at amino acid positions selected from one or more of: 7, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167; a first modification at amino acid position 27 and one or more further modifications at amino acid positions selected from one or more of: 2, 4, 6, 8, 13, 17, 23, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167; one or more additional modifications at amino acid positions selected from one or more of: 18, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167; a first modification at amino acid position 29 and one or more additional modifications at amino acid positions 2, 4, 6, 8, 13, 17, 23, 27, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165 , 166, and 167; a first modification at amino acid position 100 and one or more further modifications at amino acid positions selected from one or more of 2, 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167;a first modification at amino acid position 112 and one or more additional modifications at amino acid positions selected from one or more of: 2, 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167; a first modification at amino acid position 114 and one or more additional modifications at amino acid positions selected from one or more of: 2, 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167; a first modification at amino acid position 115 and one or more further modifications at amino acid positions selected from one or more of: 2, 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167; a first modification at amino acid position 162 and one or more further modifications at amino acid positions selected from one or more of: 2, 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 165, 166, and 167;or a first modification at amino acid position 165 and one or more additional modifications at amino acid positions selected from one or more of: 2, 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 166, and 167;

[0038] In any of the above aspects or embodiments thereof, the modifications are selected from the group consisting of S2H, V4K, V4S, V4T, V4Y, F6G, F6H, F6Y, H8Q, R13G, T17A, T17W, R23Q, E27C, E27G, E27H, E27K, E27Q, E27S, E27G, P29A, P29G, P29K, V30F, V30I, V30L, R47G, R47S, A48G, I49K, I49M, I49N, I49Q, I49T, G67W, I76H, I76R, I76W, I76Y, Y76H, Y76I ... adenosine deaminase, or a corresponding amino acid position in another adenosine deaminase. In any of the above aspects or embodiments thereof, the modification is selected from those listed in any of Tables 1A-1F.

[0039] In any of the above aspects or embodiments thereof, the modifications include E27H, Y76I, and F84M; E27H, I49K, and Y76I; E27S, I49K, and Y76I; E27S, I49K, Y76I, and A162N; E27K and D119N; E27H and Y76I; E27S, I49K, and G67W; I49T, G67W, and H96N; E27C, Y76I, and D119N; R13G, E27Q, and N127K; T17A, E27H, I49M, Y76I, and M118L; I49Q, Y76I, and G115M; of an amino acid sequence having at least about 70% or greater identity to SEQ ID NO:1. S2H, I49K, Y76I, and G112H; R47S and R107C; H8Q, I49Q, and Y76I; T17A, A48G, S82T, and A142E; E27G and I49N; E27G, D77G, and S165P; E27S, I49K, and S82T; E27S, I49K, S82T, and G115M; E27S, V30I, I49K, and S82T; E27S, V30F, I49K, S82T, F84A, R107C, and A142E; E27S, V30F, I49K, S82T, F84A, G112H, and A142E; E27S, V30F, I49K, S82T, F84A, G115M, and A142E; E27S, I49K, S82T, F84L, and R107C; E27S, I49K, S82T, F84L, and G112H; E27S, I49K, S82T, F84L, and G115M; E27S, I49K, S82T, F84L, R107C, and G112H; E27S, I49K, S82T, F84L, R107C, and G115M; E27S, I49K, S82T, F84L, R107C, and A142E; E27S, I49K, S82T, F84L, G112H, and A142E; E27S, I49K, S82T, F84L, G115M, and A142E; E27S, I49K, S82T, F84L, R107C, G112H, G115M, and A142E; E27S, V30I, I49K, S82T, and F84L; E27S, P29G, I49K, and S82T; E27S, P29G, I49K, S82T, and G115M; E27S, P29G, I49K, S82T, and A142E;P29G, I49K, and S82T; E27G, I49K, and S82T; E27G, I49K, S82T, R107C, and A142E; V4K, E27H, I49K, Y76I, and A114C; V4K, E27H, I49K, Y76I, and D77G; F6Y, E27H, I49K, Y76I, G100A, and H122R; V4T, E27H, I49K, Y76R, and H122G; F6Y, E27H, I49K, and Y76W; F6Y, E27H, I49K, Y76I, and D119N; F6Y, E27H, I49K, Y76I, and A114C; F6Y, E27H, I49K, and Y76I; V4K, E27H, I49K, Y76W, and H122T; F6G, E27H, I49K, Y76R, and G100K; F6H, E27H, I49K, Y76I, and H122N; E27H, I49K, Y76I, and A114C; F6Y, E27H, I49K, Y76H, H122R, and T166I; E27H, I49K, Y76I, and N127P; R23Q, E27H, I49K, and Y76R; E27H, I49K, Y76H, H122R, and A158V; F6Y, E27H, I49K, Y76I, and T111H; E27H, I49K, Y76I, and R147H; E27H, I49K, Y76I, and A143E; F6Y, E27H, I49K, and Y76R; T17W, E27H, I49K, Y76H, H122G, and A158V; V4S, E27H, I49K, A143E, and Q159S; E27H, I49K, Y76I, N127I, and A162Q; T17A, E27H, and A48G; T17A, E27K, and A48G; T17A, E27S, and A48G; T17A, E27S, A48G, and I49K; T17A, E27G, and A48G; T17A, A48G, and I49N; T17A, E27G, A48G, and I49N; T17A, E27Q, and A48G; E27S, I49K, S82T, and R107C; E27S, I49K, S82T, and G112H; E27S, I49K, S82T, and A142E; E27S, I49K, S82T, R107C, and G112H; E27S, I49K, S82T, R107C, and G115M;E27S, I49K, S82T, R107C, and A142E; E27S, I49K, S82T, G112H, and A142E; E27S, I49K, S82T, G115M, and A142E; E27S, I49K, S82T, R107C, G112H, G115M, and A142E; E27S, V30I, I49K, S82T, and R107C; E27S, V30I, I49K, S82T, and G112H; E27S, V30I, I49K, S82T, and G115M; E27S, V30I, I49K, S82T, and A142E; E27S, V30I, I49K, S82T, R107C, and G112H; E27S, V30I, I49K, S82T, R107C, and G115M; E27S, V30I, I49K, S82T, R107C, and A142E; E27S, V30I, I49K, S82T, G112H, and A142E; E27S, V30I, I49K, S82T, G115M, and A142E; E27S, V30I, I49K, S82T, R107C, G112H, G115M, and A142E; E27S, V30L, I49K, and S82T; E27S, V30L, I49K, S82T, and R107C; E27S, V30L, I49K, S82T, and G112H; E27S, V30L, I49K, S82T, and G115M; E27S, V30L, I49K, S82T, and A142E; E27S, V30L, I49K, S82T, R107C, and G112H; E27S, V30L, I49K, S82T, R107C, and G115M; E27S, V30L, I49K, S82T, R107C, and A142E; E27S, V30L, I49K, S82T, G112H, and A142E; E27S, V30L, I49K, S82T, G115M, and A142E; E27S, V30L, I49K, S82T, R107C, G112H, G115M, and A142E; E27S, V30F, I49K, S82T, and F84A; E27S, V30F, I49K, S82T, F84A, and R107C; E27S, V30F, I49K, S82T, F84A, and G112H; E27S, V30F, I49K, S82T, F84A, and G115M; E27S, V30F, I49K, S82T, F84A, and A142E;E27S, V30F, I49K, S82T, F84A, R107C, and G112H; E27S, V30F, I49K, S82T, F84A, R107C, and G115M; E27S, V30F, I49K, S82T, F84A, R107C, G112H, G115M, and A142E; E27S, I49K, S82T, and F84L; E27S, I49K, S82T, F84L, and A142E; E27S, V30I, I49K, S82T, F84L, and R107C; E27S, V30I, I49K, S82T, F84L, and G112H; E27S, V30I, I49K, S82T, F84L, and G115M; E27S, V30I, I49K, S82T, F84L, and A142E; E27S, V30I, I49K, S82T, F84L, R107C, and G112H; E27S, V30I, I49K, S82T, F84L, R107C, and G115M; E27S, V30I, I49K, S82T, F84L, R107C, and A142E; E27S, V30I, I49K, S82T, F84L, G112H, and A142E; E27S, V30I, I49K, S82T, F84L, G115M, and A142E; E27S, V30I, I49K, S82T, F84L, R107C, G112H, G115M, and A142E; E27S, P29G, I49K, S82T, and R107C; E27S, P29G, I49K, S82T, and G112H; E27S, P29G, I49K, S82T, R107C, and G112H; E27S, P29G, I49K, S82T, R107C, and G115M; E27S, P29G, I49K, S82T, R107C, and A142E; E27S, P29G, I49K, S82T, G112H, and A142E; E27S, P29G, I49K, S82T, G115M, and A142E; E27S, P29G, I49K, S82T, R107C, G112H, G115M, and A142E; P29G, I49K, S82T, and R107C; P29G, I49K, S82T, and G112H; P29G, I49K, S82T, and G115M; P29G, I49K, S82T, and A142E; P29G, I49K, S82T, R107C, and G112H;P29G, I49K, S82T, R107C, and G115M; P29G, I49K, S82T, R107C, and A142E; P29G, I49K, S82T, G112H, and A142E; P29G, I49K, S82T, G115M, and A142E; P29G, I49K, S82T, R107C, G112H, G115M, and A142E; P29K, I49K, and S82T; P29K, I49K, S82T, and R107C; P29K, I49K, S82T, and G112H; P29K, I49K, S82T, and G115M; P29K, I49K, S82T, and A142E; P29K, I49K, S82T, R107C, and G112H; P29K, I49K, S82T, R107C, and G115M; P29K, I49K, S82T, R107C, and A142E; P29K, I49K, S82T, G112H, and A142E; P29K, I49K, S82T, G115M, and A142E; P29K, I49K, S82T, R107C, G112H, G115M, and A142E; P29K, V30I, I49K, and S82T; P29K, V30I, I49K, S82T, and R107C; P29K, V30I, I49K, S82T, and G112H; P29K, V30I, I49K, S82T, and G115M; P29K, V30I, I49K, S82T, and A142E; P29K, V30I, I49K, S82T, R107C, and G112H; P29K, V30I, I49K, S82T, R107C, and G115M; P29K, V30I, I49K, S82T, R107C, and A142E; P29K, V30I, I49K, S82T, G112H, and A142E; P29K, V30I, I49K, S82T, G115M, and A142E; P29K, V30I, I49K, S82T, R107C, G112H, G115M, and A142E; P29K, I49K, S82T, and F84L; P29K, I49K, S82T, F84L, and R107C; P29K, I49K, S82T, F84L, and G112H; P29K, I49K, S82T, F84L, and G115M; P29K, I49K, S82T, F84L, and A142E;P29K, I49K, S82T, F84L, R107C, and G112H; P29K, I49K, S82T, F84L, R107C, and G115M; P29K, I49K, S82T, F84L, R107C, and A142E; P29K, I49K, S82T, F84L, G112H, and A142E; P29K, I49K, S82T, F84L, R107C, G112H, G115M, and A142E; P29K, I49K, S82T, F84L, R107C, G112H, G115M, and A142E; P29K, V30I, I49K, S82T, and F84L; P29K, V30I, I49K, S82T, F84L, and R107C; P29K, V30I, I49K, S82T, F84L, and G112H; P29K, V30I, I49K, S82T, F84L, and G115M; P29K, V30I, I49K, S82T, F84L, and A142E; P29K, V30I, I49K, S82T, F84L, R107C, and G112H; P29K, V30I, I49K, S82T, F84L, R107C, and G115M; P29K, V30I, I49K, S82T, F84L, R107C, and A142E; P29K, V30I, I49K, S82T, F84L, G112H, and A142E; P29K, V30I, I49K, S82T, F84L, G115M, and A142E; P29K, V30I, I49K, S82T, F84L, R107C, G112H, G115M, and A142E; E27G, I49K, S82T, and R107C; E27G, I49K, S82T, and G112H; E27G, I49K, S82T, and G115M; E27G, I49K, S82T, and A142E; E27G, I49K, S82T, R107C, and G112H; E27G, I49K, S82T, R107C, and G115M; E27G, I49K, S82T, G112H, and A142E; E27G, I49K, S82T, G115M, and A142E; E27G, I49K, S82T, R107C, G112H, G115M, and A142E; E27H, I49K, and S82T; E27H, I49K, S82T, and R107C; E27H, I49K, S82T, and G112H; E27H, I49K, S82T, and G115M;E27H, I49K, S82T, and A142E; E27H, I49K, S82T, R107C, and G112H; E27H, I49K, S82T, R107C, and G115M; E27H, I49K, S82T, R107C, and A142E; E27H, I49K, S82T, G112H, and A142E; E27H, I49K, S82T, G115M, and A142E; E27H, I49K, S82T, R107C, G112H, G115M, and A142E; E27S, and S82T; E27S, S82T, and R107C; E27S, S82T, and G112H; E27S, S82T, and G115M; E27S, S82T, and A142E; E27S, S82T, R107C, and G112H; E27S, S82T, R107C, and G115M; E27S, S82T, R107C, and A142E; E27S, S82T, G112H, and A142E; E27S, S82T, G115M, and A142E; E27S, S82T, R107C, G112H, G115M, and A142E; P29A and S82T; P29A, S82T, and R107C; P29A, S82T, and G112H; P29A, S82T, and G115M; P29A, S82T, and A142E; P29A, S82T, R107C, and G112H; P29A, S82T, R107C, and G115M; P29A, S82T, R107C, and A142E; P29A, S82T, G112H, and A142E; P29A, S82T, G115M, and A142E; P29A, S82T, R107C, G112H, G115M, and A142E; E27S, V30I, and S82T; E27S, V30I, S82T, and R107C; E27S, V30I, S82T, and G112H; E27S, V30I, S82T, and G115M; E27S, V30I, S82T, and A142E; E27S, V30I, S82T, R107C, and G112H; E27S, V30I, S82T, R107C, and G115M; E27S, V30I, S82T, R107C, and A142E; E27S, V30I, S82T, G112H, and A142E; E27S, V30I, S82T, G115M, and A142E;E27S, V30I, S82T, R107C, G112H, G115M, and A142E; P29A, V30I, S82T, and F84L; P29A, V30I, S82T, F84L, and R107C; P29A, V30I, S82T, F84L, and G112H; P29A, V30I, S82T, F84L, and G115M; P29A, V30I, S82T, F84L, and A142E; P29A, V30I, S82T, F84L, R107C, and G112H; P29A, V30I, S82T, F84L, R107C, and G115M; P29A, V30I, S82T, F84L, R107C, and A142E; P29A, V30I, S82T, F84L, G112H, and A142E; P29A, V30I, S82T, F84L, G115M, and A142E; P29A, V30I, S82T, F84L, R107C, G112H, G115M, and A142E; E27S, P29A, V30L, I49K, S82T, F84L, R107C, G112H, G115M, and A142E; V4K and A114C; V4K and D77G; F6Y, G100A, and H122R; V4T, I76R, and H122G; F6Y and I76W; F6Y and D119N; F6Y and A114C; V4K, I76W, and H122T; F6G, I76R, and G100K; F6H and H122N; F6Y, I76H, H122R, and T166I; R23Q and I76R; I76H, H122R, and A158V; F6Y and T111H; T111H, H122G, and A162C; F6Y and I76R; T17W, I76H, H122G, and A158V; V4S, I76Y, A143E, and Q159S; N127I and A162Q; E27H, Y76I, F84M, and F149Y; E27H, I49K, Y76I, and F149Y; T17A, E27H, I49M, Y76I, M118L, and F149Y; T17A, A48G, S82T, A142E, and F149Y; E27G, and F149Y; E27G, I49N, and F149Y; E27H, Y76I, F84M, Y147D, F149Y, T166I, and D167N;E27H, I49K, Y76I, Y147D, F149Y, T166I, D167N; T17A, E27H, I49M, Y76I, M118L, Y147D, F149Y, T166I, and D167N; T17A, A48G, S82T, A142E, Y147D, F149Y, T166I, and D167N; E27G, Y147D, F149Y, T166I, and D167N; E27G, I49N, Y147D, F149Y, T166I, and D167N; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, and A142E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, D119N, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, H122G, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, N127P, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, A142E, and A143E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, and A143E; F6; Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, D119N, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, H122G, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, N127P, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, A142E, and A143E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, and A143E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, A114C, G115M, and A142E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, D119N, and A142E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, H122G, and A142E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, N127P, and A142E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, A142E, and A143E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, and A143E; F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, D119N, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, H122G, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, N127P, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, D119N, H122G, and A142E;F6Y, E27H, I49K, S82T, R107C, G112H, G115M, D119N, N127P, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, H122G, N127P, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, D119N, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, H122G, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, N127P, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, A142E, and A143E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, and A143E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, H122G, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, N127P, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, A142E, and A143E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, and A143E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, D119N, H122G, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, D119N, N127P, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, H122G, N127P, and A142E;F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, H122G, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, N127P, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, H122G, N127P, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, H122G, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, N127P, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, A142E, and A143E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, and A143E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, H122G, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, N127P, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, H122G, N127P, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, A142E, and A143E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, and A143E;F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, A142E, and A143E; and F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, and A143E, or corresponding amino acid positions in another adenosine deaminase. In any of the above aspects or embodiments thereof, the modifications are E27S, I49K, and S82T; E27S, V30I, I49K, S82T, and F84L; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, and A142E; and a combination of modifications selected from one or more of F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, H122G, and A142E. In any of the above aspects or embodiments thereof, the modifications include a combination of modifications selected from those listed in any of Tables 1A-1F.

[0040] In any of the above aspects or embodiments thereof, the one or more modifications increase cytidine deaminase activity and / or cytidine deaminase specificity relative to a reference adenosine deaminase.

[0041] In any of the above aspects or embodiments thereof, the adenosine deaminase variant is a TadA deaminase variant or a fragment thereof. In embodiments, the TadA deaminase or fragment thereof is a bacterial TadA deaminase.

[0042] In any of the above aspects or embodiments thereof, the adenosine deaminase variant comprises a combination of modifications selected from one or more of E27H, Y76I, and F84M, and E27H, I49K, and Y76I, of an amino acid sequence having at least about 70% or more identity to SEQ ID NO: 1, or a corresponding combination of modifications in another adenosine deaminase.

[0043] In any of the above aspects or embodiments thereof, the adenosine deaminase variant further comprises an R at amino acid position 166 of SEQ ID NO: 1. In any of the above aspects or embodiments thereof, the adenosine deaminase variant does not comprise an R amino acid at position 48 of SEQ ID NO: 1.

[0044] In any of the above aspects or embodiments thereof, the adenosine deaminase variant exhibits an increase in cytidine deaminase activity and / or cytidine deaminase specificity of at least about 30-fold or more over the reference adenosine deaminase. In any of the above aspects or embodiments thereof, the adenosine deaminase variant exhibits an increase in cytidine deaminase activity and / or cytidine deaminase specificity of at least about 50-fold or more over the reference adenosine deaminase. In any of the above aspects or embodiments thereof, the adenosine deaminase variant exhibits an increase in cytidine deaminase activity and / or cytidine deaminase specificity of at least about 70-fold or more over the reference adenosine deaminase. In any of the above aspects or embodiments thereof, the adenosine deaminase variant retains at least about 30% or more of the adenosine deaminase activity of the reference adenosine deaminase. In any of the above aspects or embodiments thereof, the adenosine deaminase variant maintains at least about 50% or more of the adenosine deaminase activity of the reference adenosine deaminase. In any of the above aspects or embodiments thereof, the adenosine deaminase variant maintains at least about 70% or more of the adenosine deaminase activity of the reference adenosine deaminase.

[0045] In any of the above aspects or embodiments thereof, the reference adenosine deaminase is TadA*8.20 or TadA*8.19.

[0046] In any of the above aspects or embodiments thereof, the adenosine deaminase variant is capable of deaminating cytidine and adenine in a single-stranded or double-stranded target polynucleotide. In embodiments, the target polynucleotide is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

[0047] In any of the above aspects or embodiments thereof, the adenosine deaminase variant has a ratio of cytidine to adenine deamination activity of at least about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In any of the above aspects or embodiments thereof, the modification increases selectivity for deaminating cytidine compared to a reference adenosine deaminase.

[0048] In any of the above aspects or embodiments thereof, the polynucleotide-programmable DNA-binding domain is a Cas9, Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, or Cas12j / CasΦ domain. In any of the above aspects or embodiments thereof, the polynucleotide-programmable DNA-binding domain is Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), Streptococcus pyogenes Cas9 (SpCas9), or a variant thereof. In any of the above aspects or embodiments thereof, the polynucleotide-programmable DNA-binding domain comprises a modified SaCas9 with altered protospacer adjacent motif (PAM) specificity. In any of the above aspects or embodiments thereof, the polynucleotide-programmable DNA-binding domain comprises a variant of SpCas9 with altered protospacer adjacent motif (PAM) specificity. In any of the above aspects or embodiments thereof, the polynucleotide-programmable DNA-binding domain is a nuclease-inactive variant or a nickase variant.

[0049] In any of the above aspects or embodiments thereof, the fusion protein comprises a linker between the polynucleotide-programmable DNA-binding domain and the deaminase domain. In any of the above aspects or embodiments thereof, the fusion protein comprises one or more nuclear localization signals. In any of the above aspects or embodiments thereof, the fusion protein comprises one or more uracil glycosylase inhibitor (UGI) domains.

[0050] In any of the above aspects or embodiments thereof, the base editing system has at least about 30-fold increased C to T base editing activity compared to the C to T base editing activity of the reference base editing system. In any of the above aspects or embodiments thereof, the base editing system has at least about 50-fold increased C to T base editing activity compared to the C to T base editing activity of the reference base editing system. In any of the above aspects or embodiments thereof, the base editing system has at least about 70-fold increased C to T base editing activity compared to the C to T base editing activity of the reference base editing system. In any of the above aspects or embodiments thereof, the base editing system maintains A to G base editing activity that is at least about 30% of the activity of the reference base editing system. In any of the above aspects or embodiments thereof, the base editing system maintains A to G base editing activity that is at least about 50% of the activity of the reference base editing system. In any of the above aspects or embodiments thereof, the base editing system maintains A to G base editing activity that is at least about 70% of the activity of the reference base editing system. In any of the above aspects or embodiments thereof, the base editing system has at least about 30% C to T editing activity at the target polynucleotide. In any of the above aspects or embodiments thereof, the base editing system has at least about 50% C to T editing activity at the target polynucleotide. In any of the above aspects or embodiments thereof, the base editing system has at least about 70% C to T editing activity at the target polynucleotide.

[0051] In any of the above aspects or embodiments thereof, the reference base editing scheme is ABE8.20, ABE8.19, B93, B88, variant 1.17 (Table 1A), or variant 1.2 (Table 1A).

[0052] In any of the above aspects or embodiments thereof, the target polynucleotide is double-stranded or single-stranded. In any of the above aspects or embodiments thereof, the target polynucleotide is DNA or RNA. In any of the above aspects or embodiments thereof, the target polynucleotide is present in the genome of a cell.

[0053] In any of the above aspects or embodiments thereof, the A to G edit and / or the C to T edit in the target polynucleotide is associated with a genetic disease.

[0054] In any of the above aspects or embodiments thereof, the vector is a mammalian expression vector. In any of the above aspects or embodiments thereof, the vector is a viral vector. In embodiments, the viral vector is selected from one or more of an adeno-associated virus (AAV), a retroviral vector, an adenoviral vector, a lentiviral vector, a Sendai virus vector, and a herpes virus vector. In any of the above aspects or embodiments thereof, the vector comprises a promoter.

[0055] In any of the above aspects or embodiments thereof, the cell is a human cell. In any of the above aspects or embodiments thereof, the cell is in vitro or in vivo. In any of the above aspects or embodiments thereof, the cell is a bacterial, yeast, fungal, insect, plant, or mammalian cell.

[0056] In any of the above aspects or embodiments thereof, the composition further comprises a pharmaceutically acceptable excipient, diluent, or carrier.

[0057] In any of the above aspects or embodiments thereof, the subject is a mammal. In embodiments, the mammal is a human.

[0058] In any of the above aspects or embodiments thereof, the adenosine deaminase variant comprises an amino acid modification at one or more amino acid positions selected from 139 to 167. In embodiments, the modification at one or more amino acid positions selected from 139 to 167 is associated with unwinding of a portion of alpha helix 5.

[0059] In any of the above aspects or embodiments thereof, the adenosine deaminase variant containing the modification has at least about 70% or more amino acid sequence identity to SEQ ID NO:1.

[0060] In any of the above aspects or embodiments thereof, the modification in region A alters the active site of the deaminase. In any of the above aspects or embodiments thereof, the modification(s) in region B are present in one or more of loop 1, comprising residues 25-30, loop 3, comprising residues 46-47, or helix 2, comprising residues 48-51. In any of the above aspects or embodiments thereof, the modification(s) are present in loop 1. In any of the above aspects or embodiments thereof, the modification(s) are present in loop 3. In any of the above aspects or embodiments thereof, the modification(s) are present in the C-terminal helix, comprising residues 139-167. In any of the above aspects or embodiments thereof, the modification(s) are associated with helix unwinding. In embodiments, the helix unwinds between residues 145-155. In embodiments, the helix unwinds at about residue 150.

[0061] In any of the above aspects or embodiments thereof, the adenosine deaminase variant retains at least about 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the adenosine deaminase activity of the reference adenosine deaminase. In any of the above aspects or embodiments thereof, the adenosine deaminase variant retains less than about 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the adenosine deaminase activity of the reference adenosine deaminase. In any of the above aspects or embodiments thereof, the adenosine deaminase variant has cytidine deaminase activity and adenosine deaminase activity, and the cytidine deaminase activity is about, or at least about, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, or more times the adenosine deaminase activity. In any of the above aspects or embodiments thereof, the adenosine deaminase variant lacks significant adenosine deaminase activity. In any of the above aspects or embodiments thereof, the adenosine deaminase variant lacks detectable adenosine deaminase activity.

[0062] In various embodiments of any of the above aspects, the adenosine deaminase variant does not include an amino acid position selected from the group consisting of 30, 47-49, 82-84, 107-111, 139, 142, 143, 146-149, 151-161, 166, and 167, or does not include an amino acid position selected from the group consisting of V30I, V30L, V30, R47F, R47M, R47Q, R47 W, P48A, P48D, P48E, P48H, P48K, P48L, P48R, P48S, P48T, I49V, V82G, V82S, V82T, L84F, L84I, R1 07A, R107C, R107H, R107K, R107N, R107P, D108A, D108E, D108F, D108G, D108I, D108K, D108L, D10 8M, D108N, D108Q, D108S, D108V, D108W, D108Y, A109S, K110I, T111R, D139L, D139M, A142N, A143 D, A143E, A143G, A143L, S146C, S146R, S146T, D147A, D147R, D147T, D147Y, F148A, F149A, F149N , F149Y, M151V, R152H, R152P, R153C, Q154H, Q154L, Q154R, Q154S, E155D, E155G, E155V, I156D, I156F, I156Y, K157N, A158K, Q159L, K160E, K161T, T166I, T166R, and D167N.

[0063] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the following meanings unless otherwise specified.

[0064] "Adenine" or "9H-purin-6-amine" has the molecular formula C5H5N5 and the structure [ka] and refers to the purine nucleobase having the CAS number 73-24-5.

[0065] "Adenosine" or "4-amino-1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2(1H)-one" means [ka] It refers to an adenine molecule attached to a ribose sugar via a glycosidic bond, having the structure: 10 H 13 It is N5O4.

[0066] "Adenosine deaminase" or "adenine deaminase" refers to a polypeptide or fragment thereof capable of catalyzing the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine to inosine or deoxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g., modified adenosine deaminases, evolved adenosine deaminases) can be derived from any organism, such as bacteria. In some embodiments, the adenosine deaminase is an adenosine deaminase variant having one or more modifications and capable of deaminating both adenine and cytosine in a target polynucleotide (e.g., DNA). In some embodiments, the target polynucleotide is single-stranded or double-stranded. In some embodiments, the adenosine deaminase variant can deaminate both adenine and cytosine in DNA. In some embodiments, the adenosine deaminase variant can deaminate both adenine and cytosine in single-stranded DNA. In some embodiments, the adenosine deaminase variant can deaminate both adenine and cytosine in RNA.

[0067] "Adenosine deaminase activity" refers to catalyzing the deamination of adenine to guanine in a polynucleotide. In some embodiments, the adenosine deaminase variants provided herein maintain adenosine deaminase activity (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the activity of a reference adenosine deaminase (e.g., TadA*8.20 or TadA*8.19)). In some embodiments, the adenosine deaminase variants have primarily cytidine deaminase activity and retain less than about 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, or 20% of the adenosine deaminase activity. In some embodiments, adenosine deaminase variants have approximately equivalent adenosine deaminase activity and cytidine deaminase activity (e.g., activities within about, or at least about, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, or 50% of each other). In some examples, an adenosine deaminase variant has a cytosine deaminase activity that is about, or at least about 10%, 20%, 30%, 40%, 50%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 10,000-fold, or more times greater than the adenosine deaminase activity of the variant. In some embodiments, the adenosine deaminase variant has primarily cytosine deaminase activity and little, if any, adenosine deaminase activity, hi some embodiments, the adenosine deaminase variant has cytosine deaminase activity but no significant or detectable adenosine deaminase activity.

[0068] "Adenosine base editor (ABE)" means a base editor that includes adenosine deaminase.

[0069] "Adenosine base editor (ABE) polynucleotide" means a polynucleotide that encodes an ABE.

[0070] "Adenosine deaminase base editor 8 (ABE8) polypeptide" or "ABE8" means a base editor as defined herein, comprising an adenosine deaminase or adenosine deaminase variant, comprising one or more of the modifications listed in Table 14, one of the combinations of modifications listed in Table 14, or a modification at one or more of the amino acid positions listed in Table 14, wherein such modifications are relative to the following reference sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 2). In some embodiments, ABE8 contains additional modifications compared to a reference sequence, as described herein. In some embodiments, these additional modifications in the adenosine deaminase domain of ABE8 confer greater (e.g., at least about 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or more) C to T editing activity compared to the C to T editing activity in a reference ABE8 (e.g., ABE8.20).

[0071] "Adenosine deaminase base editor 8 (ABE8) polynucleotide" means a polynucleotide encoding ABE8.

[0072] "Administering" is referred to herein as providing one or more compositions described herein to a patient or subject.

[0073] By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof.

[0074] "Alteration" refers to a change (increase or decrease) in the level, structure, or activity of an analyte, gene, or polypeptide as detected by standard art-known methods, such as those described herein. As used herein, alteration includes a 10% change, a 25% change, a 40% change, and a 50% or greater change in expression level. In some embodiments, alteration includes an insertion, deletion, or substitution of a nucleic acid base or amino acid.

[0075] "Ameliorate" means to reduce, suppress, attenuate, decrease, arrest, or stabilize the onset or progression of a disease.

[0076] "Analog" refers to a molecule that is not identical but has similar functional or structural characteristics. For example, a polypeptide analog retains the biological activity of the corresponding native polypeptide while possessing certain biochemical modifications that enhance the analog's function compared to the native polypeptide. Such biochemical modifications can, for example, increase the analog's protease resistance, membrane permeability, or half-life without altering ligand binding. Analogs may also include unnatural amino acids.

[0077] "Base editor (BE)" or "nucleobase editor polypeptide (NBE)" refers to an agent that binds to a polynucleotide and has nucleobase-modifying activity. In various embodiments, the base editor comprises a nucleobase-modifying polypeptide (e.g., an adenosine deaminase variant) and a polynucleotide-programmable nucleotide-binding domain (e.g., Cas9 or Cpf1) in conjunction with a guide polynucleotide (e.g., a guide RNA (gRNA)). Representative nucleic acid and protein sequences of base editors are provided in the Sequence Listing as SEQ ID NOs: 3-12.

[0078] Examples of base editors include cytidine or cytosine base editors (CBEs) and adenine or adenosine base editors (ABEs). Non-limiting examples of cytidine base editors (CBEs) include BE1 (APOBEC1-XTEN-dCas9), BE2 (APOBEC1-XTEN-dCas9-UGI), BE3 (APOBEC1-XTEN-dCas9(A840H)-UGI), BE3-Gam, saBE3, saBE4-Gam, BE4, BE4-Gam, saBE4, or saB4E-Gam. BE4 extends the APOBEC1-Cas9n(D10A) linker to 32 amino acids, extends the Cas9n-UGI linker to 9 amino acids, and adds a second copy of UGI to the C-terminus of the construct using another 9-amino acid linker, resulting in a single base editor construct. The base editors saBE3 and saBE4 replace S. pyogenes Cas9n(D10A) with the smaller S. aureus Cas9n(D10A). BE3-Gam, saBE3-Gam, BE4-Gam, and saBE4-Gam have the 174-residue Gam protein fused to the N-terminus of BE3, saBE3, BE4, and saBE4 via a 16-amino acid XTEN linker.

[0079] Non-limiting examples of adenosine base editors include base editors comprising TadA deaminase. In some embodiments, the adenine or adenosine base editor (ABE) comprises a TadA deaminase variant (e.g., a TadA*8 variant). In some embodiments, the adenine or adenosine base editor (ABE) comprises a bacterial TadA deaminase variant (e.g., ecTadA). In some embodiments, the adenine or adenosine base editor (ABE) comprises a truncated TadA deaminase variant. In some embodiments, the adenine or adenosine base editor (ABE) comprises a fragment of a TadA deaminase variant. In some embodiments, the adenine or adenosine base editor (ABE) comprises a TadA*8.20 variant. In some embodiments, the adenine or adenosine base editor (ABE) is an ABE8 variant. In some embodiments, the ABE8 variant is an ABE8.20 variant. In some embodiments, the base editor is an adenine or adenosine base editor (ABE) comprising an adenosine deaminase variant that has both adenine and cytosine deamination activity. In some embodiments, base editing systems comprising an ABE variant provided herein (e.g., an ABE8.20 variant) have both A to G base editing activity and C to T base editing activity.

[0080] "Base editing activity" means acting to chemically modify a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytosine or cytidine deaminase activity, for example, converting a target C·G to T·A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, for example, converting A·T to G·C. In some embodiments, base editing systems comprising adenosine deaminase variants provided herein have C to T base editing activity and A to G base editing activity. In some embodiments, the base editing systems provided herein have at least about 30%, 40%, 50%, 60%, 70%, or more C to T base editing activity compared to a reference base editing system (such as ABE8.20 or ABE8.19).

[0081] The term "base editing system" refers to an intermolecular complex for editing nucleobases of a target nucleotide sequence. In various embodiments, a base editor (BE) system comprises (1) a polynucleotide-programmable nucleotide-binding domain, a deaminase domain (e.g., cytidine deaminase or adenosine deaminase) for deaminating nucleobases in a target nucleotide sequence, and (2) one or more guide polynucleotides (e.g., guide RNAs) bound to the polynucleotide-programmable nucleotide-binding domain. In various embodiments, the base editor (BE) system comprises a nucleobase editor domain (e.g., an adenosine deaminase variant domain) and a domain with nucleic acid sequence-specific binding activity. In some embodiments, the base editing system comprises (1) a base editor (BE) comprising a polynucleotide-programmable DNA-binding domain and a deaminase domain (e.g., an adenosine deaminase variant domain) for deaminating one or more nucleobases in a target nucleotide sequence, and (2) one or more guide RNAs bound to the polynucleotide-programmable DNA-binding domain. In some embodiments, the polynucleotide-programmable nucleotide-binding domain is a polynucleotide-programmable DNA-binding domain. In some embodiments, the base editing system comprises an adenosine deaminase variant having adenine and cytosine deaminase activity. In some embodiments, base editing systems comprising the adenosine deaminase variants provided herein have at least about 30%, 40%, 50%, 60%, 70%, or more C to T editing activity in a target polynucleotide (e.g., DNA). In some embodiments, base editing systems comprising the adenosine deaminase variants provided herein have increased C to T base editing activity (e.g., at least about 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or more) compared to the C to T base editing activity of a base editing system comprising a reference adenosine deaminase (e.g., ABE8.20 or ABE8.19).

[0082] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease comprising a Cas9 protein or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA-cleavage domain of Cas9 and / or a gRNA-binding domain of Cas9). Cas9 nucleases are also sometimes referred to as casnl nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)-associated nucleases.

[0083] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid with another amino acid that shares common properties. A functional method for defining common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analysis, groups of amino acids can be defined when amino acids within the group are preferentially exchanged with each other, and therefore have the most similar effects on the overall protein structure (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conservative mutations include amino acid substitutions, such as arginine to lysine, which maintains a positive charge, and vice versa, aspartic acid to glutamic acid, which maintains a negative charge, and threonine to serine, which maintains a free -OH, and asparagine to glutamine, which maintains a free -NH.

[0084] The terms "coding sequence" or "protein-coding sequence," as used interchangeably herein, refer to a segment of a polynucleotide that encodes a protein. A coding sequence may also be referred to as an open reading frame. A region or sequence is bounded proximal to the 5' end by a start codon and proximal to the 3' end by a stop codon. Stop codons useful in the base editors described herein include: [ka]

[0085] A "complex" refers to a combination of two or more molecules whose interaction relies on intermolecular forces. Non-limiting examples of intermolecular forces include covalent and non-covalent interactions. Non-limiting examples of non-covalent interactions include hydrogen bonds, ionic bonds, halogen bonds, hydrophobic bonds, van der Waals interactions (e.g., dipole-dipole interactions, dipole-induced dipole interactions, and London dispersion forces), and the π effect. In one embodiment, the complex comprises a polypeptide, a polynucleotide, or a combination of one or more polypeptides and one or more polynucleotides. In one embodiment, the complex comprises one or more polypeptides that bind to form a base editor (e.g., a base editor comprising a nucleic acid-programmable DNA-binding protein such as Cas9 and a deaminase) and a polynucleotide (e.g., a guide RNA). In one embodiment, the complex is held together by hydrogen bonds. It should be understood that one or more components of the base editor (e.g., a deaminase or a nucleic acid-programmable DNA-binding protein) can be bound by covalent or non-covalent bonds. As an example, a base editor may comprise a deaminase covalently linked (e.g., by a peptide bond) to a nucleic acid-programmable DNA-binding protein. Alternatively, a base editor may comprise a deaminase and a nucleic acid-programmable DNA-binding protein that are non-covalently associated (e.g., when one or more components of the base editor are provided in trans and associated directly or via another molecule, such as a protein or nucleic acid). In one embodiment, one or more components of the complex are held together by hydrogen bonds.

[0086] "Cytosine" or "4-aminopyrimidin-2(1H)-one" has the molecular formula C4H5N3O and the structure [ka] and corresponds to CAS number 71-30-7.

[0087] "Cytidine" is attached to the ribose sugar via a glycosidic bond, and has the structure [ka] and corresponds to CAS number 65-46-3. Its molecular formula is CH 13 It is N3O5.

[0088] "Cytidine base editor (CBE)" means a base editor that includes cytidine deaminase.

[0089] By "cytidine base editor (CBE) polynucleotide" is meant a polynucleotide that encodes a CBE.

[0090] "Cytidine deaminase" refers to a polypeptide or fragment thereof capable of catalyzing a deamination reaction that converts the amino group of cytidine to a carbonyl group. In one embodiment, cytidine deaminase converts cytosine to uracil or 5-methylcytosine to thymine. The terms "cytidine deaminase" and "cytosine deaminase" are used interchangeably throughout this application. PmCDA1 (SEQ ID NOS: 13-14) from Petromyzon marinus (Petromyzon marinus cytosine deaminase 1, "PmCDA1"), AID (activation-induced cytidine deaminase; AICDA) from mammals (e.g., human, pig, cow, horse, monkey, etc.) (exemplary AID polypeptide sequences are provided in the Sequence Listing as SEQ ID NOS: 15-21), and APOBEC (exemplary APOBEC polypeptide sequences are provided in the Sequence Listing as SEQ ID NOS: 22-62) are exemplary cytidine deaminases. Further exemplary cytidine deaminase (CDA) sequences are provided in the Sequence Listing as SEQ ID NOs: 63-67. Further exemplary cytidine deaminase sequences, including APOBEC polypeptide sequences, are provided in the Sequence Listing as SEQ ID NOs: 68-190.

[0091] "Cytosine" means a pyrimidine nucleobase having the molecular formula C4H5N3O.

[0092] "Cytosine deaminase activity" refers to catalyzing the deamination of cytosine in a polynucleotide, thereby converting an amino group to a carbonyl group. In one embodiment, a polypeptide having cytosine deaminase activity converts cytosine to uracil (i.e., C to U) or 5-methylcytosine to thymine (i.e., 5mC to T). In some embodiments, the adenosine deaminase variants provided herein have increased cytosine deaminase activity (e.g., at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more) compared to a reference adenosine deaminase (e.g., TadA*8.20 or TadA*8.19). In some embodiments, the cytosine deaminase is derived from a reference adenosine deaminase. In some embodiments, the adenosine deaminase variant has primarily cytidine deaminase activity and retains less than about 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, or 20% of the adenosine deaminase activity. In some embodiments, the adenosine deaminase variant has approximately equivalent adenosine and cytidine deaminase activity (e.g., within about, or at least about, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, or 50% of each other). In some examples, an adenosine deaminase variant has about, or at least about, 10%, 20%, 30%, 40%, 50%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 10,000-fold, or more cytosine deaminase activity compared to the adenosine deaminase activity of the variant. In some embodiments, an adenosine deaminase variant has primarily cytosine deaminase activity and little, if any, adenosine deaminase activity. In some embodiments, an adenosine deaminase variant has cytosine deaminase activity but no significant or detectable adenosine deaminase activity. As used herein, the term "deaminase" or "deaminase domain" refers to a protein or enzyme that catalyzes a deamination reaction.

[0093] "Detection" refers to identifying the presence, absence, or amount of the analyte being detected. In one embodiment, a sequence alteration in a polynucleotide or polypeptide is detected. In another embodiment, the presence of an indel is detected.

[0094] "Detectable label" refers to a composition that, when linked to a molecule of interest, renders the molecule of interest detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., enzymes commonly used in enzyme-linked immunosorbent assays (ELISAs)), biotin, digoxigenin, or haptens.

[0095] By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

[0096] "Dual editing activity" means having adenosine deaminase activity and cytidine deaminase activity. In one embodiment, a base editor with dual editing activity has both A→G and C→T activity, and the two activities are approximately equal or within about 10% or 20% of each other. In another embodiment, the dual editor has A→G activity that is no more than about 10% or 20% greater than C→T activity. In another embodiment, the dual editor has A→G activity that is no more than about 10% or 20% less than C→T activity. In some embodiments, the adenosine deaminase variant has primarily cytosine deaminase activity and little, if any, adenosine deaminase activity. In some embodiments, the adenosine deaminase variant has cytosine deaminase activity but no significant or detectable adenosine deaminase activity.

[0097] An "effective amount" refers to the amount of a drug or active compound, such as a base editor described herein, required to ameliorate disease symptoms, or the amount of a drug or active compound sufficient to induce a desired biological response, compared to an untreated patient or an individual not suffering from the disease, i.e., a healthy individual. The effective amount of the active compound(s) used to practice the present invention for the therapeutic treatment of a disease will vary depending on the method of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and administration regimen. Such an amount is referred to as an "effective" amount. In one embodiment, an effective amount is the amount of a base editor of the present invention sufficient to introduce a modification into a gene of interest in a cell (e.g., a cell in vitro or in vivo). In one embodiment, an effective amount is the amount of a base editor required to achieve a therapeutic effect. Such a therapeutic effect need not be sufficient to modify pathogenic genes in all cells of a subject, tissue, or organ; modifying pathogenic genes in approximately 1%, 5%, 10%, 25%, 50%, 75%, or more of the cells present in the subject, tissue, or organ is sufficient. In one embodiment, the effective amount is sufficient to ameliorate one or more symptoms of the disease.

[0098] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule, which portion comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. Fragments can comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0099] Generally, a "gene" is a polynucleotide that can be transcribed into RNA that has a regulatory function, a catalytic function, and / or encodes a protein. In embodiments, the polynucleotide is present in the genome of a cell. Eukaryotic genes typically contain introns and exons that can be organized to generate different RNA splice variants that encode alternative versions of the mature protein. Those skilled in the art will understand that the present disclosure encompasses all transcripts encoding a polypeptide of interest, including splice variants, allelic variants, and transcripts resulting from alternative promoter sites or alternative polyadenylation sites. Thus, a "full-length" gene or RNA encompasses any naturally occurring splice variants, allelic variants, other alternative transcripts, splice variants produced by recombinant techniques that perform the same function as the naturally occurring variant, and the resulting RNA molecules.

[0100] As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising protein domains from at least two different proteins. Those skilled in the art will understand that any protein that functions when fused will also function in the unfused state, i.e., in the context of a multimolecular complex.

[0101] "Guide RNA" or "gRNA" refers to a polynucleotide or polynucleotide complex that is specific for a target sequence and can form a complex with a polynucleotide-programmable nucleotide-binding domain protein (e.g., Cas9 or Cpf1). In one embodiment, the guide polynucleotide is a guide RNA (gRNA). The gRNA can exist as a complex of two or more RNAs or as a single RNA molecule.

[0102] "Hybridization" refers to hydrogen bonding between complementary nucleobases, which can be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

[0103] By "increase" is meant a positive change of at least about 10%, 25%, 50%, 75%, or 100%. In some embodiments, the increase is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100-fold compared to the reference.

[0104] The terms "inhibitor of base repair," "base repair inhibitor," "IBR," or grammatical equivalents thereof refer to proteins that are capable of inhibiting the activity of nucleic acid repair enzymes, such as base excision repair enzymes.

[0105] An "intein" is a fragment of a protein that can excise itself and join the remaining fragment (an extein) via a peptide bond in a process known as protein splicing.

[0106] The terms "isolated," "purified," or "biologically pure" refer to material that is free, to varying degrees, from components that normally accompany it in its natural state. "Isolated" refers to a degree of separation from its original source or surrounding environment. "Purified" refers to a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free from other substances so that impurities do not significantly affect the protein's biological properties or cause other adverse effects. That is, the nucleic acids or peptides of the invention are purified when, if produced by recombinant DNA technology, they are substantially free of cellular material, viral material, or culture medium, or when chemically synthesized, they are substantially free of chemical precursors or other chemicals. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can mean that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For example, in the case of proteins that can be modified, such as phosphorylation or glycosylation, different modifications may produce different isolated proteins, which can be individually purified.

[0107] An "isolated polynucleotide" refers to a nucleic acid (e.g., DNA) that is free of the genes that flank it in the naturally occurring genome of the organism from which the nucleic acid molecule of the present invention is derived. Thus, the term includes, for example, recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, or virus, or into the genomic DNA of a prokaryote or eukaryote, or that exists as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments generated by PCR or restriction endonuclease digestion). Furthermore, the term includes not only RNA molecules transcribed from DNA molecules, but also recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

[0108] By "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Ordinarily, a polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. Isolated polypeptides of the invention may be obtained, for example, by extraction from a natural source, by expressing a recombinant nucleic acid encoding such a polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0109] As used herein, the term "linker" refers to a molecule that connects two moieties. In one embodiment, the term "linker" refers to a covalent linker (e.g., a covalent bond) or a non-covalent linker.

[0110] By "marker" is meant any protein or polynucleotide that exhibits altered expression levels or activity associated with a disease or disorder.

[0111] As used herein, the term "mutation" refers to the substitution of a residue in a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or the deletion or insertion of one or more residues in a sequence. Mutations are generally described herein by identifying the original residue, followed by the position of the residue in the sequence, and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art and can be found, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)).

[0112] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides, are linear molecules in which adjacent nucleotides are linked to each other via phosphodiester bonds. In some embodiments, "nucleic acid" refers to an individual nucleic acid residue (e.g., a nucleotide and / or a nucleoside). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, "nucleic acid" encompasses RNA and single- and / or double-stranded DNA. Nucleic acids can be naturally occurring, e.g., in relation to a genome, a transcript, mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, a cosmid, a chromosome, a chromatid, or other naturally occurring nucleic acid molecule. Alternatively, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., recombinant DNA or RNA, an artificial chromosome, an altered genome, or a fragment thereof, or synthetic DNA, RNA, DNA / RNA hybrid, or non-naturally occurring nucleotides or nucleosides. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, e.g., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources or produced using recombinant expression systems, and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can include nucleoside analogs, e.g., analogs having chemically modified bases or sugars and backbone modifications. Nucleic acid sequences are presented in the 5' to 3' direction unless otherwise specified.In some embodiments, nucleic acids are selected from natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynyl-cytidine, C5-methylcytidine, and the like). The base may be or contain a base selected from the group consisting of 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine, chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (2'-e.g., fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0113] The term "nuclear localization sequence," "nuclear localization signal," or "NLS" refers to an amino acid sequence that promotes the import of proteins into the cell nucleus. Nuclear localization sequences are known in the art and are described, for example, in International PCT Application PCT / EP2000 / 011690 by Plank et al., filed November 23, 2000, and published May 31, 2001 as WO / 2001 / 038547, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS, for example, as described in Koblan et al., Nature Biotech. 2018 doi:10.1038 / nbt.4172. In some embodiments, the NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV (SEQ ID NO: 191), KRPAATKKAGQAKKKK (SEQ ID NO: 192), KKTELQTTNAENKTKKL (SEQ ID NO: 193), KRGINDRNFWRGENGRKTR (SEQ ID NO: 194), RKSGKIAAIVVKRPRK (SEQ ID NO: 195), PKKKRKV (SEQ ID NO: 196), or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 197).

[0114] The terms "nucleobase," "nitrogenous base," or "base," used interchangeably herein, refer to nitrogen-containing biological compounds that form nucleosides and are components of nucleotides. The ability of nucleobases to base pair and stack with each other directly gives rise to long-chain helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The five nucleobases, adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), are referred to as primary or standard. Adenine and guanine are derived from purines, while cytosine, uracil, and thymine are derived from pyrimidines. DNA and RNA can contain other modified (non-primary) bases. Non-limiting exemplary modified nucleobases include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrolauracil, 5-methylcytosine (5mC), and 5-hydromethylcytosine. Hypoxanthine and xanthine are produced in the presence of mutagens, but both are produced by deamination (substitution of an amine group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can be produced by deamination of cytosine. A "nucleoside" consists of a nucleobase and a five-carbon sugar (either ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of nucleosides with modified nucleobases include inosine (I), xanthosine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (5mC), and pseudouridine (Ψ). A "nucleotide" consists of a nucleobase, a five-carbon sugar (either ribose or deoxyribose), and at least one phosphate group.Non-limiting examples of modified nucleobases and / or chemical modifications that the modified nucleobases may comprise are as follows: pseudouridine, 5-methyl-cytosine, 2'-O-methyl-3'-phosphonoacetate, 2'-O-methylthioPACE (MSP), 2'-O-methyl-PACE (MP), 2'-fluoroRNA (2'-F-RNA), constrained ethyl (S-cEt), 2'-O-methyl ("M"), 2'-O-methyl-3'-phosphorothioate ("MS"), 2'-O-methyl-3'-thiophosphonoacetate ("MSP"), 5-methoxyuridine, phosphorothioate, and N1-methylpseudouridine.

[0115] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" is used interchangeably with "polynucleotide programmable nucleotide binding domain" and can refer to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or guide polynucleotide (e.g., gRNA), that directs the napDNAbp to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 protein. The Cas9 protein can bind to a guide RNA that directs the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, such as a nuclease-active Cas9, Cas9 nickase (nCas9), or nuclease-inactive Cas9 (dCas9). Non-limiting examples of nucleic acid programmable DNA binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, and Cas12j / CasΦ (Cas12j / Casphi).Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, and Cas9. (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Cas12j / CasΦ, Cpf1, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4 , Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, type II Cas effector proteins, type V Cas effector proteins, type VI Cas effector proteins, CARF, DinG, homologs thereof, or modified or altered versions thereof. Other nucleic acid programmable DNA binding proteins are within the scope of this disclosure, although they may not be specifically listed in this disclosure. See, for example, Makarova et al. "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?" CRISPR J. 2018 Oct; 1:325-336. doi: 10.1089 / crispr.2018.0033, Yan et al., "Functionally diverse type V CRISPR-Cas systems" Science. 2019 Jan 4; 363 (6422):88-91. doi: 10.1126 / science.aav7271 (the entire contents of each are incorporated herein by reference).Exemplary nucleic acid programmable DNA binding proteins and nucleic acid sequences encoding nucleic acid programmable DNA binding proteins are provided in the Sequence Listing as SEQ ID NOs: 198-231 and 390.

[0116] As used herein, the term "nucleobase editing domain" or "nucleobase editing protein" refers to a protein or enzyme that can catalyze nucleobase modifications in RNA or DNA, such as the deamination of cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine), and adenine (or adenosine) to hypoxanthine (or inosine), and the non-template addition and insertion of nucleotides. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., adenine deaminase or adenosine deaminase). In some embodiments, the nucleobase editing domain is an adenosine deaminase variant domain that has adenosine deaminase activity and cytosine deaminase activity.

[0117] As used herein, "obtaining," as in "obtaining an agent," includes synthesizing, purchasing, or otherwise obtaining an agent.

[0118] As used herein, "patient" or "subject" refers to a mammalian subject or individual who has been diagnosed with a disease or disorder, is at risk of having or developing a disease or disorder, or is suspected of having or developing a disease or disorder. In some embodiments, the term "patient" refers to a mammalian subject who has a higher-than-average likelihood of developing a disease or disorder. Exemplary patients can be humans, non-human primates, cats, dogs, pigs, cows, felines, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs), and other mammals that can benefit from the therapies disclosed herein. Exemplary human patients can be male and / or female.

[0119] A "patient in need thereof" or "subject in need thereof," as used herein, refers to a patient who has been diagnosed with, is at risk of having, has, has been pre-determined to have, or is suspected of having a disease or disorder.

[0120] The terms "pathogenic mutation," "pathogenic variant," "causing mutation," "pathogenic variant," "deleterious mutation," or "predisposing mutation" refer to a genetic change or mutation that increases an individual's susceptibility or predisposition to a particular disease or disorder. In some embodiments, a pathogenic mutation comprises at least one wild-type amino acid substituted with at least one pathogenic amino acid in a protein encoded by a gene.

[0121] The terms "protein," "peptide," "polypeptide," and their grammatical equivalents are used interchangeably herein to refer to a polymer of amino acid residues joined together by peptide (amide) bonds. A protein, peptide, or polypeptide may be natural, recombinant, or synthetic, or any combination thereof.

[0122] The term "recombinant," as used herein with respect to a protein or nucleic acid, refers to a protein or nucleic acid that does not occur in nature but is the product of human modification. For example, in some embodiments, a recombinant protein or nucleic acid molecule includes an amino acid or nucleotide sequence that contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations compared to the naturally occurring sequence.

[0123] By "decreasing" is meant a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0124] "Reference" refers to a standard or control condition. In one embodiment, the activity of an adenosine deaminase variant with a modification that confers cytosine deaminase activity is compared to the activity of a reference adenosine deaminase that lacks the modification. In one embodiment, the activity of a base editor comprising an adenosine deaminase variant with a modification that confers cytosine deaminase activity is compared to the activity of a base editor comprising a reference adenosine deaminase that lacks the modification.

[0125] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a specified sequence, for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence will generally be at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 35 amino acids, at least about 50 amino acids, or at least about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence will generally be at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, or at least about 300 nucleotides, or any integer thereabout or therebetween. In some embodiments, the reference sequence is the wild-type sequence of a protein of interest. In other embodiments, the reference sequence is a polynucleotide sequence encoding the wild-type protein.

[0126] The terms "RNA-programmable nuclease" and "RNA-guided nuclease" are used in conjunction with (e.g., bound or associated with) one or more RNA(s) that are not targets for cleavage. In some embodiments, when an RNA-programmable nuclease is complexed with an RNA, it may be referred to as a nuclease:RNA complex. Typically, the bound RNA(s) are referred to as guide RNAs (gRNAs). In some embodiments, the RNA-programmable nuclease is a (CRISPR-associated) Cas9 endonuclease, such as Cas9 from Streptococcus pyogenes (Csn1) (e.g., SEQ ID NO: 198), Cas9 from Neisseria meningitidis (NmeCas9, SEQ ID NO: 209), Nme2Cas9 (SEQ ID NO: 210), or a derivative thereof (e.g., a sequence having at least about 85% sequence identity to Cas9, such as Nme2Cas9 or spCas9).

[0127] The term "single nucleotide polymorphism (SNP)" refers to a single nucleotide variation occurring at a specific location in the genome, with each variation occurring to some appreciable extent (e.g., >1%) in a population. SNPs can occur within the coding region of a gene, within the non-coding region of a gene, or within an intergenic region (region between genes). In some embodiments, SNPs within a coding sequence do not necessarily alter the amino acid sequence of the protein produced due to the degeneracy of the genetic code. SNPs in coding regions are of two types: synonymous and non-synonymous SNPs. Synonymous SNPs do not affect the sequence of a protein, while non-synonymous SNPs do alter the amino acid sequence of a protein. Non-synonymous SNPs are of two types: missense and nonsense. SNPs not in protein-coding regions may still affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of non-coding RNA. Gene expression affected by this type of SNP is called an eSNP (expressed SNP) and can be located upstream or downstream of a gene. Single nucleotide variants (SNVs) are single nucleotide variations that occur at any frequency and can occur somatically. Somatic single nucleotide variations are also called single base changes.

[0128] "Substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence. In one embodiment, the reference sequence is a wild-type amino acid or nucleic acid sequence. In another embodiment, the reference sequence is any one of the amino acid or nucleic acid sequences described herein. In one embodiment, such a sequence is at least 60%, 80%, 85%, 90%, 95%, or even 99% identical at the amino acid or nucleic acid level to the sequence used for comparison.

[0129] Sequence identity is typically measured using sequence analysis software (e.g., sequence analysis software from the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: Glycine, Alanine, Valine, isoleucine, leucine, Aspartic acid, glutamic acid, asparagine, glutamine, Serine, threonine, Lysine, arginine, and Phenylanine, tyrosine. In an exemplary approach to determining the degree of identity, e, indicating closely related sequences, -3 ~e -100 The BLAST program may be used, using a probability score of

[0130] Using COBALT, for example, with the following parameters: a) Alignment parameters: Gap penalty -11, -1 and End-Gap penalty -5, -1; b) CDD parameters: Use RPS BLAST, Blast E-value 0.003, find and recalculate conserved columns, and c) Query clustering parameters: Use query cluster, word size 4, maximum cluster distance 0.8, Alphabet Regular. EMBOSS Needle is used, for example, with the following parameters: a) Matrix: BLOSUM62; b) GAP OPEN: 10; c) GAP EXTEND: 0.5; d) OUTPUT FORMAT: pair, e) END GAP PENALTY: false, f) END GAP OPEN: 10, and g) END GAP EXTEND: 0.5.

[0131] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence will typically be able to hybridize with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence will typically be able to hybridize with at least one strand of a double-stranded nucleic acid molecule. "Hybridizing" means that the pair forms a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof under various stringency conditions. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0132] For example, stringent salt concentrations are typically less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of organic solvents, such as formamide, while high stringency hybridization can be achieved in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions typically include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters, such as hybridization time, detergent concentration, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed. In a preferred embodiment, hybridization is carried out in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS at 30° C. In a more preferred embodiment, hybridization is carried out in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37° C. In a most preferred embodiment, hybridization is carried out in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42° C. Useful variations on these conditions will be readily apparent to those of skill in the art.

[0133] In most applications, the stringency of the washing step following hybridization also varies. Stringent wash conditions can be defined by salt concentration and temperature. As noted above, washing stringency can be increased by decreasing the salt concentration or increasing the temperature. For example, stringent salt concentrations for the washing step are preferably less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the washing step typically include a temperature of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In one embodiment, the washing step is performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another embodiment, the washing step is performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash step is carried out in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 68°C. Additional variations of these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977), Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975), Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001), Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York), and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0134] "Split" means divided into two or more pieces.

[0135] "Split Cas9 protein" or "split Cas9" refers to a Cas9 protein that is provided as an N-terminal fragment and a C-terminal fragment that are encoded by two separate nucleotide sequences. Polypeptides corresponding to the N-terminal and C-terminal portions of the Cas9 protein can be spliced ​​to form a "reconstituted" Cas9 protein.

[0136] The term "target site" refers to a site within a nucleic acid molecule that is deaminated by a deaminase (e.g., an adenine deaminase variant), or a fusion protein or multimolecular complex that includes a deaminase (e.g., a dCas9-adenosine deaminase variant fusion protein or a base editor disclosed herein).

[0137] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing and / or ameliorating a disorder and / or its associated symptoms, or achieving a desired pharmacological and / or physiological effect. It will be understood that treating a disorder or condition does not necessarily require, although does not exclude, that the disorder, condition, or its associated symptoms be completely eliminated. In some embodiments, the effect is therapeutic, i.e., the effect includes, but is not limited to, partially or completely reducing, reducing, arresting, alleviating, mitigating, lessening the intensity of, or curing, the disease and / or adverse symptoms resulting from the disease. In some embodiments, the effect is prophylactic, i.e., the effect protects against or prevents the onset or recurrence of the disease or condition. To this end, the methods disclosed herein comprise administering a therapeutically effective amount of a composition as described herein.

[0138] "Uracil glycosylase inhibitor" or "UGI" refers to an agent that inhibits the uracil excision repair system. Base editors containing cytidine deaminase convert cytosine to uracil, which is then converted to thymine during DNA replication or repair. Inclusion of an inhibitor of uracil DNA glycosylase (UGI) in a base editor prevents base excision repair of U back to C. Exemplary UGIs include the following amino acid sequences: >splP14739IUNGI_BPPB2 uracil-DNA glycosylase inhibitor MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSD APEYKPWALVIQDSNGENKIKML (SEQ ID NO: 232).

[0139] The term "vector" refers to a means for introducing a nucleic acid sequence into a cell, resulting in a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, and episomes. An "expression vector" is a nucleic acid sequence that contains a nucleotide sequence that is expressed in a recipient cell. Expression vectors may contain additional nucleic acid sequences to facilitate and / or promote expression of the introduced sequence, such as initiation sequences, termination sequences, enhancers, promoters, and secretion sequences.

[0140] It is understood that ranges provided herein are shorthand notations for all values ​​within that range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0141] The recitation of a list of chemical groups within any definition of a variable herein includes the definition of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0142] All terms are intended to be understood as understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0143] In this application, the use of the singular includes the plural unless clearly stated otherwise. It must be noted that as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "including," as well as other forms such as "include," "includes," and "including," is open-ended.

[0144] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Any embodiment designated as "comprising" particular component(s) or element(s) is also contemplated in some embodiments as "consisting of" or "consisting essentially of" the particular component(s) or element(s). It is contemplated that any embodiment referred to herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Additionally, the compositions of the present disclosure can be used to achieve the methods of the present disclosure.

[0145] The term "about" or "approximately" means within an acceptable error range for a particular value as measured by one of ordinary skill in the art. Such an acceptable range will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system.

[0146] References herein to "some embodiments," "embodiments," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments, but need not be included in all embodiments of the present disclosure. [Brief explanation of the drawings]

[0147] [Figure 1A]The figure shows the editing rates of single-stranded DNA-specific adenosine and cytidine deaminase (ssDacd) base editors 1.1 to 1.20 using the target sequence 5'-GAACACAAAGCATAGACTGCGGG-3' (SEQ ID NO: 233). The target site is shown in double-byte characters, and the PAM sequence is shown in underlined font. The adenosine base editors ABE8.20 and ABE8.2b were used as negative controls for C to non-C editing and as positive controls for A to G editing. The cytidine base editors BE4, BE4max, and BE3b were used as positive controls for C to non-C editing and as negative controls for A to G editing. Base editors (ME-1 and ME-2) containing fusion proteins with both an adenosine deaminase domain and a cytosine deaminase domain were used as controls. The figure is a graph showing the percentage of C to non-C editing at position C4 (bold font) in the absence of the UGI domain for each base edited construct. [Figure 1B] The figure shows the editing rates of single-stranded DNA-specific adenosine and cytidine deaminase (ssDacd) base editors 1.1 to 1.20 using the target sequence 5'-GAACACAAAGCATAGACTGCGGG-3' (SEQ ID NO: 233). The target site is shown in double-byte characters, and the PAM sequence is shown in underlined font. The adenosine base editors ABE8.20 and ABE8.2b were used as negative controls for C to non-C editing and as positive controls for A to G editing. The cytidine base editors BE4, BE4max, and BE3b were used as positive controls for C to non-C editing and as negative controls for A to G editing. Base editors (ME-1 and ME-2) containing fusion proteins with both an adenosine deaminase domain and a cytosine deaminase domain were used as controls. The figure is a graph showing the percentage of A to G editing at position A5 (bold font) in the absence of a UGI domain for each base edited construct. [Figure 1C]The figure shows the editing rates of single-stranded DNA-specific adenosine and cytidine deaminase (ssDacd) base editors 1.1 to 1.20 using the target sequence 5'-GAACACAAAGCATAGACTGCGGG-3' (SEQ ID NO: 233). The target site is shown in full-width characters, and the PAM sequence is shown in underlined font. The adenosine base editors ABE8.20 and ABE8.2b were used as negative controls for C to non-C editing and as positive controls for A to G editing. The cytidine base editors BE4, BE4max, and BE3b were used as positive controls for C to non-C editing and as negative controls for A to G editing. Base editors (ME-1 and ME-2) containing fusion proteins with both adenosine deaminase and cytosine deaminase domains were used as controls. The figure is a heat map showing the rate of indels for each base editor. [Figure 2A] The figure shows the percentage of editing for ssDacd1.1 to ssDacd1.20 base editors using the sequence 5'-GAACACAAAGCATAGACTGCGGG-3' (SEQ ID NO: 233). The target site is shown in full-width characters, and the PAM sequence is shown in underlined font. The adenosine base editors ABE8.20 and ABE8.2b were used as negative controls for C to non-C editing and as positive controls for A to G editing. The cytidine base editors BE4, BE4max, and BE3b were used as positive controls for C to non-C editing and as negative controls for A to G editing. ME-1 and ME-2 were used as controls. The figure is a graph showing the percentage of C to non-C editing at position C4 (bold font) in the presence of the UGI domain for each base editor. [Figure 2B]The figure shows the percentage of editing for ssDacd1.1 to ssDacd1.20 base editors using the sequence 5'-GAACACAAAGCATAGACTGCGGG-3' (SEQ ID NO: 233). The target site is shown in full-width characters, and the PAM sequence is shown in underlined font. Adenosine base editors ABE8.20 and ABE8.2b were used as negative controls for C to non-C editing and as positive controls for A to G editing. Cytidine base editors BE4, BE4max, and BE3b were used as positive controls for C to non-C editing and as negative controls for A to G editing. ME-1 and ME-2 were used as controls. The figure is a graph showing the percentage of A to G editing at position A5 (bold) in the presence of the UGI domain for each base editor. [Figure 2C] The figure shows the percentage of editing for ssDacd1.1 to ssDacd1.20 base editors using the sequence 5'-GAACACAAAGCATAGACTGCGGG-3' (SEQ ID NO: 233). The target site is shown in full-width characters, and the PAM sequence is shown in underlined font. The adenosine base editors ABE8.20 and ABE8.2b were used as negative controls for C to non-C editing and as positive controls for A to G editing. The cytidine base editors BE4, BE4max, and BE3b were used as positive controls for C to non-C editing and as negative controls for A to G editing. ME-1 and ME-2 were used as controls. The figure is a heat map showing the percentage of indels for each base editor. [Figure 3A]Figure 1 shows the percentage of editing for ssDacd1.1, ssDacd1.2, ssDacd1.9, ssDacd1.12, ssDacd1.17, ssDacd1.18, and ssDacd1.19 base editors using the Hek site 2 ("299") sequence 5'-GAACACAAAGCATAGACTGCGGG-3' (SEQ ID NO: 233). Target sites are shown in double-byte characters, and PAM sequences are shown in underlined font. The adenosine base editor ABE8.20 was used as a positive control for A to G editing and a negative control for C to non-C editing. The cytidine base editor BE4 was used as a positive control for C to non-C editing and a negative control for A to G editing. ME-1 and ME-2 were used as controls. The figure is a graph showing the rate of deamination at positions C4T, A5G, C6R, and A7G (shown in bold font) for each of the base edits. [Figure 3B] The figure shows the percentage of editing for ssDacd1.1, ssDacd1.2, ssDacd1.9, ssDacd1.12, ssDacd1.17, ssDacd1.18, and ssDacd1.19 base editors using the Hek site 2 ("299") sequence 5'-GAACACAAAGCATAGACTGCGGG-3' (SEQ ID NO: 233). The target site is shown in double-byte characters, and the PAM sequence is shown in underlined font. The adenosine base editor ABE8.20 was used as a positive control for A to G editing and a negative control for C to non-C editing. The cytidine base editor BE4 was used as a positive control for C to non-C editing and a negative control for A to G editing. ME-1 and ME-2 were used as controls. The figure is a heatmap showing the percentage of indels for each base editor. [Figure 4A]The figure shows the editing rates of the ssDacd1.1 to ssDacd1.20 base editors. The adenosine base editors ABE8.20 and ABE8.2b were used as negative controls for C to T editing and as positive controls for A to G editing. The cytidine base editors BE4, BE4max, and BE3b were used as positive controls for C to T editing and as negative controls for A to G editing. ME-1 and ME-2 were used as controls. The figure is a graph showing the deamination rates for A to G and C to Tmax editing using the RNF2 sequence 5'-GTCATCTTAGTCATTACCTGAGG-3' (SEQ ID NO: 234) in the presence of the UGI domain for each base editor. The PAM sequence is shown in underlined font. [Figure 4B] The figure shows the editing rates of ssDacd1.1 to ssDacd1.20 base editors. Adenosine base editors ABE8.20 and ABE8.2b were used as negative controls for C to T editing and as positive controls for A to G editing. Cytidine base editors BE4, BE4max, and BE3b were used as positive controls for C to T editing and as negative controls for A to G editing. ME-1 and ME-2 were used as controls. The figure is a graph showing the deamination rates of A to G and C to T editing for each base editor using the Emx1 sequence 5'-GAGTCCGAGCAGAAGAAGAAGGG-3' (SEQ ID NO: 235) in the presence of the UGI domain. [Figure 4C]The figure shows the editing rates of ssDacd1.1 to ssDacd1.20 base editors. Adenosine base editors ABE8.20 and ABE8.2b were used as negative controls for C to T editing and as positive controls for A to G editing. Cytidine base editors BE4, BE4max, and BE3b were used as positive controls for C to T editing and as negative controls for A to G editing. ME-1 and ME-2 were used as controls. The figure is a graph showing the deamination rates of A to G and C to T max editing at site 1 of the EMX1 v2 ("B415") sequence 5'-GCTCCCATCACATCAACCGGTGG-3' (SEQ ID NO: 236) in the presence of the UGI domain for each base editor. [Figure 4D] The figure shows the editing rates of ssDacd1.1 to ssDacd1.20 base editors. Adenosine base editors ABE8.20 and ABE8.2b were used as negative controls for C to T editing and as positive controls for A to G editing. Cytidine base editors BE4, BE4max, and BE3b were used as positive controls for C to T editing and as negative controls for A to G editing. ME-1 and ME-2 were used as controls. The figure is a graph showing the deamination rates of A to G and C to T editing for each base editor using the Hek2 site 3 sequence 5'-GGCCCAGACTGAGCACGTGATGG-3' (SEQ ID NO: 237) in the presence of the UGI domain. [Figure 4E] The editing rates of ssDacd1.1 to ssDacd1.20 base editors are shown. Adenosine base editors ABE8.20 and ABE8.2b were used as negative controls for C to T editing and as positive controls for A to G editing. Cytidine base editors BE4, BE4max, and BE3b were used as positive controls for C to T editing and as negative controls for A to G editing. ME-1 and ME-2 were used as controls. The figure is a heatmap showing the rate of indels for each of the base editors for RNF2, Emx1, Hek site 1, and Hek2 site 3. [Figure 5]1 is a graph showing the deamination rates for A to G and C to T editing for each of the base editors ssDacd1.1 to ssDacd1.20 using the Hek2 site 2 sequence 5'-GAACACAAAGCATAGACTGCGGG-3' (SEQ ID NO: 233) in the presence of the UGI domain. The PAM sequence is shown in underlined font. Adenosine base editors ABE8.20 and ABE8.2b were used as negative controls for C to T editing and as positive controls for A to G editing. Cytidine base editors BE4, BE4max, and BE3b were used as positive controls for C to T editing and as negative controls for A to G editing. ME-1 and ME-2 were used as controls. [Figure 6A] The figure shows the percentage of editing of ssDacd1.1 to ssDacd1.20 base editors in the presence and absence of the UGI domain at Hek site 2. Guide alone and no transfection (NoTxCtr and NoTxCtr1) were used as negative controls. B433, B434, B970, B88, B120, B802, YY-B2, B93, and B110 were used as controls. The figure is a graph showing the percentage of A to G, C to T, or C to G editing for each base editor. [Figure 6B] The figure shows the percentage of edits of ssDacd1.1 to ssDacd1.20 base editors in the presence and absence of the UGI domain at Hek site 2. Guide only and no transfection (NoTxCtr and NoTxCtr1) were used as negative controls. B433, B434, B970, B88, B120, B802, YY-B2, B93, and B110 were used as controls. The figure is a graph showing the percentage of indels in each base editor. [Figure 7A]The figure shows the percentage of editing for ssDacd1.1 to ssDacd1.20 base editors in the presence and absence of the UGI domain in EMX1 (ack115). Guide only, NoTxCtr, and NoTxCtr1 were used as negative controls. The figures are graphs showing the percentage of A to G, C to T, or C to G editing for each base editor. [Figure 7B] The figure shows the percentage of edits in the ssDacd1.1 to ssDacd1.20 base edit constructs in EMX1 (ack115) with and without the UGI domain. Guide only, NoTxCtr, and NoTxCtr1 were used as negative controls. The figures are graphs showing the percentage of indels in each base edit construct. [Figure 8A] The figure shows the percentage of editing for ssDacd1.1 to ssDacd1.20 base editors at Hek site 3 (ack115) in the presence and absence of the UGI domain. Guide only, NoTxCtr, and NoTxCtr1 were used as negative controls. The figures are graphs showing the percentage of A to G, C to T, or C to G editing for each base editor. [Figure 8B] The figure shows the percentage of edits for ssDacd1.1 to ssDacd1.20 base editors at Hek site 3 (ack115) in the presence and absence of the UGI domain. Guide only, NoTxCtr, and NoTxCtr1 were used as negative controls. B433, B434, B970, B88, B120, B802, YY-B2, B93, and B110. The figure is a graph showing the percentage of indels for each base editor. [Figure 9A]The figure shows the percentage of editing for ssDacd1.1 to ssDacd1.20 base editors in the presence and absence of the UGI domain in RNF2 (ack121). Guide only, NoTxCtr, and NoTxCtr1 were used as negative controls. The figures are graphs showing the percentage of A to G, C to T, or C to G editing for each base editor. [Figure 9B] The figure shows the percentage of edits in the ssDacd1.1 to ssDacd1.20 base edits in RNF2 (ack121) with and without the UGI domain. Guide only, NoTxCtr, and NoTxCtr1 were used as negative controls. B433, B434, B970, B88, B120, B802, YY-B2, B93, and B110. The figure is a graph showing the percentage of indels in each base edit. [Figure 10A] The figure shows the percentage of editing of ssDacd1.1 to ssDacd1.20 base editors at EMX1 site 2 (B415) in the presence and absence of the UGI domain. Guide only, NoTxCtr, and NoTxCtr1 were used as negative controls. B433, B434, B970, B88, B120, B802, YY-B2, B93, and B110. The figure is a graph showing the percentage of A to G, C to T, or C to G editing for each base editor. [Figure 10B] The figure shows the percentage of edits for ssDacd1.1 to ssDacd1.20 base editors at EMX1 site 2 (B415) in the presence and absence of the UGI domain. Guide only, NoTxCtr, and NoTxCtr1 were used as negative controls. B433, B434, B970, B88, B120, B802, YY-B2, B93, and B110. The figure is a graph showing the percentage of indels for each base editor. [Figure 11A]The figure shows the percentage of editing of ssDacd1.1 to ssDacd1.20 base editors in spA12 (YY-A12) in the presence and absence of the UGI domain. Guide only, NoTxCtr, and NoTxCtr1 were used as negative controls. The figures are graphs showing the percentage of A to G, C to T, or C to G editing for each base editor. [Figure 11B] The figure shows the percentage of edits for ssDacd1.1 to ssDacd1.20 base editors in spA12 (YY-A12) with and without the UGI domain. Guide only, NoTxCtr, and NoTxCtr1 were used as negative controls. The figures show the percentage of indels for each base editor. [Figure 12] 1 is a graph showing the maximum efficiency of adenosine base editor variants using the sequence 5'-GTATTACTATTATTATCTGAGA-3' (YY-A1) (SEQ ID NO: 238), and a heat map showing the percentage of indels for each base editor. The target sequence is underlined. [Figure 13] 1 is a graph showing the maximum efficiency of adenosine base editor variants using the sequence 5'-GTGGGACTGATCCCTTAATGTG-3' (YY-A2) (SEQ ID NO: 239), and a heat map showing the percentage of indels for each base editor. The target sequence is underlined. [Figure 14A] 1 is a graph showing editing of the YY-A2 sequence at site A6 by an adenosine base editor variant. The sequence at the top of the figure is SEQ ID NO: 239. [Figure 14B] 1 is a graph showing editing of the YY-A2 sequence at site C7 by adenosine base editor variants. The sequence at the top of the figure is SEQ ID NO: 239. [Figure 15]1 is a graph showing the maximum efficiency of adenosine base editor variants using the sequence 5'-GACCAGGTCAGCAAACATGTT-3 (YY-A6) (SEQ ID NO: 240), and a heat map showing the percentage of indels for each base editor. The target sequence is underlined. [Figure 16] 1 is a graph showing the maximum efficiency of adenosine base editor variants in the presence of a UGI domain using the sequence 5'-GACTCAGCGCCCCTGCCGGGCC-3' (YY-A7) (SEQ ID NO: 241), and a heat map showing the percentage of indels for each base editor. The target sequence is underlined. [Figure 17] 1 is a graph showing the maximum efficiency of adenosine base editor variants in the presence of a UGI domain using the sequence 5'-GCCACAGTGGGAGGGGACATG-3' (YY-A15) (SEQ ID NO: 242), and a heat map showing the percentage of indels for each base editor. The target sequence is underlined. [Figure 18] 1 is a graph showing the maximum efficiency of adenosine base editor variants in the presence of a UGI domain using the sequence 5'-GCCCAGCAATTCACTGTGAAG-3' (YY-A16) (SEQ ID NO: 243), and a heat map showing the percentage of indels for each base editor. The target sequence is underlined. [Figure 19] 1 is a graph showing the maximum efficiency of adenosine base editor variants in the presence of a UGI domain using the sequence 5'-GCCCAGCTCCAGCCTCTGATG-3' (YY-A17) (SEQ ID NO: 244), and a heat map showing the percentage of indels for each base editor. The target sequence is underlined. [Figure 20] 1 is a graph showing the maximum efficiency of adenosine base editor variants in the presence of a UGI domain using the sequence 5'-GGTCGACCCTTGGTATCCATG-3' (YY-A27) (SEQ ID NO: 245), and a heat map showing the percentage of indels for each base editor. The target sequence is underlined. [Figure 21A]1 is a graph showing editing of the YY-A27 sequence at site C4 by an adenosine base editor variant with a UGI domain. The sequence at the top of the figure is SEQ ID NO: 245. [Figure 21B] 1 is a graph showing editing of the YY-A27 sequence at site A6 by an adenosine base editor variant with a UGI domain. The sequence at the top of the figure is SEQ ID NO: 245. [Figure 21C] 1 is a graph showing editing of the YY-A27 sequence at site C7 by an adenosine base editor variant with a UGI domain. The sequence at the top of the figure is SEQ ID NO: 245. [Figure 21D] 1 is a graph showing editing of the YY-A27 sequence at site C8 by an adenosine base editor variant with a UGI domain. The sequence at the top of the figure is SEQ ID NO: 245. [Figure 22] 1 is a graph showing the maximum efficiency of adenosine base editor variants in the presence of a UGI domain using the sequence 5'-GGTCGTAGCCAGTCCGAACCC-3' (YY-A28) (SEQ ID NO: 246), and a heat map showing the percentage of indels for each base editor. The target sequence is underlined. [Figure 23A] 1 is a graph showing the maximum editing efficiency of adenosine base editor variants across nine sites (YY-A1, YY-A2, YY-A6, YY-A7, YY-A15, YY-A16, YY-A17, YY-A27, and YY-A28).The figure is a graph showing the maximum editing efficiency of adenosine base editor variant 1.1. [Figure 23B] 1 is a graph showing the maximum editing efficiency of adenosine base editor variants across nine sites (YY-A1, YY-A2, YY-A6, YY-A7, YY-A15, YY-A16, YY-A17, YY-A27, and YY-A28).The figure is a graph showing the maximum editing efficiency of adenosine base editor variant 1.2. [Figure 23C]1 is a graph showing the maximum editing efficiency of adenosine base editor variants across nine sites (YY-A1, YY-A2, YY-A6, YY-A7, YY-A15, YY-A16, YY-A17, YY-A27, and YY-A28).The figure is a graph showing the maximum editing efficiency of adenosine base editor variant 1.3. [Figure 23D] 1 is a graph showing the maximum editing efficiency of adenosine base editor variants across nine sites (YY-A1, YY-A2, YY-A6, YY-A7, YY-A15, YY-A16, YY-A17, YY-A27, and YY-A28).The figure is a graph showing the maximum editing efficiency of adenosine base editor variant 1.4. [Figure 23E] 1 is a graph showing the maximum editing efficiency of adenosine base editor variants across nine sites (YY-A1, YY-A2, YY-A6, YY-A7, YY-A15, YY-A16, YY-A17, YY-A27, and YY-A28).The figure is a graph showing the maximum editing efficiency of adenosine base editor variant 1.5. [Figure 23F] 1 is a graph showing the maximum editing efficiency of adenosine base editor variants across nine sites (YY-A1, YY-A2, YY-A6, YY-A7, YY-A15, YY-A16, YY-A17, YY-A27, and YY-A28).The figure is a graph showing the maximum editing efficiency of adenosine base editor variant 1.6. [Figure 23G] 1 is a graph showing the maximum editing efficiency of adenosine base editor variants across nine sites (YY-A1, YY-A2, YY-A6, YY-A7, YY-A15, YY-A16, YY-A17, YY-A27, and YY-A28).The figure is a graph showing the maximum editing efficiency of adenosine base editor variant 1.7. [Figure 23H] 1 is a graph showing the maximum editing efficiency of adenosine base editor variants across nine sites (YY-A1, YY-A2, YY-A6, YY-A7, YY-A15, YY-A16, YY-A17, YY-A27, and YY-A28).The figure shows the maximum editing efficiency of adenosine base editor variant 1.9. [Figure 23I]1 is a graph showing the maximum editing efficiency of adenosine base editor variants across nine sites (YY-A1, YY-A2, YY-A6, YY-A7, YY-A15, YY-A16, YY-A17, YY-A27, and YY-A28).The figure is a graph showing the maximum editing efficiency of adenosine base editor variant 1.10. [Figure 23J] 1 is a graph showing the maximum editing efficiency of adenosine base editor variants across nine sites (YY-A1, YY-A2, YY-A6, YY-A7, YY-A15, YY-A16, YY-A17, YY-A27, and YY-A28).The figure is a graph showing the maximum editing efficiency of adenosine base editor variant 1.11. [Figure 23K] 1 is a graph showing the maximum editing efficiency of adenosine base editor variants across nine sites (YY-A1, YY-A2, YY-A6, YY-A7, YY-A15, YY-A16, YY-A17, YY-A27, and YY-A28).The figure is a graph showing the maximum editing efficiency of adenosine base editor variant 1.12. [Figure 23L] 1 is a graph showing the maximum editing efficiency of adenosine base editor variants across nine sites (YY-A1, YY-A2, YY-A6, YY-A7, YY-A15, YY-A16, YY-A17, YY-A27, and YY-A28).The figure is a graph showing the maximum editing efficiency of adenosine base editor variant 1.13. [Figure 23M] 1 is a graph showing the maximum editing efficiency of adenosine base editor variants across nine sites (YY-A1, YY-A2, YY-A6, YY-A7, YY-A15, YY-A16, YY-A17, YY-A27, and YY-A28).The figure is a graph showing the maximum editing efficiency of adenosine base editor variant 1.14. [Figure 23N] 1 is a graph showing the maximum editing efficiency of adenosine base editor variants across nine sites (YY-A1, YY-A2, YY-A6, YY-A7, YY-A15, YY-A16, YY-A17, YY-A27, and YY-A28).The figure is a graph showing the maximum editing efficiency of adenosine base editor variant 1.15. [Figure 23O] 1 is a graph showing the maximum editing efficiency of adenosine base editor variants across nine sites (YY-A1, YY-A2, YY-A6, YY-A7, YY-A15, YY-A16, YY-A17, YY-A27, and YY-A28).The figure is a graph showing the maximum editing efficiency of adenosine base editor variant 1.16. [Figure 23P] 1 is a graph showing the maximum editing efficiency of adenosine base editor variants across nine sites (YY-A1, YY-A2, YY-A6, YY-A7, YY-A15, YY-A16, YY-A17, YY-A27, and YY-A28).The figure is a graph showing the maximum editing efficiency of adenosine base editor variant 1.17. [Figure 23Q] 1 is a graph showing the maximum editing efficiency of adenosine base editor variants across nine sites (YY-A1, YY-A2, YY-A6, YY-A7, YY-A15, YY-A16, YY-A17, YY-A27, and YY-A28).The figure is a graph showing the maximum editing efficiency of adenosine base editor variant 1.18. [Figure 23R] 1 is a graph showing the maximum editing efficiency of adenosine base editor variants across nine sites (YY-A1, YY-A2, YY-A6, YY-A7, YY-A15, YY-A16, YY-A17, YY-A27, and YY-A28).The figure is a graph showing the maximum editing efficiency of adenosine base editor variant 1.19. [Figure 23S] 1 is a graph showing the maximum editing efficiency of adenosine base editor variants across nine sites (YY-A1, YY-A2, YY-A6, YY-A7, YY-A15, YY-A16, YY-A17, YY-A27, and YY-A28).The figure is a graph showing the maximum editing efficiency of adenosine base editor variant 1.20. [Figure 24A] 1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with a UGI domain at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.1 with a UGI domain. [Figure 24B] 1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with a UGI domain at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.2 with a UGI domain. [Figure 24C] 1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with a UGI domain at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.3 with a UGI domain. [Figure 24D] 1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with a UGI domain at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.4 with a UGI domain. [Figure 24E] 1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with UGI domains at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.5 with a UGI domain. [Figure 24F] 1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with a UGI domain at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.6 with a UGI domain. [Figure 24G] 1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with a UGI domain at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of 1.7 adenosine base editor variants with a UGI domain. [Figure 24H]1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with UGI domains at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of 1.8 adenosine base editor variants with UGI domains. [Figure 24I] 1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with UGI domains at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of 1.9 adenosine base editor variants with UGI domains. [Figure 24J] 1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with a UGI domain at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.10 with a UGI domain. [Figure 24K] 1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with a UGI domain at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.11 with a UGI domain. [Figure 24L] 1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with a UGI domain at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.12 with a UGI domain. [Figure 24M] 1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with a UGI domain at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.13 with a UGI domain. [Figure 24N]1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with a UGI domain at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.14 with a UGI domain. [Figure 24O] 1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with UGI domains at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of 1.15 adenosine base editor variants with UGI domains. [Figure 24P] 1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with a UGI domain at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.16 with a UGI domain. [Figure 24Q] 1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with a UGI domain at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.17 with a UGI domain. [Figure 24R] 1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with a UGI domain at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.18 with a UGI domain. [Figure 24S] 1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with UGI domains at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of 1.19 adenosine base editor variants with UGI domains. [Figure 24T]1 is a boxplot graph showing the average editing efficiency (A to G or C to T) of adenosine base editor variants with a UGI domain at each window position across 18 target sites. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.20 with a UGI domain. [Figure 25A] Figure 1 is a box plot graph showing the mean editing efficiency (A to G or C to T) of selected controls. The figure is a graph showing the mean editing efficiency of ABE8.19m. [Figure 25B] Figure 1 is a boxplot graph showing the mean editing efficiency of selected controls (A to G or C to T). The figure is a graph showing the mean editing efficiency of ABE8.19m, which has a UGI domain. [Figure 25C] Figure 1 shows a boxplot graph depicting the mean editing efficiency (A to G or C to T) of selected controls. Figure 1 shows a graph depicting the mean editing efficiency of ABE8.20m. [Figure 25D] Figure 1 is a boxplot graph showing the mean editing efficiency (A to G or C to T) of selected controls. The figure is a graph showing the mean editing efficiency of ABE8.20m, which has a UGI domain. [Figure 25E] Figure 1 is a boxplot graph showing the mean editing efficiency of selected controls (A to G or C to T). The figure is a graph showing the mean editing efficiency of BE4-max. [Figure 25F] Figure 1 is a box plot graph showing the mean editing efficiency of selected controls (A to G or C to T). The figure is a graph showing the mean editing efficiency of BE4. [Figure 25G] Figure 1 is a boxplot graph showing the mean editing efficiency of selected controls (A to G or C to T). The figure is a graph showing the mean editing efficiency of combined ABE / CBE fusion proteins. [Figure 25H] Figure 1 is a boxplot graph showing the mean editing efficiency of selected controls (A to G or C to T).The figure is a graph showing the mean editing efficiency of BE3b without the UGI domain. [Figure 25I]Figure 1 is a boxplot graph showing the mean editing efficiency of selected controls (A to G or C to T). [Figure 26A] A bar graph showing the percentage of all reads exhibiting a C to T or A to G change made by each indicated base editor is shown. In the figure, for each base editor, C to T changes are shown in the left bar, and A to G changes are shown in the right bar. Target sites are shown in bold font in the figure. The sequence in the figure corresponds to the first 12 nucleotides of the nucleotide sequence of SEQ ID NO: 237. Base editors 8.20m+UGI, B93, B88 (rAPOBEC1 BE4), variant 1.17 (Table 1A), and variant 1.2 (Table 1A) were used as controls. [Figure 26B] A bar graph showing the percentage of total reads exhibiting a C to T or A to G change made by each indicated base editor is shown. In the figure, for each base editor, C to T changes are shown in the left bar, and A to G changes are shown in the right bar. Target sites are shown in bold font in the figure. The sequence in the figure corresponds to the first 12 nucleotides of the nucleotide sequence of SEQ ID NO: 239. Base editors 8.20m+UGI, B93, B88 (rAPOBEC1 BE4), variant 1.17 (Table 1A), and variant 1.2 (Table 1A) were used as controls. [Figure 26C] A bar graph showing the percentage of all reads exhibiting a C to T or A to G change made by each indicated base editor is shown. In the figure, for each base editor, C to T changes are shown in the left bar, and A to G changes are shown in the right bar. Target sites are shown in bold font in the figure. The sequence in the figure corresponds to the first 12 nucleotides of the nucleotide sequence of SEQ ID NO: 240. Base editors 8.20m+UGI, B93, B88 (rAPOBEC1 BE4), variant 1.17 (Table 1A), and variant 1.2 (Table 1A) were used as controls. [Figure 26D]A bar graph showing the percentage of all reads exhibiting a C to T or A to G change made by each indicated base editor is shown. In the figure, for each base editor, C to T changes are shown in the left bar, and A to G changes are shown in the right bar. Target sites are shown in bold font in the figure. The sequence in the figure corresponds to the first 12 nucleotides of the nucleotide sequence of SEQ ID NO: 241. Base editors 8.20m+UGI, B93, B88 (rAPOBEC1 BE4), variant 1.17 (Table 1A), and variant 1.2 (Table 1A) were used as controls. [Figure 26E] A bar graph showing the percentage of all reads exhibiting a C to T or A to G change made by each indicated base editor is shown. In the figure, for each base editor, C to T changes are shown in the left bar, and A to G changes are shown in the right bar. Target sites are shown in bold font in the figure. The sequence in the figure corresponds to the first 12 nucleotides of the nucleotide sequence of SEQ ID NO: 386. Base editors 8.20m+UGI, B93, B88 (rAPOBEC1 BE4), variant 1.17 (Table 1A), and variant 1.2 (Table 1A) were used as controls. [Figure 26F] A bar graph showing the percentage of all reads exhibiting a C to T or A to G change made by each indicated base editor is shown. In the figure, for each base editor, C to T changes are shown in the left bar, and A to G changes are shown in the right bar. Target sites are shown in bold font in the figure. The sequence in the figure corresponds to the first 12 nucleotides of the nucleotide sequence of SEQ ID NO: 244. Base editors 8.20m+UGI, B93, B88 (rAPOBEC1 BE4), variant 1.17 (Table 1A), and variant 1.2 (Table 1A) were used as controls. [Figure 27A]A bar graph showing the percentage of all reads that exhibit a C to T or A to G change made by each of the indicated base editors is shown. In the figure, for each base editor, the C to T change is shown in the left bar, and the A to G change is shown in the right bar. The target site is shown in bold font in the figure. The sequence in the figure corresponds to the first 12 nucleotides of the nucleotide sequence of SEQ ID NO: 237. Base editors B88 (rAPOBEC1 BE4), ABE8.20, B93, variant 1.2 (Table 1A), and variant 1.17 (Table 1A) were used as controls. [Figure 27B] A bar graph showing the percentage of all reads exhibiting a C to T or A to G change made by each of the indicated base editors is shown. In the figure, for each base editor, the C to T change is shown in the left bar, and the A to G change is shown in the right bar. The target site is shown in bold font in the figure. The sequence in the figure corresponds to the first 12 nucleotides of the nucleotide sequence of SEQ ID NO: 239. Base editors B88 (rAPOBEC1 BE4), ABE8.20, B93, variant 1.2 (Table 1A), and variant 1.17 (Table 1A) were used as controls. [Figure 27C] A bar graph showing the percentage of all reads that exhibit a C to T or A to G change made by each of the indicated base editors is shown. In the figure, for each base editor, the C to T change is shown in the left bar, and the A to G change is shown in the right bar. The target site is shown in bold font in the figure. The sequence in the figure corresponds to the first 12 nucleotides of the nucleotide sequence of SEQ ID NO: 240. Base editors B88 (rAPOBEC1 BE4), ABE8.20, B93, variant 1.2 (Table 1A), and variant 1.17 (Table 1A) were used as controls. [Figure 27D]A bar graph showing the percentage of all reads exhibiting a C to T or A to G change made by each of the indicated base editors is shown. In the figure, for each base editor, the C to T change is shown in the left bar, and the A to G change is shown in the right bar. The target site is shown in bold font in the figure. The sequence in the figure corresponds to the first 12 nucleotides of the nucleotide sequence of SEQ ID NO: 241. Base editors B88 (rAPOBEC1 BE4), ABE8.20, B93, variant 1.2 (Table 1A), and variant 1.17 (Table 1A) were used as controls. [Figure 27E] A bar graph showing the percentage of all reads that exhibit a C to T or A to G change made by each of the indicated base editors is shown. In the figure, for each base editor, the C to T change is shown in the left bar, and the A to G change is shown in the right bar. The target site is shown in bold font in the figure. The sequence in the figure corresponds to the first 12 nucleotides of the nucleotide sequence of SEQ ID NO: 386. Base editors B88 (rAPOBEC1 BE4), ABE8.20, B93, variant 1.2 (Table 1A), and variant 1.17 (Table 1A) were used as controls. [Figure 27F] A bar graph showing the percentage of all reads exhibiting a C to T or A to G change made by each of the indicated base editors is shown. In the figure, for each base editor, the C to T change is shown in the left bar, and the A to G change is shown in the right bar. The target site is shown in bold font in the figure. The sequence in the figure corresponds to the first 12 nucleotides of the nucleotide sequence of SEQ ID NO: 244. Base editors B88 (rAPOBEC1 BE4), ABE8.20, B93, variant 1.2 (Table 1A), and variant 1.17 (Table 1A) were used as controls. [Figure 28A]A bar graph showing the percentage of all reads showing a C to T or A to G change made by each of the indicated base editors is shown. For each base editor, the C to T change is shown in the left bar, and the A to G change is shown in the right bar. In the figure, the target site is indicated above the bar. Base editors B93, B88 (rAPOBEC1 BE4), 8.20m+UGI, variant 1.17 (Table 1A), and variant 1.2 (Table 1A) were used as controls. [Figure 28B] A bar graph showing the percentage of all reads showing a C to T or A to G change made by each of the indicated base editors is shown. For each base editor, the C to T change is shown in the left bar, and the A to G change is shown in the right bar. In the figure, the target site is indicated above the bar. Base editors B93, B88 (rAPOBEC1 BE4), 8.20m+UGI, variant 1.17 (Table 1A), and variant 1.2 (Table 1A) were used as controls. [Figure 28C] A bar graph showing the percentage of all reads showing a C to T or A to G change made by each of the indicated base editors is shown. For each base editor, the C to T change is shown in the left bar, and the A to G change is shown in the right bar. In the figure, the target site is indicated above the bar. Base editors B93, B88 (rAPOBEC1 BE4), 8.20m+UGI, variant 1.17 (Table 1A), and variant 1.2 (Table 1A) were used as controls. [Figure 28D] A bar graph showing the percentage of all reads showing a C to T or A to G change made by each of the indicated base editors is shown. For each base editor, the C to T change is shown in the left bar, and the A to G change is shown in the right bar. In the figure, the target site is indicated above the bar. Base editors B93, B88 (rAPOBEC1 BE4), 8.20m+UGI, variant 1.17 (Table 1A), and variant 1.2 (Table 1A) were used as controls. [Figure 28E]A bar graph showing the percentage of all reads showing a C to T or A to G change made by each of the indicated base editors is shown. For each base editor, the C to T change is shown in the left bar, and the A to G change is shown in the right bar. In the figure, the target site is indicated above the bar. Base editors B93, B88 (rAPOBEC1 BE4), 8.20m+UGI, variant 1.17 (Table 1A), and variant 1.2 (Table 1A) were used as controls. [Figure 28F] A bar graph showing the percentage of all reads showing a C to T or A to G change made by each of the indicated base editors is shown. For each base editor, the C to T change is shown in the left bar, and the A to G change is shown in the right bar. In the figure, the target site is indicated above the bar. Base editors B93, B88 (rAPOBEC1 BE4), 8.20m+UGI, variant 1.17 (Table 1A), and variant 1.2 (Table 1A) were used as controls. [Figure 29A] A bar graph showing the percentage of all reads exhibiting a C to T or A to G change made by each of the indicated base editors is shown. For each base editor, the C to T change is shown in the left bar, and the A to G change is shown in the right bar. The target site is indicated above the bar. Base editors B93, B88 (rAPOBEC1 BE4), 8.20m+UGI, variant 1.17 (Table 1A), and variant 1.2 (Table 1A) were used as controls. [Figure 29B] A bar graph showing the percentage of all reads exhibiting a C to T or A to G change made by each of the indicated base editors is shown. For each base editor, the C to T change is shown in the left bar, and the A to G change is shown in the right bar. The target site is indicated above the bar. Base editors B93, B88 (rAPOBEC1 BE4), 8.20m+UGI, variant 1.17 (Table 1A), and variant 1.2 (Table 1A) were used as controls. [Figure 29C]A bar graph showing the percentage of all reads exhibiting a C to T or A to G change made by each of the indicated base editors is shown. For each base editor, the C to T change is shown in the left bar, and the A to G change is shown in the right bar. The target site is indicated above the bar. Base editors B93, B88 (rAPOBEC1 BE4), 8.20m+UGI, variant 1.17 (Table 1A), and variant 1.2 (Table 1A) were used as controls. [Figure 29D] A bar graph showing the percentage of all reads exhibiting a C to T or A to G change made by each of the indicated base editors is shown. For each base editor, the C to T change is shown in the left bar, and the A to G change is shown in the right bar. The target site is indicated above the bar. Base editors B93, B88 (rAPOBEC1 BE4), 8.20m+UGI, variant 1.17 (Table 1A), and variant 1.2 (Table 1A) were used as controls. [Figure 29E] A bar graph showing the percentage of all reads exhibiting a C to T or A to G change made by each of the indicated base editors is shown. For each base editor, the C to T change is shown in the left bar, and the A to G change is shown in the right bar. The target site is indicated above the bar. Base editors B93, B88 (rAPOBEC1 BE4), 8.20m+UGI, variant 1.17 (Table 1A), and variant 1.2 (Table 1A) were used as controls. [Figure 29F] A bar graph showing the percentage of all reads exhibiting a C to T or A to G change made by each of the indicated base editors is shown. For each base editor, the C to T change is shown in the left bar, and the A to G change is shown in the right bar. The target site is indicated above the bar. Base editors B93, B88 (rAPOBEC1 BE4), 8.20m+UGI, variant 1.17 (Table 1A), and variant 1.2 (Table 1A) were used as controls. [Figure 30]

[0023] Figure 2 shows a heat map depicting A to G and C to T base editing activity for the adenosine deaminase variants listed in Table 25. In the figure, base editors are clustered based on their measured A to G and C to T activity. In the figure, darker shading indicates increased activity. [Figure 31] 31 shows a heatmap depicting A to G and C to T base editing activity for the adenosine deaminase variants listed in Table 25. In Figure 31, base editors are clustered based on measured A to G and C to T activity. In Figure 31, darker shading indicates increased activity. [Figure 32A] 2 shows bar graphs depicting C to T, A to G, and C to G editing activity for the indicated base editors (see Table 26 for a description of base editors) at the following target sites: YY-A2, YY-A6, YY-A7, YY-A12, YY-A17, and ACK119. In the figure, the bar immediately to the left of each hatch mark represents C to T activity, the bar immediately above each hatch mark represents A to G activity, and the bar immediately to the right of each hatch mark represents C to G activity. Controls used to generate the bar graphs included ABE / CBE fusion proteins. [Figure 32B] 2 shows bar graphs depicting C to T, A to G, and C to G editing activity for the indicated base editors (see Table 26 for a description of base editors) at the following target sites: YY-A2, YY-A6, YY-A7, YY-A12, YY-A17, and ACK119. In the figure, the bar immediately to the left of each hatch mark represents C to T activity, the bar immediately above each hatch mark represents A to G activity, and the bar immediately to the right of each hatch mark represents C to G activity. Controls used to generate the bar graphs included ABE / CBE fusion proteins. [Figure 32C]2 shows bar graphs depicting C to T, A to G, and C to G editing activity for the indicated base editors (see Table 26 for a description of base editors) at the following target sites: YY-A2, YY-A6, YY-A7, YY-A12, YY-A17, and ACK119. In the figure, the bar immediately to the left of each hatch mark represents C to T activity, the bar immediately above each hatch mark represents A to G activity, and the bar immediately to the right of each hatch mark represents C to G activity. Controls used to generate the bar graphs included ABE / CBE fusion proteins. [Figure 32D] 2 shows bar graphs depicting C to T, A to G, and C to G editing activity for the indicated base editors (see Table 26 for a description of base editors) at the following target sites: YY-A2, YY-A6, YY-A7, YY-A12, YY-A17, and ACK119. In the figure, the bar immediately to the left of each hatch mark represents C to T activity, the bar immediately above each hatch mark represents A to G activity, and the bar immediately to the right of each hatch mark represents C to G activity. Controls used to generate the bar graphs included ABE / CBE fusion proteins. [Figure 32E] 2 shows bar graphs depicting C to T, A to G, and C to G editing activity for the indicated base editors (see Table 26 for a description of base editors) at the following target sites: YY-A2, YY-A6, YY-A7, YY-A12, YY-A17, and ACK119. In the figure, the bar immediately to the left of each hatch mark represents C to T activity, the bar immediately above each hatch mark represents A to G activity, and the bar immediately to the right of each hatch mark represents C to G activity. Controls used to generate the bar graphs included ABE / CBE fusion proteins. [Figure 32F]2 shows bar graphs depicting C to T, A to G, and C to G editing activity for the indicated base editors (see Table 26 for a description of base editors) at the following target sites: YY-A2, YY-A6, YY-A7, YY-A12, YY-A17, and ACK119. In the figure, the bar immediately to the left of each hatch mark represents C to T activity, the bar immediately above each hatch mark represents A to G activity, and the bar immediately to the right of each hatch mark represents C to G activity. Controls used to generate the bar graphs included ABE / CBE fusion proteins. [Figure 33A]

[0023] Figure 2 shows a heatmap depicting the frequency of indel formation associated with the indicated base editors (see Table 26 for a description of base editors) at the following target sites: YY-A2, YY-A6, YY-A7, YY-A12, YY-A17, and ACK119. Darker shades of gray in the figure indicate higher relative frequencies of indel formation. Controls used to generate the heatmap included the ABE / CBE fusion protein. [Figure 33B]

[0023] Figure 2 shows a heatmap depicting the frequency of indel formation associated with the indicated base editors (see Table 26 for a description of base editors) at the following target sites: YY-A2, YY-A6, YY-A7, YY-A12, YY-A17, and ACK119. Darker shades of gray in the figure indicate higher relative frequencies of indel formation. Controls used to generate the heatmap included the ABE / CBE fusion protein. [Figure 33C]

[0023] Figure 2 shows a heatmap depicting the frequency of indel formation associated with the indicated base editors (see Table 26 for a description of base editors) at the following target sites: YY-A2, YY-A6, YY-A7, YY-A12, YY-A17, and ACK119. Darker shades of gray in the figure indicate higher relative frequencies of indel formation. Controls used to generate the heatmap included the ABE / CBE fusion protein. [Figure 33D]

[0023] Figure 2 shows a heatmap depicting the frequency of indel formation associated with the indicated base editors (see Table 26 for a description of base editors) at the following target sites: YY-A2, YY-A6, YY-A7, YY-A12, YY-A17, and ACK119. Darker shades of gray in the figure indicate higher relative frequencies of indel formation. Controls used to generate the heatmap included the ABE / CBE fusion protein. [Figure 33E]

[0023] Figure 2 shows a heatmap depicting the frequency of indel formation associated with the indicated base editors (see Table 26 for a description of base editors) at the following target sites: YY-A2, YY-A6, YY-A7, YY-A12, YY-A17, and ACK119. Darker shades of gray in the figure indicate higher relative frequencies of indel formation. Controls used to generate the heatmap included the ABE / CBE fusion protein. [Figure 33F]

[0023] Figure 2 shows a heatmap depicting the frequency of indel formation associated with the indicated base editors (see Table 26 for a description of base editors) at the following target sites: YY-A2, YY-A6, YY-A7, YY-A12, YY-A17, and ACK119. Darker shades of gray in the figure indicate higher relative frequencies of indel formation. Controls used to generate the heatmap included the ABE / CBE fusion protein. [Figure 34A] 1 shows a bar graph depicting C to T, A to G, and C to G editing activity for the indicated base editors (see Table 26 for a description of base editors) at the following target sites: YY-A2 and Hek site 3. In the figure, the bar immediately to the left of each hatch mark represents C to T activity, the bar immediately above each hatch mark represents A to G activity, and the bar immediately to the right of each hatch mark represents C to G activity. In the figure, unlabeled bars correspond to combined ABE / CBE fusion proteins. [Figure 34B]1 shows a bar graph depicting C to T, A to G, and C to G editing activity for the indicated base editors (see Table 26 for a description of base editors) at the following target sites: YY-A2 and Hek site 3. In the figure, the bar immediately to the left of each hatch mark represents C to T activity, the bar immediately above each hatch mark represents A to G activity, and the bar immediately to the right of each hatch mark represents C to G activity. In the figure, unlabeled bars correspond to combined ABE / CBE fusion proteins. [Figure 35A]

[0033] Figure 2 shows a heatmap depicting the frequency of indel formation associated with the indicated base editors (see Table 26 for a description of base editors) at the following target sites: HEK site 3 and YY-A2. In the figure, darker shades of gray indicate higher relative frequencies of indel formation. In the figure, unlabeled cells correspond to the combined ABE / CBE fusion proteins. [Figure 35B]

[0033] Figure 2 shows a heatmap depicting the frequency of indel formation associated with the indicated base editors (see Table 26 for a description of base editors) at the following target sites: HEK site 3 and YY-A2. In the figure, darker shades of gray indicate higher relative frequencies of indel formation. In the figure, unlabeled cells correspond to the combined ABE / CBE fusion proteins. [Figure 36A] Figures 36A-36S show plots showing the average editing efficiency (A to G or C to T) of the adenosine base editor variants in Figures 24A-24T. Figures 36A-36S each show the same data as the corresponding data in Figures 24A-24T, but in a different format. [Figure 36B] Figures 36A-36S show plots showing the average editing efficiency (A to G or C to T) of the adenosine base editor variants in Figures 24A-24T. Figures 36A-36S each show the same data as the corresponding data in Figures 24A-24T, but in a different format. [Figure 36C]Figures 36A-36S show plots showing the average editing efficiency (A to G or C to T) of the adenosine base editor variants in Figures 24A-24T. Figures 36A-36S each show the same data as the corresponding data in Figures 24A-24T, but in a different format. [Figure 36D] Figures 36A-36S show plots showing the average editing efficiency (A to G or C to T) of the adenosine base editor variants in Figures 24A-24T. Figures 36A-36S each show the same data as the corresponding data in Figures 24A-24T, but in a different format. [Figure 36E] Figures 36A-36S show plots showing the average editing efficiency (A to G or C to T) of the adenosine base editor variants in Figures 24A-24T. Figures 36A-36S each show the same data as the corresponding data in Figures 24A-24T, but in a different format. [Figure 36F] Figures 36A-36S show plots showing the average editing efficiency (A to G or C to T) of the adenosine base editor variants in Figures 24A-24T. Figures 36A-36S each show the same data as the corresponding data in Figures 24A-24T, but in a different format. [Figure 36G] Figures 36A-36S show plots showing the average editing efficiency (A to G or C to T) of the adenosine base editor variants in Figures 24A-24T. Figures 36A-36S each show the same data as the corresponding data in Figures 24A-24T, but in a different format. [Figure 36H] Figures 36A-36S show plots showing the average editing efficiency (A to G or C to T) of the adenosine base editor variants in Figures 24A-24T. Figures 36A-36S each show the same data as the corresponding data in Figures 24A-24T, but in a different format. [Figure 36I] Figures 36A-36S show plots showing the average editing efficiency (A to G or C to T) of the adenosine base editor variants in Figures 24A-24T. Figures 36A-36S each show the same data as the corresponding data in Figures 24A-24T, but in a different format. [Figure 36J]Figures 36A-36S show plots showing the average editing efficiency (A to G or C to T) of the adenosine base editor variants in Figures 24A-24T. Figures 36A-36S each show the same data as the corresponding data in Figures 24A-24T, but in a different format. [Figure 36K] Figures 36A-36S show plots showing the average editing efficiency (A to G or C to T) of the adenosine base editor variants in Figures 24A-24T. Figures 36A-36S each show the same data as the corresponding data in Figures 24A-24T, but in a different format. [Figure 36L] Figures 36A-36S show plots showing the average editing efficiency (A to G or C to T) of the adenosine base editor variants in Figures 24A-24T. Figures 36A-36S each show the same data as the corresponding data in Figures 24A-24T, but in a different format. [Figure 36M] Figures 36A-36S show plots showing the average editing efficiency (A to G or C to T) of the adenosine base editor variants in Figures 24A-24T. Figures 36A-36S each show the same data as the corresponding data in Figures 24A-24T, but in a different format. [Figure 36N] Figures 36A-36S show plots showing the average editing efficiency (A to G or C to T) of the adenosine base editor variants in Figures 24A-24T. Figures 36A-36S each show the same data as the corresponding data in Figures 24A-24T, but in a different format. [Figure 36O] Figures 36A-36S show plots showing the average editing efficiency (A to G or C to T) of the adenosine base editor variants in Figures 24A-24T. Figures 36A-36S each show the same data as the corresponding data in Figures 24A-24T, but in a different format. [Figure 36P] Figures 36A-36S show plots showing the average editing efficiency (A to G or C to T) of the adenosine base editor variants in Figures 24A-24T. Figures 36A-36S each show the same data as the corresponding data in Figures 24A-24T, but in a different format. [Figure 36Q]Figures 36A-36S show plots showing the average editing efficiency (A to G or C to T) of the adenosine base editor variants in Figures 24A-24T. Figures 36A-36S each show the same data as the corresponding data in Figures 24A-24T, but in a different format. [Figure 36R] Figures 36A-36S show plots showing the average editing efficiency (A to G or C to T) of the adenosine base editor variants in Figures 24A-24T. Figures 36A-36S each show the same data as the corresponding data in Figures 24A-24T, but in a different format. [Figure 36S] Figures 36A-36S show plots showing the average editing efficiency (A to G or C to T) of the adenosine base editor variants in Figures 24A-24T. Figures 36A-36S each show the same data as the corresponding data in Figures 24A-24T, but in a different format. [Figure 37A] Figures 37A-37E show plots showing the mean editing efficiency (A to G or C to T) of selected controls from Figures 25A-25I. Figures 37A-37E each show similar or identical data to the data shown in the corresponding Figures 25A-25I, but in a different format. [Figure 37B] Figures 37A-37E show plots showing the mean editing efficiency (A to G or C to T) of selected controls from Figures 25A-25I. Figures 37A-37E each show similar or identical data to the data shown in the corresponding Figures 25A-25I, but in a different format. [Figure 37C] Figures 37A-37E show plots showing the mean editing efficiency (A to G or C to T) of selected controls from Figures 25A-25I. Figures 37A-37E each show similar or identical data to the data shown in the corresponding Figures 25A-25I, but in a different format. [Figure 37D] Figures 37A-37E show plots showing the mean editing efficiency (A to G or C to T) of selected controls from Figures 25A-25I. Figures 37A-37E each show similar or identical data to the data shown in the corresponding Figures 25A-25I, but in a different format. [Figure 37E]Figures 37A-37E show plots showing the mean editing efficiency (A to G or C to T) of selected controls from Figures 25A-25I. Figures 37A-37E each show similar or identical data to the data shown in the corresponding Figures 25A-25I, but in a different format. [Figure 38]

[0023] Figure 1 shows a heatmap depicting the maximum percent AT to GC editing for the indicated base editor variants (see Table 1E for variant descriptions) at the indicated target sites (see Table 24 for target site descriptions). Base editor variants 879 and 882 were associated with increased on-target activity and minimal guide-independent off-target activity. [Figure 39A]

[0033] Figure 2 shows a plot showing the average editing efficiency (A to G or C to T) of adenosine base editor variants (see Tables 1A and 1F) at each window position across six target sites (see 299, ACK119, ACK115, ACK121, B415, and sA12 in Table 24), or, as indicated, across all 18 target sites listed in Table 24. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.12. [Figure 39B]

[0033] Figure 2 shows a plot showing the average editing efficiency (A to G or C to T) of adenosine base editor variants (see Tables 1A and 1F) at each window position across six target sites (see 299, ACK119, ACK115, ACK121, B415, and sA12 in Table 24), or, as indicated, across all 18 target sites listed in Table 24. The figure is a graph showing the average editing efficiency of the adenosine base editor variant 1.12+8e (B869). [Figure 39C]

[0033] Figure 2 shows a plot showing the average editing efficiency (A to G or C to T) of adenosine base editor variants (see Tables 1A and 1F) at each window position across six target sites (see 299, ACK119, ACK115, ACK121, B415, and sA12 in Table 24), or, as indicated, across all 18 target sites listed in Table 24. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.12+8e (B882). [Figure 39D]

[0033] Figure 2 shows a plot showing the average editing efficiency (A to G or C to T) of adenosine base editor variants (see Tables 1A and 1F) at each window position across six target sites (see 299, ACK119, ACK115, ACK121, B415, and sA12 in Table 24), or, as indicated, across all 18 target sites listed in Table 24. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.17. [Figure 39E]

[0033] Figure 2 shows a plot showing the average editing efficiency (A to G or C to T) of adenosine base editor variants (see Tables 1A and 1F) at each window position across six target sites (see 299, ACK119, ACK115, ACK121, B415, and sA12 in Table 24), or, as indicated, across all 18 target sites listed in Table 24. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.17+8e (B869). [Figure 39F]

[0033] Figure 2 shows a plot showing the average editing efficiency (A to G or C to T) of adenosine base editor variants (see Tables 1A and 1F) at each window position across six target sites (see 299, ACK119, ACK115, ACK121, B415, and sA12 in Table 24), or, as indicated, across all 18 target sites listed in Table 24. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.17+8e (B882). [Figure 39G]

[0033] Figure 2 shows a plot showing the average editing efficiency (A to G or C to T) of adenosine base editor variants (see Tables 1A and 1F) at each window position across six target sites (see 299, ACK119, ACK115, ACK121, B415, and sA12 in Table 24), or, as indicated, across all 18 target sites listed in Table 24. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.18. [Figure 39H]

[0033] Figure 2 shows a plot showing the average editing efficiency (A to G or C to T) of adenosine base editor variants (see Tables 1A and 1F) at each window position across six target sites (see 299, ACK119, ACK115, ACK121, B415, and sA12 in Table 24), or, as indicated, across all 18 target sites listed in Table 24. The figure is a graph showing the average editing efficiency of the adenosine base editor variant 1.18+8e (B869). [Figure 39I]

[0033] Figure 2 shows a plot showing the average editing efficiency (A to G or C to T) of adenosine base editor variants (see Tables 1A and 1F) at each window position across six target sites (see 299, ACK119, ACK115, ACK121, B415, and sA12 in Table 24), or, as indicated, across all 18 target sites listed in Table 24. The figure is a graph showing the average editing efficiency of the adenosine base editor variant 1.18+8e (B882). [Figure 39J]

[0033] Figure 2 shows a plot showing the average editing efficiency (A to G or C to T) of adenosine base editor variants (see Tables 1A and 1F) at each window position across six target sites (see 299, ACK119, ACK115, ACK121, B415, and sA12 in Table 24), or, as indicated, across all 18 target sites listed in Table 24. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.19. [Figure 39K]

[0033] Figure 2 shows a plot showing the average editing efficiency (A to G or C to T) of adenosine base editor variants (see Tables 1A and 1F) at each window position across six target sites (see 299, ACK119, ACK115, ACK121, B415, and sA12 in Table 24), or, as indicated, across all 18 target sites listed in Table 24. The figure is a graph showing the average editing efficiency of the adenosine base editor variant 1.19+8e (B869). [Figure 39L]

[0033] Figure 2 shows a plot showing the average editing efficiency (A to G or C to T) of adenosine base editor variants (see Tables 1A and 1F) at each window position across six target sites (see 299, ACK119, ACK115, ACK121, B415, and sA12 in Table 24), or, as indicated, across all 18 target sites listed in Table 24. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.19+8e (B882). [Figure 39M]

[0033] Figure 2 shows a plot showing the average editing efficiency (A to G or C to T) of adenosine base editor variants (see Tables 1A and 1F) at each window position across six target sites (see 299, ACK119, ACK115, ACK121, B415, and sA12 in Table 24), or, as indicated, across all 18 target sites listed in Table 24. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.1. [Figure 39N]

[0033] Figure 2 shows a plot showing the average editing efficiency (A to G or C to T) of adenosine base editor variants (see Tables 1A and 1F) at each window position across six target sites (see 299, ACK119, ACK115, ACK121, B415, and sA12 in Table 24), or, as indicated, across all 18 target sites listed in Table 24. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.1+8e (B869). [Figure 39O]

[0033] Figure 2 shows a plot showing the average editing efficiency (A to G or C to T) of adenosine base editor variants (see Tables 1A and 1F) at each window position across six target sites (see 299, ACK119, ACK115, ACK121, B415, and sA12 in Table 24), or, as indicated, across all 18 target sites listed in Table 24. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.1+8e (B882). [Figure 39P]

[0033] Figure 2 shows a plot showing the average editing efficiency (A to G or C to T) of adenosine base editor variants (see Tables 1A and 1F) at each window position across six target sites (see 299, ACK119, ACK115, ACK121, B415, and sA12 in Table 24), or, as indicated, across all 18 target sites listed in Table 24. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.2. [Figure 39Q]

[0033] Figure 2 shows a plot showing the average editing efficiency (A to G or C to T) of adenosine base editor variants (see Tables 1A and 1F) at each window position across six target sites (see 299, ACK119, ACK115, ACK121, B415, and sA12 in Table 24), or, as indicated, across all 18 target sites listed in Table 24. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.2+8e (B869). [Figure 39R]

[0033] Figure 2 shows a plot showing the average editing efficiency (A to G or C to T) of adenosine base editor variants (see Tables 1A and 1F) at each window position across six target sites (see 299, ACK119, ACK115, ACK121, B415, and sA12 in Table 24), or, as indicated, across all 18 target sites listed in Table 24. The figure is a graph showing the average editing efficiency of adenosine base editor variant 1.2+8e (B882). [Figure 40]FIG. 1 shows bar graphs depicting C to T, A to G, and C to G editing activity for the indicated base editors (see Table 1D for a description of base editors) at the following target site: YY-A2. In the figure, the bar immediately to the left of each hatch mark represents C to T activity, the bar immediately above each hatch mark represents A to G activity, and the bar immediately to the right of each hatch mark represents C to G activity. Controls used to generate the bar graphs included an ABE / CBE fusion protein. Candidate editor S2.20 failed to demonstrate high levels of C→T-specific activity at the evaluated target site and is therefore omitted from the figure. [Figure 41]

[0023] Figure 1 shows a schematic outlining experiments performed to develop TadA* polypeptides with increased cytosine deaminase activity. In the figure, TADAC stands for "TadA* acting on DNA adenine and cytosine," TADC stands for "TadA* acting on DNA cytosine," and TadA* stands for "tRNA-acting adenosine deaminase A variant." In embodiments, TADAC and TADC are engineered variants of TadA that, when ligated to Cas9, can generate both A·T to G·C and C·G to T·A or C·G to T·A mutations in DNA. [Figure 42] A schematic diagram including a ribbon diagram of the protein structure showing the relationships between the different TadA* polypeptides is shown (see Table 1A). [Figure 43A] Ribbon diagrams of protein structures and chemical structures are shown. The figure shows a ribbon diagram of the crystal structure of TadA*8.20. The crystal structure was prepared using the sequence 5'-GCTCGGCT / d8AZ / CGGA-3' (SEQ ID NO: 411), shown in Figures 43A and 43B. The chemical structure in Figures 43A and 43B shows how 2'-deoxy-8-azambularine (d8AZ) was used to capture the transition state analog associated with adenosine deamination. Without being bound by theory, the structure of TadA*8.20 provides structural insight into the basis of protein binding to single-stranded DNA (ssDNA), demonstrating that mutations introduced during directed evolution altered the C-terminal protein structure, favoring ssDNA binding over tRNA. [Figure 43B] Ribbon diagrams of protein structures, chemical structures shown. The figure shows ribbon diagrams of the crystal structures of variants 1.17, 1.14, and 1.19 in Table 1A. [Figure 43C] 1 shows a bar graph depicting C→T and A→G activity measured at target site YY-A2 for ABE8.20 and variants 1.14, 1.17, and 1.19. In the figure, the bar to the left of each hash mark represents C→T activity, and the bar to the right of each hash mark represents A→G activity. [Figure 44] Figure 1 shows a ribbon diagram showing the structural superposition of variant 1.17 and TadA*8.20 from Table 1A. Without being bound by theory, the S82T and A142E substitutions may be related to the small amount of C to T editing in TadA variant 1.17. [Figure 45] Figure 1 shows a ribbon diagram showing the superposition of variants 1.14 and TadA*8.20 from Table 1A. Without being bound by theory, the G112H substitution in the 1.14 variant induced a structural disorder (dashed line) and conformational change in loop-5. [Figure 46]

[0033] Figure 1 shows a ribbon diagram showing that the amino acid substitutions corresponding to variant 1.14 in Table 1A affected ssDNA binding. Without being bound by theory, substitutions after loop-5 in the protein structure affected ssDNA binding. [Figure 47] 1 shows a ribbon diagram showing the superposition of the crystal structures of variants 1.19 and TadA*8.20 from Table 1A. [Figure 48] 1 shows a ribbon diagram showing the superposition of the crystal structures of variant 1.19 and TadA*8.20 in Table 1A. Without being bound by theory, the amino acid substitution corresponding to variant 1.19 in Table 1A induced structural conformational changes (loop-1 and α-1) and unfolding of the C-terminal α-helix. [Figure 49]

[0039] Figure 1 shows the crystal structure of variant 1.19 from Table 1A, and a ribbon diagram showing how the amino acid substitutions affect ssDNA binding. Without being bound by theory, subsequent substitutions in the protein structure (loop-1, α-1, and C-terminus) affected ssDNA binding. [Figure 50] 1 shows a ribbon diagram showing the superposition between the crystal structures of variants 1.14, 1.17, and 1.19 in Table 1A. Amino acid substitutions at positions 27, 49, 82, 112, and / or 142 affected DNA binding. [Figure 51A] A ribbon diagram of TadA*8.20 is shown, with the modified sites indicated by spheres. The figure shows the ribbon diagram of the 10 sites selected for combinatorial screening. The sites corresponding to variants 1.19, 1.17, and 1.14 in Table 1A are circled, corresponding to three regions. The sites corresponding to the modifications are indicated by spheres. Mutation(s) from any one region were sufficient to increase the specificity of cytosine deamination. Directed evolution did not identify any variants containing mutations in all three regions, and very few variants contained mutations in more than one region. A combinatorial library of 199 variants was created, each containing at least one mutation in each of the three regions: one to two altered sites in region A, one to two altered sites in region B, and one to four altered sites in region C. [Figure 51B] A ribbon diagram of TadA*8.20 is shown, with modifications to the TadA*8.20 polypeptide corresponding to variant S1.154 in Table 1B shown as dark gray circles, and the locations of the eight modification sites identified in the second round of directed evolution screening shown as light gray circles (see Table 1C). [Figure 52] Stacked bar plots showing the maximum percentage of C→T, A→G, and C→G editing observed at target site YY-A2 for variants S2.14, S2.46, and S2.52 in Table 1D are shown. [Figure 53]2 shows a heatmap showing the rate of indel formation for variants S2.14, S2.46, and S2.52 from Table 1D, as well as BE4, ABE8 control, nCas9, and negative control at target sites YY-A2, YY-A17, and HEK site 3 (see Table 24). [Figure 54A]

[0033] Figure 2 shows a boxplot graph depicting the mean editing efficiency (A→G or C→T) of adenosine base editor variants S2.14, S2.46, and S2.52, BE4, and the ABE8 control from Table 1D evaluated at target sites YY-A2, YY-A6, YY-A7, YY-A12, YY-A17, and Hek site 3 (see Table 24). The base editing window sizes for the variants listed in Table 1D ranged from about 4 bases to about 8 bases. [Figure 54B]

[0033] Figure 2 shows a boxplot graph depicting the mean editing efficiency (A→G or C→T) of adenosine base editor variants S2.14, S2.46, and S2.52, BE4, and the ABE8 control from Table 1D evaluated at target sites YY-A2, YY-A6, YY-A7, YY-A12, YY-A17, and Hek site 3 (see Table 24). The base editing window sizes for the variants listed in Table 1D ranged from about 4 bases to about 8 bases. [Figure 54C]

[0033] Figure 2 shows a boxplot graph depicting the mean editing efficiency (A→G or C→T) of adenosine base editor variants S2.14, S2.46, and S2.52, BE4, and the ABE8 control from Table 1D evaluated at target sites YY-A2, YY-A6, YY-A7, YY-A12, YY-A17, and Hek site 3 (see Table 24). The base editing window sizes for the variants listed in Table 1D ranged from about 4 bases to about 8 bases. [Figure 54D]

[0033] Figure 2 shows a boxplot graph depicting the mean editing efficiency (A→G or C→T) of adenosine base editor variants S2.14, S2.46, and S2.52, BE4, and the ABE8 control from Table 1D evaluated at target sites YY-A2, YY-A6, YY-A7, YY-A12, YY-A17, and Hek site 3 (see Table 24). The base editing window sizes for the variants listed in Table 1D ranged from about 4 bases to about 8 bases. [Figure 54E]

[0033] Figure 2 shows a boxplot graph depicting the mean editing efficiency (A→G or C→T) of adenosine base editor variants S2.14, S2.46, and S2.52, BE4, and the ABE8 control from Table 1D evaluated at target sites YY-A2, YY-A6, YY-A7, YY-A12, YY-A17, and Hek site 3 (see Table 24). The base editing window sizes for the variants listed in Table 1D ranged from about 4 bases to about 8 bases. [Figure 55] Figure 2 shows a stacked bar plot showing the allele distribution (i.e., distribution of specific bases edited) of base editor variants S2.14, S2.46, and S2.52 from Table 1D, BE4, the ABE8 control, nCas9, and the negative control at target site YY-A2 (see Table 24). The variants in Table 1D had a tighter allele distribution compared to BE4, consistent with the observation of variants with tighter editing windows (i.e., approximately 4 to approximately 8 bases). The S2.14, S2.46, and S2.52 variants showed high specificity for editing at position 7, while BE4 had a much broader editing window, containing many alleles with C edited at position 12. [Figure 56A] Figure 2 shows a heatmap depicting the results of an R-loop assay demonstrating that variants S2.14, S2.46, and S1.52 in Table 1D exhibited reduced A→G guide-independent off-target activity at the indicated target sites (see A2, A6, A15, A16, A17, and A27 in Table 24) compared to BE4, the ABE8 control, and nCas9. The S2.14, S2.46, and S1.52 variants exhibited very low to no A→G editing activity in cis or trans, low to no C→T editing activity in trans, and high C→T editing activity in cis. These data suggest that the variants in Table 1D have reduced guide-independent and off-target properties and are therefore superior to BE4. [Figure 56B]Figure 2 shows a heatmap depicting the results of an R-loop assay demonstrating that variants S2.14, S2.46, and S1.52 in Table 1D exhibited reduced A→G guide-independent off-target activity at the indicated target sites (see A2, A6, A15, A16, A17, and A27 in Table 24) compared to BE4, the ABE8 control, and nCas9. The S2.14, S2.46, and S1.52 variants exhibited very low to no A→G editing activity in cis or in trans, low to no C→T editing activity in trans, and high C→T editing activity in cis. These data suggest that the variants in Table 1D have reduced guide-independent and off-target properties and are therefore superior to BE4. [Figure 57] A ribbon diagram and shaded chart illustrating regions A, B, and C used in the rational design of candidate base editors in Example 4 are shown. DETAILED DESCRIPTION OF THE INVENTION

[0148] The present disclosure provides adenosine deaminase variants that have adenine deaminase activity and cytosine deaminase activity, or that have increased cytosine deaminase activity and decreased adenosine deaminase activity. The disclosure also provides fusion proteins, multimolecular complexes, base editors, and base editing systems that include the adenosine deaminase variants, and methods of using such variants.

[0149] In one aspect, the present invention is based, at least in part, on the discovery that adenosine deaminase can be modified to deaminate cytosines in a target polynucleotide (e.g., DNA) while retaining adenine deamination activity. In particular, a base editing system comprising an adenosine deaminase variant, when expressed in a cell (e.g., a mammalian cell), can convert A to G and C to T in a target polynucleotide (e.g., DNA). Thus, editing of C to T and A to G can be catalyzed by a single deaminase. In some embodiments, the adenosine deaminase variants described herein have approximately equal adenosine deaminase activity and cytidine deaminase activity. Base editors comprising deaminase variants having adenosine deaminase activity and cytidine deaminase activity (e.g., approximately equal activity) are referred to as having "dual editing activity." Without being bound by theory, the adenosine deaminase variants described herein have the potential to offer various advantages, including smaller double C-to-T and A-to-G editors compared to currently available double rAPOBEC+TadA editors, superior properties of TadA as a deaminase compared to APOBEC (e.g., lower off-target editing, use in inlaid base editors (IBEs)), more uniform allele distribution compared to two-deaminase-based systems, and enhanced applications for multiplex editing using both A and C targets in cell engineering.

[0150] In some embodiments, the adenosine deaminase variant has predominantly cytosine deaminase activity, with little, if any, adenosine deaminase activity. In some embodiments, the adenosine deaminase variant has cytosine deaminase activity, but does not have significant or detectable adenosine deaminase activity. In embodiments, the adenosine deaminase activity is less than about 0.01%, 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% of the cytosine deaminase activity.

[0151] In another aspect, the present invention is based, at least in part, on the discovery that adenosine deaminase can be modified to deaminate cytosines in a target polynucleotide (e.g., DNA) while minimizing adenine deamination activity. In particular, a base editing system comprising an adenosine deaminase variant, when expressed in a cell (e.g., a mammalian cell), can convert C to T in a target polynucleotide, but does not substantially convert A to G in the target polynucleotide (e.g., DNA). In some embodiments, the adenosine deaminase variants described herein have primarily C to T editing activity, i.e., have 30% or more cytidine deaminase activity compared to adenosine deaminase activity. Without being bound by theory, the adenosine deaminase variants described herein have the potential to offer various advantages, including smaller size relative to APOBEC proteins and therefore smaller C-to-T editors relative to currently available rAPOBEC base editors, superior properties of TadA as a deaminase relative to APOBEC (e.g., lower off-target editing, use with inlaid base editors (IBEs)), and an expanded repertoire of base editing tools for C-to-T applications and cellular modification.

[0152] The adenosine deaminase base editor variants of the invention are particularly useful for targeted editing of DNA, for example, to introduce mutations that alter the activity of regulatory sequences (e.g., splice sites, enhancers, and transcriptional regulatory elements) or alter the activity of an encoded protein (e.g., antibody complementarity-determining regions (CDRs)). In embodiments, the adenosine base editor variants of the invention have reduced guide-independent off-target editing properties compared to a reference CBE (e.g., rAPOBEC or BE4), are compatible with inlaid base editor (IBE) architectures, have narrower editing windows compared to APOBEC-based CBEs (e.g., BE4), and / or can be multiplexed with increased on-target editing compared to a reference CBE (e.g., BE4).

[0153] Targeted gene editing The present invention provides adenosine deaminase variants having adenine deaminase activity and cytosine deaminase activity. Compositions comprising the adenosine deaminase variants described herein are used to deaminate adenine and cytosine in a target polynucleotide (e.g., DNA). In some embodiments, the target polynucleotide is single-stranded or double-stranded. In some embodiments, the adenosine deaminase variants deaminate adenine and cytosine in DNA. In some embodiments, the adenosine deaminase variants deaminate adenine and cytosine in single-stranded DNA. In some embodiments, the adenosine deaminase variants deaminate adenine and cytosine in RNA. In some embodiments, the adenosine deaminase variant primarily deaminates cytosine in DNA and / or RNA (e.g., greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of all deamidations catalyzed by the adenosine deaminase variant, or the number of cytosine deamidations catalyzed by the variant is about or at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, 500-fold, or 1,000-fold greater than the number of adenine deamidations catalyzed by the variant). In some embodiments, the adenosine deaminase variant has approximately equal cytosine and adenosine deaminase activities (e.g., the two activities are within about 10% or 20% of each other). In some embodiments, the adenosine deaminase variant has predominantly cytosine deaminase activity, with little, if any, adenosine deaminase activity. In some embodiments, the adenosine deaminase variant has cytosine deaminase activity, but no significant or detectable adenosine deaminase activity. In some embodiments, the target polynucleotide is present in a cell in vitro or in vivo. In some embodiments, the cell is a bacterial, yeast, fungal, insect, plant, or mammalian cell.

[0154] In some embodiments, the adenine or adenosine base editor (ABE) comprises a bacterial TadA deaminase variant (e.g., ecTadA). In some embodiments, the adenine or adenosine base editor (ABE) comprises a truncated TadA deaminase variant. In some embodiments, the adenine or adenosine base editor (ABE) comprises a fragment of a TadA deaminase variant. In some embodiments, the adenine or adenosine base editor (ABE) comprises a TadA*8.20 variant.

[0155] In some embodiments, the adenosine deaminase variants of the present invention comprise one or more modifications. In some embodiments, the adenosine deaminase variants of the present invention are TadA adenosine deaminase variants that comprise one or more modifications that increase cytosine deaminase activity (e.g., by at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or more) while maintaining adenosine deaminase activity (e.g., at least about 30%, 40%, 50%, or more of the activity of a reference adenosine deaminase (e.g., TadA*8.20 or TadA*8.19)). In some embodiments, the adenosine deaminase variant is a bacterial TadA deaminase variant (e.g., ecTadA). In some embodiments, the adenosine deaminase variant is a truncated TadA deaminase variant. In some embodiments, the adenosine deaminase variant is a fragment of a TadA deaminase variant. In some embodiments, the adenosine deaminase variant is a TadA*8 variant that includes one or more modifications that increase cytosine deaminase activity (e.g., by at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or more) while maintaining at least about 30%, 40%, 50%, or more of the adenosine deaminase activity of a reference adenosine deaminase (e.g., TadA*8.20 or TadA*8.19). In some embodiments, the adenosine deaminase variant is a TadA*8.20 adenosine deaminase that includes one or more modifications that increase cytosine deaminase activity (e.g., by at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or more) while maintaining an adenosine deaminase activity that is at least 30%, 40%, 50%, or more of the activity of a reference adenosine deaminase (e.g., TadA*8.20 or TadA*8.19).In some embodiments, the adenosine deaminase variants provided herein have at least about a 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more increase in cytosine deaminase activity compared to a reference adenosine deaminase (e.g., TadA*8.20 or TadA*8.19). In some embodiments, the adenosine deaminase variants provided herein maintain an adenosine deaminase activity level that is at least about 30%, 40%, 50%, 60%, or 70% of the activity of the reference adenosine deaminase (e.g., TadA*8.20 or TadA*8.19). In some embodiments, the reference adenosine deaminase is TadA*8.20 or TadA*8.19.

[0156] In some embodiments, the adenosine deaminase variant is an adenosine deaminase that includes one or more modifications that increase cytosine deaminase activity, such as the following SEQ ID NO:1: 10 20 30 40 50 MSEVEFSHEY WMRHALTLAK RARDEREVPV GAVLVLNNRV IGEGWNRAIG 60 70 80 90 100 LHDPTAHAEI MALRQGGLVM QNYRLYDATL YSTFEPCVMC AGAMIHSRIG 110 120 130 140 150 RVVFGVRNAK TGAAGSLMDV LHHPGMNHRV EITEGILADE CAALLCRFFR 160 MPRRVFNAQK KAQSSTD (SEQ ID NO: 1) having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identity to

[0157] In some embodiments, the adenosine deaminase variant is an adenosine deaminase comprising the amino acid sequence of SEQ ID NO: 1 and one or more modifications that increase cytosine deaminase activity. In various embodiments, the one or more modifications of the invention do not comprise an R amino acid at position 48 of SEQ ID NO: 1 or a corresponding mutation in another adenosine deaminase.

[0158] In some embodiments, the adenosine deaminase variant is an adenosine deaminase that contains one or more modifications at amino acid positions selected from 2, 4, 6, 13, 27, 29, 100, 112, 114, 115, 162, and 165 of an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identity to SEQ ID NO: 1, or the corresponding modifications in another deaminase. In some embodiments, the adenosine deaminase variant is an adenosine deaminase that contains two or more modifications at amino acid positions selected from the group consisting of 2, 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167 of an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identity to SEQ ID NO:1, or the corresponding modifications in another deaminase. In some embodiments, the two or more modifications are selected from the group consisting of S2X, V4X, F6X, H8X, R13X, T17X, ​​R23X, E27X, P29X, V30X, R47X, A48X, I49X, G67X, Y76X, D77X, S82X, F84X, and the like, of an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identity to SEQ ID NO:1. The modification is at an amino acid position selected from the group consisting of H96X, G100X, R107X, G112X, A114X, G115X, M118X, D119X, H122X, N127X, A142X, A143X, R147X, Y147X, F149X, A158X, Q159X, A162X, S165X, T166X, and D167X, or a corresponding modification in another deaminase. In various embodiments, the modification of the present invention does not include the 48R mutation.In some embodiments, the adenosine deaminase variant is an adenosine deaminase that contains one or more modifications at amino acid positions selected from 2, 4, 6, 13, 27, 29, 100, 112, 114, 115, 162, and 165 of an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identity to SEQ ID NO: 1, or the corresponding modifications in another deaminase.

[0159] In some embodiments, the adenosine deaminase variants are selected from the group consisting of S2H, V4K, V4S, V4T, V4Y, F6G, F6H, F6Y, H8Q, R13G, T17A, T17W, R23Q, E27C, E27G, E27H, E27K, E27Q, E27S, E27G, P29A, P29G, P29K, V30F, V30I, R47G, R47S, A48G, I49K, I49M, I49N ... 9Q, I49T, G67W, I76H, I76R, I76W, Y76H, Y76R, Y76W, F84A, F84M, H96N, G100A, G100K, T111H, G112H, A114C, G115M, M118L, H122G, H122R, H122T, N127I, N127K, N127P, A142E, R147H, A158V, Q159S, A162C, A162N, A162Q, and S165P, or the corresponding modification in another deaminase.

[0160] In some embodiments, the adenosine deaminase variant has an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identity to SEQ ID NO:1: E27H, Y76I, and F84M; E27H, I49K, and Y76I; E27S, I49K, Y76I, and A162N; E27K and D119N; E27H and Y76I; E27S, I49K, and G67W; E27S, I49K, and Y76I; I49T, G67W, and H96N; E27C, Y76I, and D119N; R13G, E27Q, and N127K; T17A, E27H, I49M, Y76I, and M118L; I49Q, Y76I, and G115M; S2H, I49K, Y76I, and G112H; R47S and R107C; H8Q, I49Q, and Y76I; T17A, A48G, S82T, and A142E; E27G and I49N; E27G, D77G, and S165P; E27S, I49K, and S82T; E27S, I49K, S82T, and G115M; E27S, V30I, I49K, and S82T; E27S, V30F, I49K, S82T, F84A, R107C, and A142E; E27S, V30F, I49K, S82T, F84A, G112H, and A142E; E27S, V30F, I49K, S82T, F84A, G115M, and A142E; E27S, I49K, S82T, F84L, and R107C; E27S, I49K, S82T, F84L, and G112H; E27S, I49K, S82T, F84L, and G115M; E27S, I49K, S82T, F84L, R107C, and G112H; E27S, I49K, S82T, F84L, R107C, and G115M; E27S, I49K, S82T, F84L, R107C, and A142E; E27S, I49K, S82T, F84L, G112H, and A142E; E27S, I49K, S82T, F84L, G115M, and A142E; E27S, I49K, S82T, F84L, R107C, G112H, G115M, and A142E; E27S, V30I, I49K, S82T, and F84L; E27S, P29G, I49K, and S82T; E27S, P29G, I49K, S82T, and G115M; E27S, P29G, I49K, S82T, and A142E; P29G, I49K, and S82T; E27G, I49K, and S82T; E27G, I49K, S82T, R107C, and A142E;V4K, E27H, I49K, Y76I, and A114C; V4K, E27H, I49K, Y76I, and D77G; F6Y, E27H, I49K, Y76I, G100A, and H122R; V4T, E27H, I49K, Y76R, and H122G; F6Y, E27H, I49K, and Y76W; F6Y, E27H, I49K, Y76I, and D119N; F6Y, E27H, I49K, Y76I, and A114C; F6Y, E27H, I49K, and Y76I; V4K, E27H, I49K, Y76W, and H122T; F6G, E27H, I49K, Y76R, and G100K; F6H, E27H, I49K, Y76I, and H122N; E27H, I49K, Y76I, and A114C; F6Y, E27H, I49K, Y76H, H122R, and T166I; E27H, I49K, Y76I, and N127P; R23Q, E27H, I49K, and Y76R; E27H, I49K, Y76H, H122R, and A158V; F6Y, E27H, I49K, Y76I, and T111H; E27H, I49K, Y76I, and R147H; E27H, I49K, Y76I, and A143E; F6Y, E27H, I49K, and Y76R; T17W, E27H, I49K, Y76H, H122G, and A158V; V4S, E27H, I49K, A143E, and Q159S; E27H, I49K, Y76I, N127I, and A162Q; T17A, E27H, and A48G; T17A, E27K, and A48G; T17A, E27S, and A48G; T17A, E27S, A48G, and I49K; T17A, E27G, and A48G; T17A, A48G, and I49N; T17A, E27G, A48G, and I49N; T17A, E27Q, and A48G; E27S, I49K, S82T, and R107C; E27S, I49K, S82T, and G112H; E27S, I49K, S82T, and A142E; E27S, I49K, S82T, R107C, and G112H; E27S, I49K, S82T, R107C, and G115M; E27S, I49K, S82T, R107C, and A142E; E27S, I49K, S82T, G112H, and A142E;E27S, I49K, S82T, G115M, and A142E; E27S, I49K, S82T, R107C, G112H, G115M, and A142E; E27S, V30I, I49K, S82T, and R107C; E27S, V30I, I49K, S82T, and G112H; E27S, V30I, I49K, S82T, and G115M; E27S, V30I, I49K, S82T, and A142E; E27S, V30I, I49K, S82T, R107C, and G112H; E27S, V30I, I49K, S82T, and A142E; E27S, V30I, I49K, S82T, R107C, and G115M; E27S, V30I, I49K, S82T, R107C, and A142E; E27S, V30I, I49K, S82T, G112H, and A142E; E27S, V30I, I49K, S82T, G115M, and A142E; E27S, V30I, I49K, S82T, R107C, G112H, G115M, and A142E; E27S, V30L, I49K, and S82T; E27S, V30L, I49K, S82T, and R107C; E27S, V30L, I49K, S82T, and G112H; E27S, V30L, I49K, S82T, and G115M; E27S, V30L, I49K, S82T, and A142E; E27S, V30L, I49K, S82T, R107C, and G112H; E27S, V30L, I49K, S82T, R107C, and G115M; E27S, V30L, I49K, S82T, R107C, and A142E; E27S, V30L, I49K, S82T, G112H, and A142E; E27S, V30L, I49K, S82T, G115M, and A142E; E27S, V30L, I49K, S82T, R107C, G112H, G115M, and A142E; E27S, V30F, I49K, S82T, and F84A; E27S, V30F, I49K, S82T, F84A, and R107C; E27S, V30F, I49K, S82T, F84A, and G112H; E27S, V30F, I49K, S82T, F84A, and G115M; E27S, V30F, I49K, S82T, F84A, and A142E; E27S, V30F, I49K, S82T, F84A, R107C, and G112H;E27S, V30F, I49K, S82T, F84A, R107C, and G115M; E27S, V30F, I49K, S82T, F84A, R107C, G112H, G115M, and A142E; E27S, I49K, S82T, and F84L; E27S, I49K, S82T, F84L, and A142E; E27S, V30I, I49K, S82T, F84L, and R107C; E27S, V30I, I49K, S82T, F84L, and G112H; E27S, V30I, I49K, S82T, F84L, and G115M; E27S, V30I, I49K, S82T, F84L, and A142E; E27S, V30I, I49K, S82T, F84L, R107C, and G112H; E27S, V30I, I49K, S82T, F84L, R107C, and G115M; E27S, V30I, I49K, S82T, F84L, R107C, and A142E; E27S, V30I, I49K, S82T, F84L, G112H, and A142E; E27S, V30I, I49K, S82T, F84L, G115M, and A142E; E27S, V30I, I49K, S82T, F84L, R107C, G112H, G115M, and A142E; E27S, P29G, I49K, S82T, and R107C; E27S, P29G, I49K, S82T, and G112H; E27S, P29G, I49K, S82T, R107C, and G112H; E27S, P29G, I49K, S82T, R107C, and G115M; E27S, P29G, I49K, S82T, R107C, and A142E; E27S, P29G, I49K, S82T, G112H, and A142E; E27S, P29G, I49K, S82T, G115M, and A142E; E27S, P29G, I49K, S82T, R107C, G112H, G115M, and A142E; P29G, I49K, S82T, and R107C; P29G, I49K, S82T, and G112H; P29G, I49K, S82T, and G115M; P29G, I49K, S82T, and A142E; P29G, I49K, S82T, R107C, and G112H; P29G, I49K, S82T, R107C, and G115M; P29G, I49K, S82T, R107C, and A142E;P29G, I49K, S82T, G112H, and A142E; P29G, I49K, S82T, G115M, and A142E; P29G, I49K, S82T, R107C, G112H, G115M, and A142E; P29K, I49K, and S82T; P29K, I49K, S82T, and R107C; P29K, I49K, S82T, and G112H; P29K, I49K, S82T, and G115M; P29K, I49K, S82T, and A142E; P29K, I49K, S82T, R107C, and G112H; P29K, I49K, S82T, R107C, and G115M; P29K, I49K, S82T, R107C, and A142E; P29K, I49K, S82T, G112H, and A142E; P29K, I49K, S82T, G115M, and A142E; P29K, I49K, S82T, R107C, G112H, G115M, and A142E; P29K, V30I, I49K, and S82T; P29K, V30I, I49K, S82T, and R107C; P29K, V30I, I49K, S82T, and G112H; P29K, V30I, I49K, S82T, and G115M; P29K, V30I, I49K, S82T, and A142E; P29K, V30I, I49K, S82T, R107C, and G112H; P29K, V30I, I49K, S82T, R107C, and G115M; P29K, V30I, I49K, S82T, R107C, and A142E; P29K, V30I, I49K, S82T, G112H, and A142E; P29K, V30I, I49K, S82T, G115M, and A142E; P29K, V30I, I49K, S82T, R107C, G112H, G115M, and A142E; P29K, I49K, S82T, and F84L; P29K, I49K, S82T, F84L, and R107C; P29K, I49K, S82T, F84L, and G112H; P29K, I49K, S82T, F84L, and G115M; P29K, I49K, S82T, F84L, and A142E; P29K, I49K, S82T, F84L, R107C, and G112H; P29K, I49K, S82T, F84L, R107C, and G115M;P29K, I49K, S82T, F84L, R107C, and A142E; P29K, I49K, S82T, F84L, G112H, and A142E; P29K, I49K, S82T, F84L, G115M, and A142E; P29K, I49K, S82T, F84L, R107C, G112H, G115M, and A142E; P29K, V30I, I49K, S82T, and F84L; P29K, V30I, I49K, S82T, F84L, and R107C; P29K, V30I, I49K, S82T, F84L, and G112H; P29K, V30I, I49K, S82T, F84L, and G115M; P29K, V30I, I49K, S82T, F84L, and A142E; P29K, V30I, I49K, S82T, F84L, R107C, and G112H; P29K, V30I, I49K, S82T, F84L, R107C, and G115M; P29K, V30I, I49K, S82T, F84L, R107C, and A142E; P29K, V30I, I49K, S82T, F84L, G112H, and A142E; P29K, V30I, I49K, S82T, F84L, G115M, and A142E; P29K, V30I, I49K, S82T, F84L, R107C, G112H, G115M, and A142E; E27G, I49K, S82T, and R107C; E27G, I49K, S82T, and G112H; E27G, I49K, S82T, and G115M; E27G, I49K, S82T, and A142E; E27G, I49K, S82T, R107C, and G112H; E27G, I49K, S82T, R107C, and G115M; E27G, I49K, S82T, G112H, and A142E; E27G, I49K, S82T, G115M, and A142E; E27G, I49K, S82T, R107C, G112H, G115M, and A142E; E27H, I49K, and S82T; E27H, I49K, S82T, and R107C; E27H, I49K, S82T, and G112H; E27H, I49K, S82T, and G115M; E27H, I49K, S82T, and A142E; E27H, I49K, S82T, R107C, and G112H; E27H, I49K, S82T, R107C, and G115M;E27H, I49K, S82T, R107C, and A142E; E27H, I49K, S82T, G112H, and A142E; E27H, I49K, S82T, G115M, and A142E; E27H, I49K, S82T, R107C, G112H, G115M, and A142E; E27S, and S82T; E27S, S82T, and R107C; E27S, S82T, and G112H; E27S, S82T, and G115M; E27S, S82T, and A142E; E27S, S82T, R107C, and G112H; E27S, S82T, R107C, and G115M; E27S, S82T, R107C, and A142E; E27S, S82T, G112H, and A142E; E27S, S82T, G115M, and A142E; E27S, S82T, R107C, G112H, G115M, and A142E; P29A, and S82T; P29A, S82T, and R107C; P29A, S82T, and G112H; P29A, S82T, and G115M; P29A, S82T, and A142E; P29A, S82T, R107C, and G112H; P29A, S82T, R107C, and G115M; P29A, S82T, R107C, and A142E; P29A, S82T, G112H, and A142E; P29A, S82T, G115M, and A142E; P29A, S82T, R107C, G112H, G115M, and A142E; E27S, V30I, and S82T; E27S, V30I, S82T, and R107C; E27S, V30I, S82T, and G112H; E27S, V30I, S82T, and G115M; E27S, V30I, S82T, and A142E; E27S, V30I, S82T, R107C, and G112H; E27S, V30I, S82T, R107C, and G115M; E27S, V30I, S82T, R107C, and A142E; E27S, V30I, S82T, G112H, and A142E; E27S, V30I, S82T, G115M, and A142E; E27S, V30I, S82T, R107C, G112H, G115M, and A142E; P29A, V30I, S82T, and F84L; P29A, V30I, S82T, F84L, and R107C;P29A, V30I, S82T, F84L, and G112H; P29A, V30I, S82T, F84L, and G115M; P29A, V30I, S82T, F84L, and A142E; P29A, V30I, S82T, F84L, R107C, and G112H; P29A, V30I, S82T, F84L, R107C, and G115M; P29A, V30I, S82T, F84L, R107C, and A142E; P29A, V30I, S82T, F84L, G112H, and A142E; P29A, V30I, S82T, F84L, G115M, and A142E; P29A, V30I, S82T, F84L, R107C, G112H, G115M, and A142E; E27S, P29A, V30L, I49K, S82T, F84L, R107C, G112H, G115M, and A142E; V4K and A114C; V4K and D77G; F6Y, G100A, and H122R; V4T, I76R, and H122G; F6Y and I76W; F6Y and D119N; F6Y and A114C; V4K, I76W, and H122T; F6G, I76R, and G100K; F6H and H122N; F6Y, I76H, H122R, and T166I; R23Q, and I76R; I76H, H122R, and A158V; F6Y, and T111H; T111H, H122G, and A162C; F6Y, and I76R; T17W, I76H, H122G, and A158V; V4S, I76Y, A143E, and Q159S; N127I, A162Q; E27H, Y76I, F84M, and F149Y; E27H, I49K, Y76I, and F149Y; T17A, E27H, I49M, Y76I, M118L, and F149Y; T17A, A48G, S82T, A142E, and F149Y; E27G, and F149Y; E27G, I49N, and F149Y; E27H, Y76I, F84M, Y147D, F149Y, T166I, and D167N; E27H, I49K, Y76I, Y147D, F149Y, T166I, D167N; T17A, E27H, I49M, Y76I, M118L, Y147D, F149Y, T166I, and D167N;T17A, A48G, S82T, A142E, Y147D, F149Y, T166I, and D167N; E27G, Y147D, F149Y, T166I, and D167N; E27G, I49N, Y147D, F149Y, T166I, and D167N; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, and A142E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, D119N, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, H122G, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, N127P, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, A142E, and A143E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, and A143E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, and A1; 42E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, D119N, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, H122G, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, N127P, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, A142E, and A143E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, and A143E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, A114C, G115M, and A142E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, D119N, and A142E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, H122G, and A142E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, N127P, and A142E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, A142E, and A143E; F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, and A143E; F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, D119N, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, H122G, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, N127P, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, D119N, H122G, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, D119N, N127P, and A142E; F6Y, E27H, I49K, S82T, R107C, G112H, G115M, H122G, N127P, and A142E;F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, D119N, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, H122G, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, N127P, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, A142E, and A143E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, and A143E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, H122G, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, N127P, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, A142E, and A143E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, and A143E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, D119N, H122G, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, D119N, N127P, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, H122G, N127P, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, H122G, and A142E;F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, N127P, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, H122G, N127P, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, H122G, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, N127P, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, A142E, and A143E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, and A143E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, H122G, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, N127P, and A142E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, H122G, N127P, and A142E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, A142E, and A143E; F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, and A143E; F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, A142E, and A143E;and F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, and A143E; or a combination of corresponding modifications in another deaminase.

[0161] In some embodiments, the adenosine deaminase variant is an adenosine deaminase that includes an amino acid modification or combination of amino acid modifications selected from those listed in any of Tables 1A-1F.

[0162] The residue identities of exemplary adenosine deaminase variants capable of deaminating adenine and / or cytidine in a target polynucleotide (e.g., DNA) are shown in Tables 1A-1F below. Further examples of adenosine deaminase variants include the following variants of 1.17 (see Table 1A): 1.17+E27H; 1.17+E27K; 1.17+E27S; 1.17+E27S+I49K; 1.17+E27G; 1.17+I49N; 1.17+E27G+I49N; and 1.17+E27Q. In some embodiments, any of the amino acid modifications provided herein are substituted with conservative amino acids. Additional mutations known in the art can be further added to any of the adenosine deaminase variants provided herein.

[0163] In some embodiments, base editing is performed to induce therapeutic changes in the genome of cells of a subject (e.g., a human). The cells are removed from the subject and contacted with one or more guide RNAs and a nucleic acid base editor polypeptide comprising a nucleic acid programmable DNA binding protein (napDNAbp) (e.g., Cas9) and an adenosine deaminase variant capable of deaminating both adenine and cytosine in a target polynucleotide (e.g., DNA). In some embodiments, the cells are contacted with one or more guide RNAs and a fusion protein comprising a nucleic acid programmable DNA binding protein (napDNAbp) (e.g., Cas9) and an adenosine deaminase variant capable of deaminating both adenine and cytosine in a target polynucleotide (e.g., DNA). In some embodiments, the napDNAbp is Cas9.

[0164] In some embodiments, the cell is contacted with a multimolecular complex. In some embodiments, the cell is contacted with a base editing system provided herein. In some embodiments, the base editing system provided herein comprises an adenosine base editor (ABE) variant. In some embodiments, the ABE variant is an ABE8 variant. In some embodiments, the ABE8 variant is an ABE8.20 variant. In some embodiments, a base editing system comprising an ABE variant provided herein (e.g., an ABE8.20 variant) has both A-to-G base editing activity and C-to-T base editing activity. Thus, multiple edits may be introduced into a subject's (e.g., human) genome. The ability to target both A-to-G and C-to-T base editing activity allows for versatile targeting of polynucleotides within a subject's genome to treat genetic diseases or disorders.

[0165] In some embodiments, a base editing system comprising an adenosine deaminase variant provided herein has at least about 30%, 40%, 50%, 60%, 70%, or more C to T editing activity in a target polynucleotide (e.g., DNA). In some embodiments, a base editing system comprising an adenosine deaminase variant provided herein has increased C to T base editing activity (e.g., at least about 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or more) compared to a reference base editing system comprising a reference adenosine deaminase (e.g., TadA*8.20 or TadA*8.19).

[0166] In various instances, it is advantageous to include 5' and / or 3' "G" nucleotides in the spacer sequence of a guide RNA. In some cases, for example, any spacer sequence or guide polynucleotide provided herein includes or further includes a 5' "G", where in some embodiments, the 5' "G" is complementary or not complementary to the target sequence. In some embodiments, a 5' "G" is added to a spacer sequence that does not already include a 5' "G". For example, because the U6 promoter prefers a "G" at the transcription start site, it may be advantageous to include a 5'-terminal "G" in the guide RNA when the guide RNA is expressed under the control of, for example, a U6 promoter (see Cong, L. et al. "Multiplex genome engineering using CRISPR / Cas systems. Science 339:819-823 (2013) doi: 10.1126 / science.1231143). In some cases, a 5'-terminal "G" is added to a guide polynucleotide (e.g., a guide RNA) that is expressed under the control of a promoter, but is optionally not added to the guide polynucleotide when the guide polynucleotide is not expressed under the control of a promoter.

[0167] [Table 1]

[0168]

Table 2

[0169]

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

[0170]

Table 4

[0171]

Table 5-1

Table 5-2

Table 5-3

[0172]

Table 6-1

Table 6-2

[0173]

Table 7

[0174] In certain embodiments, the fusion proteins and multimolecular complexes provided herein comprise one or more features that improve the base editing activity of the fusion protein or multimolecular complex. For example, any of the fusion proteins or multimolecular complexes provided herein may comprise a Cas9 domain with reduced nuclease activity. In some embodiments, any of the fusion proteins provided herein may comprise a Cas9 domain with no nuclease activity (dCas9) or a Cas9 domain that cleaves one strand of a double-stranded DNA molecule, referred to as a Cas9 nickase (nCas9). Without being bound by any particular theory, the presence of a catalytic residue (e.g., H840) maintains the activity of Cas9 to cleave the unedited (e.g., unmethylated) strand opposite the targeted nucleobase. Mutation of the catalytic residue (e.g., D10 to A10) prevents cleavage of the edited strand containing the targeted A residue. Such Cas9 variants generate single-stranded DNA breaks (nicks) at specific locations based on the target sequence specified by the gRNA, triggering repair of the non-edited strand and ultimately resulting in nucleobase changes on the non-edited strand.

[0175] Nucleic acid base editor Nucleobase editors that edit, modify, or alter target nucleotide sequences of polynucleotides are useful in the methods and compositions described herein. The nucleobase editors described herein typically comprise a polynucleotide-programmable nucleotide-binding domain and a nucleobase-editing domain (e.g., an adenosine deaminase variant domain). The polynucleotide-programmable nucleotide-binding domain, when combined with an associated guide polynucleotide (e.g., a gRNA), specifically binds to the target polynucleotide sequence, thereby allowing the base editor to localize to the target nucleic acid sequence to be edited.

[0176] Polynucleotide-programmable nucleotide-binding domains The polynucleotide-programmable nucleotide-binding domain binds to a polynucleotide (e.g., RNA, DNA). The polynucleotide-programmable nucleotide-binding domain of a base editor may itself comprise one or more domains (e.g., one or more nuclease domains). In some embodiments, the nuclease domain of the polynucleotide-programmable nucleotide-binding domain may comprise an endonuclease or an exonuclease. An endonuclease can cleave one strand of a double-stranded nucleic acid molecule or both strands of a double-stranded nucleic acid molecule. In some embodiments, the nuclease domain of the polynucleotide-programmable nucleotide-binding domain can cleave zero, one, or two strands of a target polynucleotide.

[0177] Non-limiting examples of polynucleotide-programmable nucleotide-binding domains that can be incorporated into base editors include domains derived from CRISPR proteins, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs). In some embodiments, the base editors contain a polynucleotide-programmable nucleotide-binding domain comprising a natural or modified protein or portion thereof, and can bind to a nucleic acid sequence via an attached guide nucleic acid during CRISPR (i.e., clustered regularly interspaced short palindromic repeats)-mediated nucleic acid modification. Such proteins are referred to herein as "CRISPR proteins." Accordingly, base editors containing a polynucleotide-programmable nucleotide-binding domain comprising all or a portion of a CRISPR protein (i.e., base editors comprising all or a portion of a CRISPR protein as a domain, also referred to as the "CRISPR protein-derived domain" of the base editor) are disclosed herein. The CRISPR protein-derived domain incorporated into the base editor can be modified compared to a wild-type or natural version of the CRISPR protein. For example, as described below, a domain derived from a CRISPR protein can contain one or more mutations, insertions, deletions, rearrangements, and / or recombinations compared to a wild-type or naturally occurring version of the CRISPR protein.

[0178] Cas proteins that may be used herein include class 1 and class 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csx1, Csx2, Csx3, Csx4, Csx5e, Csx1, Csx2, Csx5, Csx ...3, Csx4, Csx5e, Csx1, Csx2, Csx5, Csx6, Csx1, Csx2, Csx3, Csx4, Csx5e, Csx1, Csx2, Csx3, Csx4, Csx5 Examples include sb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas12a / Cpf1, Cas12b / C2c1 (e.g., SEQ ID NO: 247), Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, and Cas12j / CasΦ, CARF, DinG, homologs thereof, or modified or altered versions thereof. CRISPR enzymes can direct cleavage of one or both strands at a target sequence, e.g., within the target sequence and / or within a sequence complementary to the target sequence. For example, CRISPR enzymes can direct cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence.

[0179] A vector encoding a CRISPR enzyme mutated relative to the corresponding wild-type enzyme can be used, such that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. A Cas protein (e.g., Cas9, Cas12) or Cas domain (e.g., Cas9, Cas12) can refer to a polypeptide or domain that has at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or homology to a wild-type exemplary Cas polypeptide or Cas domain. Cas (e.g., Cas9, Cas12) can refer to wild-type or modified forms of Cas proteins that can include amino acid changes such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0180] In some embodiments, the CRISPR protein-derived domains of the base editors are derived from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1), Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1), Spiroplasma syrphidicola (NCBI Ref: NC_021284.1), Prevotella intermedia (NCBI Ref: NC_017861.1), Spiroplasma taiwanense (NCBI Ref: NC_021846.1), Streptococcus iniae (NCBI Ref: NC_021314.1), Belliella baltica (NCBI Ref: NC_018010.1), Psychroflexus torquis (NCBI Ref: NC_018721.1), Streptococcus thermophilus (NCBI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1), Neisseria meningitidis (NCBI Ref: YP_002342100.1), Streptococcus pyogenes, or Staphylococcus aureus.

[0181] The sequence and structure of Cas9 nuclease are well known to those of skill in the art (see, e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes," Ferretti et al., Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III," Deltcheva E., et al., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity," Jinek M., et al., Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on the present disclosure, including Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference.

[0182] High-fidelity Cas9 domain Some aspects of the present disclosure provide high-fidelity Cas9 domains. High-fidelity Cas9 domains are known in the art and are described, for example, in Kleinstiver, BP, et al. "High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects," Nature 529, 490-495 (2016) and Slaymaker, IM, et al. "Rationally engineered Cas9 nucleases with improved specificity," Science 351, 84-88 (2015), the entire contents of each of which are incorporated herein by reference. An exemplary high-fidelity Cas9 domain is set forth in the Sequence Listing as SEQ ID NO: 248. In some embodiments, the high-fidelity Cas9 domain is a modified Cas9 domain containing one or more mutations that reduce the electrostatic interaction between the Cas9 domain and the sugar-phosphate backbone of DNA compared to the corresponding wild-type Cas9 domain. High-fidelity Cas9 domains that reduce electrostatic interactions with the sugar-phosphate backbone of DNA result in fewer off-target effects. In some embodiments, the Cas9 domain (e.g., a wild-type Cas9 domain (SEQ ID NOS: 198 and 201)) comprises one or more mutations that reduce the association of the Cas9 domain with the sugar-phosphate backbone of DNA. In some embodiments, the Cas9 domain comprises one or more mutations that reduce the association of the Cas9 domain with the sugar-phosphate backbone of DNA by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70%.

[0183] In some embodiments, any of the Cas9 fusion proteins provided herein contain one or more of the following mutations: D10A, N497X, R661X, Q695X, and / or Q926X, or corresponding mutations in any of the amino acid sequences provided herein, where X is any amino acid. In some embodiments, the high-fidelity Cas9 enzyme is SpCas9(K855A), eSpCas9(1.1), SpCas9-HF1, or an ultra-high-precision Cas9 variant (HypaCas9). In some embodiments, the modified Cas9 eSpCas9(1.1) contains an alanine substitution that weakens the interaction between the HNH / RuvC groove and non-target DNA strands, preventing strand separation and cleavage at off-target sites. Similarly, SpCas9-HF1 reduces off-target editing through an alanine substitution that disrupts the interaction of Cas9 with the DNA phosphate backbone. HypaCas9 contains mutations in the REC3 domain (SpCas9 N692A / M694A / Q695A / H698A) that improve Cas9 proofreading and target discrimination. All three high-fidelity enzymes generate fewer off-target edits compared to wild-type Cas9.

[0184] Reduced exclusivity Cas9 domains Typically, Cas9 proteins, such as S. pyogenes Cas9 (SpCas9), require a "protospacer adjacent motif (PAM)" or PAM-like motif, a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease of the CRISPR bacterial adaptive immune system. The presence of the NGG PAM sequence is required to bind to specific nucleic acid regions, where the "N" in "NGG" is adenosine (A), thymidine (T), or cytosine (C), and the "G" is guanosine. This can limit their ability to edit a target base in the genome. In some embodiments, the base-editing fusion proteins provided herein may need to be placed at a precise location, e.g., in the region containing the target base upstream of the PAM. See, e.g., Komor, AC, et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage," Nature 533, 420-424 (2016), the entire contents of which are incorporated herein by reference. Exemplary polypeptide sequences of spCas9 proteins capable of binding to PAM sequences are set forth in the Sequence Listing as SEQ ID NOS: 198, 202, and 249-252. Thus, in some embodiments, any of the fusion proteins provided herein can contain a Cas9 domain capable of binding to a nucleotide sequence that does not contain a standard (e.g., NGG) PAM sequence. Cas9 domains that bind to non-standard PAM sequences have been described in the art and would be apparent to one of skill in the art.For example, Cas9 domains that bind to non-canonical PAM sequences are described in Kleinstiver, BP, et al., "Engineered CRISPR-Cas9 nucleases with altered PAM specificities" Nature 523, 481-485 (2015), and Kleinstiver, BP, et al., "Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition" Nature Biotechnology 33, 1293-1298 (2015), the entire contents of each of which are incorporated herein by reference.

[0185] Nickase In some embodiments, a polynucleotide-programmable nucleotide-binding domain can comprise a nickase domain. As used herein, the term "nickase" refers to a polynucleotide-programmable nucleotide-binding domain, including a nuclease domain, that can cleave only one strand of a double-stranded nucleic acid molecule (e.g., DNA). In some embodiments, a nickase can be derived from a fully catalytically active (e.g., native) form of a polynucleotide-programmable nucleotide-binding domain by introducing one or more mutations into the active form of the polynucleotide-programmable nucleotide-binding domain. For example, if the polynucleotide-programmable nucleotide-binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain can comprise a D10A mutation and a histidine at position 840. In such an embodiment, residue H840 retains catalytic activity, thereby enabling cleavage of one strand of a nucleic acid duplex. In another example, a Cas9-derived nickase domain can comprise an H840A mutation, but the amino acid residue at position 10 remains D. In some embodiments, a nickase can be derived from a fully catalytically active (e.g., native) form of a polynucleotide-programmable nucleotide-binding domain by removing all or part of a nuclease domain that is not required for nickase activity. For example, if the polynucleotide-programmable nucleotide-binding domain comprises a nickase domain from Cas9, the nickase domain from Cas9 can comprise a deletion of all or part of the RuvC domain or the HNH domain.

[0186] In some embodiments, the wild-type Cas9 corresponds to or comprises the following amino acid sequence: TIFF2024511163000028.tif173165 (SEQ ID NO: 198) (Single underline: HNH domain; double underline: RuvC domain)

[0187] In some embodiments, the strand of a nucleic acid double-stranded target polynucleotide sequence cleaved by a base editor containing a nickase domain (e.g., a nickase domain from Cas9, a nickase domain from Cas12) is the strand that is not edited by the base editor (i.e., the strand cleaved by the base editor is opposite the strand containing the edited base). In other embodiments, a base editor containing a nickase domain (e.g., a nickase domain from Cas9, a nickase domain from Cas12) can cleave the strand of a DNA molecule that is targeted for editing. In such embodiments, the non-target strand is not cleaved.

[0188] In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, i.e., the Cas9 is a nickase referred to as a "nCas9" protein (for "nickase" Cas9). The Cas9 nickase can be a Cas9 protein that can cleave only one strand of a double-stranded nucleic acid molecule (e.g., a double-stranded DNA molecule). In some embodiments, the Cas9 nickase cleaves the target strand of the double-stranded nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is base-paired (complementary) to the gRNA (e.g., sgRNA) that binds to the Cas9. In some embodiments, the Cas9 nickase comprises a D10A mutation and has a histidine at position 840. In some embodiments, the Cas9 nickase cleaves the non-target, unbase-edited strand of the double-stranded nucleic acid molecule, meaning that the Cas9 nickase cleaves the strand that is not base-paired to the gRNA (e.g., sgRNA) that binds to the Cas9. In some embodiments, the Cas9 nickase comprises an H840A mutation and an aspartic acid residue at position 10, or a corresponding mutation. In some embodiments, the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 nickases provided herein. Additional suitable Cas9 nickases will be apparent to those of skill in the art based on this disclosure and knowledge in the art, and are within the scope of this disclosure.

[0189] The amino acid sequence of an exemplary catalytic Cas9 nickase (nCas9) is as follows:

[0190] The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Upon target binding, Cas9 undergoes a conformational change that positions the nuclease domains to cleave opposite strands of the target DNA. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (approximately 3–4 nucleotides upstream of the PAM sequence). The resulting DSB is then repaired by one of two general repair pathways: (1) the efficient but error-prone non-homologous end joining (NHEJ) pathway, or (2) the less efficient but high-fidelity homology-directed repair (HDR) pathway.

[0191] The "efficiency" of non-homologous end joining (NHEJ) and / or homology-directed repair (HDR) can be calculated in any convenient manner. For example, in some embodiments, efficiency can be expressed in terms of the percentage of successful HDR. For example, a surveyor nuclease assay can be used to generate cleavage products, and the ratio of product to substrate can be used to calculate the percentage. For example, a surveyor nuclease enzyme can be used to directly cleave DNA containing the newly incorporated restriction sequence as a result of successful HDR. More cleaved substrate indicates a higher percentage of HDR (higher HDR efficiency). As an illustrative example, the percentage of HDR can be calculated using the following formula: [(cleavage product) / (substrate + cleavage product)] (e.g., (b + c) / (a + b + c), where "a" is the band intensity of the DNA substrate and "b" and "c" are the cleavage products).

[0192] In some embodiments, efficiency can be expressed in terms of the percentage of successful NHEJ. For example, a T7 endonuclease I assay can be used to generate cleavage products, and the product-to-substrate ratio can be used to calculate the percentage of NHEJ. T7 endonuclease I cleaves mismatched heteroduplex DNA resulting from hybridization of wild-type and mutant DNA strands (NHEJ generates small random insertions or deletions (indels) at the original cleavage site). More cleavages indicate a higher percentage of NHEJ (more efficient NHEJ). As an illustrative example, the percentage of NHEJ can be calculated using the following formula: (1-(1-(b+c) / (a+b+c)) 1 / 2 ) × 100, where "a" is the band intensity of the DNA substrate, and "b" and "c" are the cleavage products (Ran et al., Cell. 2013 Sep. 12; 154 (6):1380-9, and Ran et al., Nat Protoc. 2013 Nov.; 8 (11): 2281-2308).

[0193] The NHEJ repair pathway is the most active repair mechanism, frequently causing small nucleotide insertions or deletions (indels) at DSB sites. The random nature of NHEJ-mediated DSB repair has practical implications, as a diverse range of mutations can be generated in cell populations expressing Cas9 and gRNA or guide polynucleotides. In most embodiments, NHEJ generates small indels within the target DNA, resulting in amino acid deletions, insertions, or frameshift mutations, resulting in premature stop codons within the open reading frame (ORF) of the target gene. The ideal end result is a loss-of-function mutation within the target gene.

[0194] While NHEJ-mediated DSB repair often disrupts the open reading frame of a gene, homology-directed repair (HDR) can be used to generate specific nucleotide changes, ranging from single nucleotide changes to larger insertions, such as the addition of fluorophores or tags.

[0195] To utilize HDR for gene editing, a DNA repair template containing the desired sequence can be delivered into a cell type of interest using gRNA(s) and Cas9 or Cas9 nickase. The repair template can contain not only the desired edit but also additional homologous sequences (called left and right homology arms) immediately upstream and downstream of the target. The length of each homology arm can depend on the size of the alteration to be introduced, with longer homology arms required for larger insertions. The repair template can be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid. HDR efficiency is generally low (<10% of modified alleles) even in cells expressing Cas9, gRNA, and an exogenous repair template. Because HDR occurs during the S and G2 phases of the cell cycle, HDR efficiency can be increased by synchronizing cells. Chemical or genetic inhibition of genes involved in NHEJ can increase the frequency of HDR.

[0196] In some embodiments, the Cas9 is a modified Cas9. A given gRNA targeting sequence may have additional sites of partial homology throughout the genome. These sites are called off-targets and must be considered when designing the gRNA. In addition to optimizing gRNA design, Cas9 can also be modified to enhance CRISPR specificity. Cas9 combines the activities of two nuclease domains, RuvC and HNH, to generate double-strand breaks (DSBs). The Cas9 nickase, a D10A variant of SpCas9, retains one nuclease domain and generates DNA nicks rather than DSBs. The nickase system can also be combined with HDR-mediated gene editing for specific gene editing.

[0197] Catalytically inactive nucleases Also provided herein are base editors comprising catalytically inactive (i.e., unable to cleave a target polynucleotide sequence) polynucleotide-programmable nucleotide-binding domains. As used herein, the terms "catalytically inactive" and "nuclease-inactive" are used interchangeably to refer to polynucleotide-programmable nucleotide-binding domains with one or more mutations and / or deletions that result in an inability to cleave a strand of nucleic acid. In some embodiments, catalytically inactive polynucleotide-programmable nucleotide-binding domain base editors may lack nuclease activity as a result of specific point mutations in one or more nuclease domains. For example, in the case of a base editor comprising a Cas9 domain, Cas9 may contain both the D10A and H840A mutations. Such mutations inactivate both nuclease domains, thereby resulting in loss of nuclease activity. In other embodiments, catalytically inactive polynucleotide-programmable nucleotide-binding domains may contain one or more deletions of all or part of a catalytic domain (e.g., the RuvC1 and / or HNH domains). In further embodiments, the catalytically inactive polynucleotide-programmable nucleotide-binding domain comprises a point mutation (e.g., D10A or H840A) and a deletion of all or part of the nuclease domain. dCas9 domains are known in the art and are described, for example, in Qi et al., "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression." Cell. 2013; 152 (5):1173-83, the entire contents of which are incorporated herein by reference.

[0198] Additional suitable nuclease-inactive dCas9 domains will be apparent to those of skill in the art based on this disclosure and knowledge in the art and are within the scope of this disclosure. Exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, the D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31 (9): 833-838, the entire contents of which are incorporated herein by reference).

[0199] In some embodiments, the dCas9 corresponds to, or partially or entirely comprises, a Cas9 amino acid sequence with one or more mutations that inactivate Cas9 nuclease activity. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10X mutation and a H840X mutation in the amino acid sequence described herein, or a corresponding mutation in any amino acid sequence provided herein, where X is any amino acid change. In some embodiments, the nuclease-inactive dCas9 domain comprises a D10A mutation and a H840A mutation in the amino acid sequence described herein, or a corresponding mutation in any amino acid sequence provided herein. In one embodiment, the nuclease-inactive Cas9 domain comprises the amino acid sequence described in the cloning vector pPlatTET-gRNA2 (accession number BAV54124).

[0200] In some embodiments, the variant Cas9 protein is capable of cleaving the complementary strand of a guide target sequence but has a reduced ability to cleave the non-complementary strand of a double-stranded guide target sequence. For example, the variant Cas9 protein may have a mutation (amino acid substitution) that reduces the function of the RuvC domain. As a non-limiting example, in some embodiments, the variant Cas9 protein has D10A (aspartic acid to alanine at amino acid position 10), and thus is capable of cleaving the complementary strand of a double-stranded guide target sequence but has a reduced ability to cleave the non-complementary strand of a double-stranded guide target sequence (thus generating a single-strand break (SSB) instead of a double-strand break (DSB) when the variant Cas9 protein cleaves a double-stranded target nucleic acid) (see, e.g., Jinek et al., Science. 2012 Aug. 17; 337 (6096):816-21).

[0201] In some embodiments, the variant Cas9 protein can cleave the non-complementary strand of a double-stranded guide target sequence, but has a reduced ability to cleave the complementary strand of the guide target sequence. For example, the variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC / HNH / RuvC domain motif). As a non-limiting example, in some embodiments, the variant Cas9 protein has an H840A (histidine to alanine at amino acid position 840) mutation, and thus can cleave the non-complementary strand of a guide target sequence, but has a reduced ability to cleave the complementary strand of a guide target sequence (thus generating an SSB rather than a DSB when the variant Cas9 protein cleaves a double-stranded guide target sequence). Such a Cas9 protein has a reduced ability to cleave a guide target sequence (e.g., a single-stranded guide target sequence), but retains the ability to bind to the guide target sequence (e.g., a single-stranded guide target sequence).

[0202] As another non-limiting example, in some embodiments, a variant Cas9 protein comprises a W476A and a W1126A mutation, thereby reducing the ability of the polypeptide to cleave target DNA. Such a Cas9 protein has a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retains the ability to bind to target DNA (e.g., single-stranded target DNA).

[0203] As another non-limiting example, in some embodiments, a variant Cas9 protein has P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, thereby reducing the ability of the polypeptide to cleave target DNA. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0204] As another non-limiting example, in some embodiments, a variant Cas9 protein has H840A, W476A, and W1126A mutations, thereby reducing the polypeptide's ability to cleave target DNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA). As another non-limiting example, in some embodiments, a variant Cas9 protein has H840A, D10A, W476A, and W1126A mutations, thereby reducing the polypeptide's ability to cleave target DNA. Such Cas9 proteins have reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA). In some embodiments, a variant Cas9 has a catalytic His residue restored at position 840 of the Cas9 HNH domain (A840H).

[0205] As another non-limiting example, in some embodiments, a variant Cas9 protein has H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, thereby reducing the ability of the polypeptide to cleave target DNA. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA). As another non-limiting example, in some embodiments, a variant Cas9 protein has D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, thereby reducing the ability of the polypeptide to cleave target DNA. Such Cas9 proteins have a reduced ability to cleave target DNA (e.g., single-stranded target DNA) but retain the ability to bind to target DNA (e.g., single-stranded target DNA). In some embodiments, when a variant Cas9 protein contains the W476A and W1126A mutations, or when a variant Cas9 protein contains the P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, the variant Cas9 protein does not efficiently bind to PAM sequences. Thus, in some such embodiments, when such a variant Cas9 protein is used in a binding method, the method does not require a PAM sequence. In other words, in some embodiments, when such a variant Cas9 protein is used in a binding method, the method can include a guide RNA, but can be performed without a PAM sequence (and binding specificity is therefore provided by the targeting segment of the guide RNA). Other residues can be mutated to achieve the above effects (i.e., to inactivate one or other nuclease moieties). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 can be modified (i.e., substituted). Mutations other than alanine substitutions are also suitable.

[0206] In some embodiments, variant Cas9 proteins with reduced catalytic activity (e.g., when the Cas9 protein has a mutation at D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987, e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A) can still bind to target DNA in a site-specific manner (because it is still guided to the target DNA sequence by the guide RNA), so long as the variant Cas9 protein retains the ability to interact with the guide RNA.

[0207] In some embodiments, the variant Cas protein can be spCas9, spCas9-VRQR, spCas9-VRER, xCas9 (sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9-LRVSQL.

[0208] In some embodiments, the Cas9 domain is a Cas9 domain from Staphylococcus aureus (SaCas9). In some embodiments, the SaCas9 domain is a nuclease-active SaCas9, a nuclease-inactive SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, the SaCas9 comprises an N579A mutation or a corresponding mutation in any of the amino acid sequences set forth in the Sequence Listing submitted herewith.

[0209] In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence having a non-canonical PAM. In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence having an NNGRRT or NNGRRV PAM sequence. In some embodiments, the SaCas9 domain comprises one or more of an E781X, N967X, and R1014X mutation, or a corresponding mutation in any of the amino acid sequences provided herein, where X is any amino acid. In some embodiments, the SaCas9 domain comprises one or more of an E781K, N967K, and R1014H mutation, or a corresponding mutation in any of the amino acid sequences provided herein. In some embodiments, the SaCas9 domain comprises an E781K, N967K, or R1014H mutation, or a corresponding mutation in any of the amino acid sequences provided herein.

[0210] In some embodiments, one of the Cas9 domains present in the fusion protein may be replaced with a DNA-binding protein domain that is programmable with a guide nucleotide sequence that does not require a PAM sequence. In some embodiments, the Cas9 is SaCas9. Residue A579 of SaCas9 can be mutated from N579 to generate SaCas9 nickase. Residues K781, K967, and H1014 can be mutated from E781, N967, and R1014 to generate SaKKH Cas9.

[0211] In some embodiments, a modified SpCas9 was used that contains the amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER) and has specificity for the modified PAM5'-NGC-3'.

[0212] Alternatives to S. pyogenes Cas9 include RNA-guided endonucleases of the Cpf1 family that exhibit cleavage activity in mammalian cells. CRISPR from Prevotella and Francisella 1 (CRISPR / Cpf1) is a DNA editing technology similar to the CRISPR / Cas9 system. Cpf1 is an RNA-guided endonuclease of a class II CRISPR / Cas system. This adaptive immune mechanism is observed in the bacteria Prevotella and Francisella. The Cpf1 gene is associated with the CRISPR locus and encodes an endonuclease that uses guide RNA to locate and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the limitations of the CRISPR / Cas9 system. Unlike Cas9 nuclease, Cpf1-mediated DNA cleavage results in double-strand breaks with short 3' overhangs. The staggered cleavage pattern of Cpf1 may open up the possibility of directed gene transfer, which could increase the efficiency of gene editing, similar to conventional restriction enzyme cloning. Like the Cas9 variants and orthologs mentioned above, Cpf1 can also expand the number of CRISPR targetable sites to AT-rich regions or AT-rich genomes lacking the NGG PAM site preferred by SpCas9. The Cpf1 locus contains a mixed α / β domain, RuvC-I, followed by a helical region, RuvC-II, and a zinc finger-like domain. The Cpf1 protein possesses a RuvC-like endonuclease domain similar to the RuvC domain of Cas9.

[0213] Furthermore, unlike Cas9, Cpf1 lacks the HNH endonuclease domain and the α-helical recognition lobe of Cas9 at its N-terminus. The Cpf1 CRISPR-Cas domain architecture indicates that Cpf1 is functionally unique and falls within the Class 2, Type V CRISPR system. The Cpf1 locus encodes Cas1, Cas2, and Cas4 proteins, which are more similar to Type I and Type III CRISPR systems than Type II systems. Functional Cpf1 does not require trans-activating CRISPR RNA (tracrRNA) and therefore requires only CRISPR (crRNA). Cpf1 is not only smaller than Cas9, but also possesses a smaller sgRNA molecule (approximately half the nucleotides of Cas9), which is advantageous for genome editing. The Cpf1-crRNA complex cleaves target DNA or RNA by recognizing the protospacer adjacent motif 5'-YTN-3' or 5'-TTN-3', in contrast to the G-rich PAM targeted by Cas9. After recognizing the PAM, Cpf1 introduces sticky-end-like DNA double-strand breaks with 4- or 5-nucleotide overhangs.

[0214] In some embodiments, the Cas9 is a Cas9 variant with specificity for an engineered PAM sequence. In some embodiments, additional Cas9 variants and PAM sequences are described in Miller, SM, et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs, Nat. Biotechnol. (2020), incorporated herein by reference in its entirety. In some embodiments, the Cas9 variant does not have a specific PAM requirement. In some embodiments, the Cas9 variant, e.g., an SpCas9 variant, has specificity for the NRNH PAM, where R is A or G and H is A, C, or T. In some embodiments, the SpCas9 variant has specificity for the PAM sequence AAA, TAA, CAA, GAA, TAT, GAT, or CAC. In some embodiments, SpCas9 variants comprise an amino acid substitution at, or a corresponding position to, 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1218, 1219, 1221, 1249, 1256, 1264, 1290, 1318, 1317, 1320, 1321, 1323, 1332, 1333, 1335, 1337, or 1339. In some embodiments, SpCas9 variants comprise an amino acid substitution at, or a corresponding position to, 1114, 1135, 1218, 1219, 1221, 1249, 1320, 1321, 1323, 1332, 1333, 1335, or 1337. In some embodiments, the SpCas9 variant comprises an amino acid substitution at positions 1114, 1134, 1135, 1137, 1139, 1151, 1180, 1188, 1211, 1219, 1221, 1256, 1264, 1290, 1318, 1317, 1320, 1323, 1333, or the corresponding positions.In some embodiments, SpCas9 variants comprise amino acid substitutions at positions 1114, 1131, 1135, 1150, 1156, 1180, 1191, 1218, 1219, 1221, 1227, 1249, 1253, 1286, 1293, 1320, 1321, 1332, 1335, 1339, or their corresponding positions. In some embodiments, SpCas9 variants comprise amino acid substitutions at positions 1114, 1127, 1135, 1180, 1207, 1219, 1234, 1286, 1301, 1332, 1335, 1337, 1338, 1349, or their corresponding positions. Exemplary amino acid substitutions and PAM specificities of SpCas9 variants are shown in Tables 2A-2D.

[0215] [Table 8]

[0216] [Table 9]

[0217] [Table 10]

[0218] [Table 11]

[0219] Additional exemplary Cas9 (e.g., SaCas9) polypeptides with modified PAM recognition are described in Kleinstiver, et al. "Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition," Nature Biotechnology, 33:1293-1298 (2015) DOI: 10.1038 / nbt.3404, the disclosure of which is incorporated herein by reference in its entirety for all purposes. In some embodiments, Cas9 variants (e.g., SaCas9 variants) comprising one or more of the modifications E782K, N929R, N968K, and / or R1015H are specific for or associated with increased editing activity at NNNRRT or NNHRRT PAM sequences compared to a reference polypeptide (e.g., SaCas9), where N represents any nucleotide, H represents any nucleotide other than G (i.e., "non-G"), and R represents a purine. In embodiments, the Cas9 variant (e.g., a SaCas9 variant) comprises the modifications E782K, N968K, and R1015H, or the modifications E782K, K929R, and R1015H.

[0220] In some embodiments, the nucleic acid programmable DNA-binding protein (napDNAbp) is the single effector of the microbial CRISPR-Cas system. Single effectors of the microbial CRISPR-Cas system include, but are not limited to, Cas9, Cpf1, Cas12b / C2c1, and Cas12c / C2c3. Microbial CRISPR-Cas systems are generally divided into class 1 and class 2 systems. Class 1 systems have a multi-subunit effector complex, while class 2 systems have a single protein effector. For example, Cas9 and Cpf1 are class 2 effectors. In addition to Cas9 and Cpf1, three distinct Class 2 CRISPR-Cas systems (Cas12b / C2c1 and Cas12c / C2c3) have been described by Shmakov et al., "Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems," Mol. Cell, 2015 Nov. 5; 60(3):385-397, the entire contents of which are incorporated herein by reference. Two of the effectors in the systems, Cas12b / C2c1 and Cas12c / C2c3, contain a RuvC-like endonuclease domain related to Cpf1. The third system contains an effector with two predicted HEPN RNase domains. Unlike CRISPR RNA production by Cas12b / C2c1, production of mature CRISPR RNA is independent of tracrRNA. Cas12b / C2c1 depends on both CRISPR RNA and tracrRNA for DNA cleavage.

[0221] In some embodiments, the napDNAbp is a circular permutation (eg, SEQ ID NO: 253).

[0222] The crystal structure of Alicyclobacillus acidoterrastris Cas12b / C2c1 (AacC2c1) complexed with a chimeric single-molecule guide RNA (sgRNA) has been reported. See, for example, Liu et al., "C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism," Mol. Cell, 2017 Jan. 19; 65 (2):310-322 (the entire contents of which are incorporated herein by reference). A crystal structure has also been reported for Alicyclobacillus acidoterrestris C2c1 bound to target DNA as a ternary complex. See, e.g., Yang et al., "PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease," Cell, 2016 Dec. 15; 167 (7):1814-1828, the entire contents of which are incorporated herein by reference. Both target and non-target DNA strands capture catalytically competent conformations of AacC2c1, which are independently positioned within a single RuvC catalytic pocket, resulting in a staggered seven-nucleotide cleavage of the target DNA upon Cas12b / C2c1-mediated cleavage. Structural comparison of the Cas12b / C2c1 ternary complex with previously identified Cas9 and Cpf1 counterparts demonstrates the diversity of mechanisms employed by the CRISPR-Cas9 system.

[0223] In some embodiments, the nucleic acid programmable DNA-binding protein (napDNAbp) of any of the fusion proteins provided herein can be a Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the napDNAbp is a Cas12b / C2c1 protein. In some embodiments, the napDNAbp is a Cas12c / C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the napDNAbp is a naturally occurring Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the napDNAbp sequences provided herein. It should be understood that Cas12b / C2c1 or Cas12c / C2c3 from other bacterial species may also be used in accordance with the present disclosure.

[0224] In some embodiments, napDNAbp refers to Cas12c. In some embodiments, the Cas12c protein is Cas12c1 (SEQ ID NO: 254) or a variant of Cas12c1. In some embodiments, the Cas12 protein is Cas12c2 (SEQ ID NO: 255) or a variant of Cas12c2. In some embodiments, the Cas12 protein is a Cas12c protein from Oleiphilus species HI0009 (i.e., OspCas12c, SEQ ID NO: 256) or a variant of OspCas12c. These Cas12c molecules are described in Yan et al., "Functionally Diverse Type V CRISPR-Cas Systems," Science, 2019 Jan. 4; 363: 88-91, the entire contents of which are incorporated herein by reference. In some embodiments, the napDNAbp comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring Cas12c1, Cas12c2, or OspCas12c protein. In some embodiments, the napDNAbp is a naturally occurring Cas12c1, Cas12c2, or OspCas12c protein. In some embodiments, the napDNAbp comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any Cas12c1, Cas12c2, or OspCas12c protein described herein. It should be understood that Cas12c1, Cas12c2, or OspCas12c from other bacterial species may also be used in accordance with the present disclosure.

[0225] In some embodiments, napDNAbp refers to Cas12g, Cas12h, or Cas12i, e.g., as described in Yan et al., "Functionally Diverse Type V CRISPR-Cas Systems," Science, 2019 Jan. 4; 363: 88-91, the entire contents of each of which are incorporated herein by reference. Exemplary Cas12g, Cas12h, and Cas12i polypeptide sequences are set forth in the Sequence Listing as SEQ ID NOs: 257-260. By aggregating over 10 terabytes of sequence data, new classes of type V Cas proteins, such as Cas12g, Cas12h, and Cas12i, have been identified that show weak similarity to previously characterized class V proteins. In some embodiments, the Cas12 protein is Cas12g or a variant of Cas12g. In some embodiments, the Cas12 protein is Cas12h or a variant of Cas12h. In some embodiments, the Cas12 protein is Cas12i or a variant of Cas12i. It should be understood that other RNA-guided DNA-binding proteins may also be used as napDNAbp and are within the scope of the present disclosure. In some embodiments, the napDNAbp comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring Cas12g, Cas12h, or Cas12i protein. In some embodiments, the napDNAbp is a naturally occurring Cas12g, Cas12h, or Cas12i protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any Cas12g, Cas12h, or Cas12i protein described herein.It should be understood that Cas12g, Cas12h, or Cas12i from other bacterial species may also be used in accordance with the present disclosure. In some embodiments, the Cas12i is Cas12i1 or Cas12i2.

[0226] In some embodiments, the nucleic acid-programmable DNA-binding protein (napDNAbp) of any of the fusion proteins provided herein can be a Cas12j / CasΦ protein. Cas12j / CasΦ is described in Pausch et al., "CRISPR-CasΦ from huge phages is a hypercompact genome editor," Science, July 17, 2020, Vol. 369, Issue 6501, pp. 333-337, which is incorporated herein by reference in its entirety. In some embodiments, the napDNAbp comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a native Cas12j / CasΦ protein. In some embodiments, the napDNAbp is a native Cas12j / CasΦ protein. In some embodiments, the napDNAbp is a nuclease-inactive ("dead") Cas12j / CasΦ protein. It should be understood that Cas12j / CasΦ from other species may also be used in accordance with the present disclosure.

[0227] Fusion proteins with internal insertions Provided herein are nucleic acid programmable nucleic acid binding proteins, e.g., fusion proteins comprising a heterologous polypeptide fused to a napDNAbp. The heterologous polypeptide can be a polypeptide not found in the native, i.e., wild-type, napDNAbp polypeptide sequence. The heterologous polypeptide can be fused to the napDNAbp at the C-terminus of the napDNAbp, the N-terminus of the napDNAbp, or inserted at an internal position of the napDNAbp. In some embodiments, the heterologous polypeptide is a deaminase (e.g., an adenosine deaminase variant) or a functional fragment thereof. For example, the fusion protein can include a deaminase flanked by N- and C-terminal fragments of a Cas9 or Cas12 (e.g., Cas12b / C2c1) polypeptide. In some embodiments, the adenosine deaminase variant is a TadA variant (e.g., a TadA*8 variant). In some embodiments, TadA is a TadA*8 variant. In some embodiments, TadA*8 is TadA*8.20, which contains one or more modifications that increase cytidine deamination activity. The TadA sequences described herein (e.g., TadA*8) are suitable deaminases for the fusion proteins described above.

[0228] In some embodiments, the fusion protein has the following structure: NH2-[N-terminal fragment of napDNAbp]-[deaminase]-[C-terminal fragment of napDNAbp]-COOH, NH2-[N-terminal fragment of Cas9]-[adenosine deaminase]-[C-terminal fragment of Cas9]-COOH, NH2-[N-terminal fragment of Cas12]-[adenosine deaminase]-[C-terminal fragment of Cas12]-COOH, where each instance of "]-[" is an optional linker.

[0229] The deaminase can be a circularly substituted deaminase. For example, the deaminase can be a circularly substituted adenosine deaminase. In some embodiments, the deaminase is a circularly substituted TadA that is circularly substituted at amino acid residue 116, 136, or 65 as numbered in the TadA reference sequence.

[0230] The fusion protein may contain multiple deaminases. The fusion protein may contain, for example, 1, 2, 3, 4, 5, or more deaminases. In some embodiments, the fusion protein contains one or two deaminases. The two or more deaminases may be homodimers or heterodimers. The two or more deaminases may be inserted in tandem into the napDNAbp. In some embodiments, the two or more deaminases may not be present in tandem in the napDNAbp.

[0231] In some embodiments, the napDNAbp in the fusion protein is a Cas9 polypeptide or a fragment thereof. The Cas9 polypeptide can be a variant Cas9 polypeptide. In some embodiments, the Cas9 polypeptide is a Cas9 nickase (nCas9) polypeptide or a fragment thereof. In some embodiments, the Cas9 polypeptide is a nuclease-inactive Cas9 (dCas9) polypeptide or a fragment thereof. The Cas9 polypeptide in the fusion protein can be a full-length Cas9 polypeptide. In some cases, the Cas9 polypeptide in the fusion protein may not be a full-length Cas9 polypeptide. The Cas9 polypeptide can be truncated, for example, at the N-terminus or C-terminus compared to a native Cas9 protein. The Cas9 polypeptide can be a circularly permuted Cas9 protein. The Cas9 polypeptide can be a fragment, portion, or domain of a Cas9 polypeptide, but it can still bind to a target polynucleotide and a guide nucleic acid sequence.

[0232] In some embodiments, the Cas9 polypeptide is Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), or a fragment or variant of any of the Cas9 polypeptides described herein.

[0233] In various embodiments, the catalytic domain has a DNA-modifying activity (e.g., deaminase activity), such as adenosine deaminase and / or cytosine deaminase activity. In various embodiments, the catalytic domain has both adenosine deaminase activity and cytosine deaminase activity. In various embodiments, the domain of the adenosine deaminase variant contains one or more modifications that increase cytosine deaminase activity.

[0234] A heterologous polypeptide (e.g., a deaminase) can be inserted into the napDNAbp (e.g., Cas9 or Cas12 (e.g., Cas12b / C2c1)) at an appropriate position, e.g., such that the napDNAbp retains the ability to bind to a target polynucleotide and a guide nucleic acid. A deaminase (e.g., an adenosine deaminase variant) can be inserted into the napDNAbp without impairing the function of the deaminase (e.g., base editing activity) or the function of the napDNAbp (e.g., the ability to bind to a target nucleic acid and a guide nucleic acid). A deaminase (e.g., an adenosine deaminase variant) can be inserted into the napDNAbp, e.g., in a denatured region or a region containing a high temperature factor or B factor, as shown in crystallographic studies. Regions of less ordered, denatured, or amorphous proteins, e.g., solvent-exposed regions and loops, can be used for insertion without impairing structure or function. The deaminase (e.g., adenosine deaminase variant) can be inserted into the napDNAbp in a flexible loop region or a solvent-exposed region. In some embodiments, the deaminase (e.g., adenosine deaminase variant) is inserted into a flexible loop of the Cas9 or Cas12b / C2c1 polypeptide.

[0235] In some embodiments, the insertion location of the deaminase (e.g., adenosine deaminase variant) is determined by B-factor analysis of the crystal structure of the Cas9 polypeptide. In some embodiments, the deaminase (e.g., adenosine deaminase variant) is inserted into a region of the Cas9 polypeptide with a higher-than-average B-factor (e.g., a higher B-factor compared to the whole protein or a protein domain including the denatured region). The B-factor or temperature factor can indicate variation of atoms from their average positions (e.g., as a result of temperature-dependent atomic vibrations or static disorder in the crystal lattice). A high B-factor (e.g., a higher-than-average B-factor) of backbone atoms can indicate a region with relatively high local mobility. Such a region can be used to insert the deaminase without compromising structure or function. The deaminase (e.g., an adenosine deaminase variant) can be inserted at a position having a residue with a Cα atom that has a B factor that is 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more than 200% higher than the average B factor for the entire protein. A deaminase (e.g., an adenosine deaminase variant) can be inserted at a position having a residue with a Cα atom that has a B-factor that is 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more than 200% higher than the average B-factor for the Cas9 protein domain containing that residue. Positions in a Cas9 polypeptide that have higher-than-average B-factors can include, for example, residues 768, 792, 1052, 1015, 1022, 1026, 1029, 1067, 1040, 1054, 1068, 1246, 1247, and 1248, as numbered in the above Cas9 reference sequence. Regions of the Cas9 polypeptide having higher than average B factors can include, for example, residues 792-872, 792-906, and 2-791, numbered in the above Cas9 reference sequence.

[0236] A heterologous polypeptide (e.g., a deaminase) can be inserted into the napDNAbp at an amino acid residue selected from the group consisting of: 768, 791, 792, 1015, 1016, 1022, 1023, 1026, 1029, 1040, 1052, 1054, 1067, 1068, 1069, 1246, 1247, and 1248, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the heterologous polypeptide is inserted between amino acid positions 768-769, 791-792, 792-793, 1015-1016, 1022-1023, 1026-1027, 1029-1030, 1040-1041, 1052-1053, 1054-1055, 1067-1068, 1068-1069, 1247-1248, or 1248-1249, or their corresponding amino acid positions, as numbered in the above Cas9 reference sequence. In some embodiments, the heterologous polypeptide is inserted between amino acid positions 769-770, 792-793, 793-794, 1016-1017, 1023-1024, 1027-1028, 1030-1031, 1041-1042, 1053-1054, 1055-1056, 1068-1069, 1069-1070, 1248-1249, or 1249-1250, or their corresponding amino acid positions, as numbered in the above Cas9 reference sequence. In some embodiments, the heterologous polypeptide replaces an amino acid residue selected from the group consisting of: 768, 791, 792, 1015, 1016, 1022, 1023, 1026, 1029, 1040, 1052, 1054, 1067, 1068, 1069, 1246, 1247, and 1248, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. It should be understood that reference to the above Cas9 reference sequence for insertion positions is for illustrative purposes.Insertions discussed herein are not limited to the Cas9 polypeptide sequences of the above Cas9 reference sequences, but include insertions at the corresponding positions of variant Cas9 polypeptides, such as Cas9 nickase (nCas9), nuclease-inactive Cas9 (dCas9), nuclease domain-deleted Cas9 variants, truncated Cas9, or Cas9 domains partially or completely lacking the HNH domain.

[0237] A heterologous polypeptide (e.g., an adenosine deaminase variant) can be inserted into the napDNAbp at an amino acid residue selected from the group consisting of: 768, 792, 1022, 1026, 1040, 1068, and 1247, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the heterologous polypeptide is inserted between amino acid positions 768-769, 792-793, 1022-1023, 1026-1027, 1029-1030, 1040-1041, 1068-1069, or 1247-1248, as numbered in the above Cas9 reference sequence, or their corresponding amino acid positions. In some embodiments, the heterologous polypeptide is inserted between amino acid positions 769-770, 793-794, 1023-1024, 1027-1028, 1030-1031, 1041-1042, 1069-1070, or 1248-1249, as numbered in the above Cas9 reference sequence, or their corresponding amino acid positions. In some embodiments, the heterologous polypeptide replaces an amino acid residue selected from the group consisting of: 768, 792, 1022, 1026, 1040, 1068, and 1247, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide.

[0238] A heterologous polypeptide (e.g., an adenosine deaminase variant) can be inserted into the napDNAbp at an amino acid residue described herein, or the corresponding amino acid residue of another Cas9 polypeptide. In one embodiment, the heterologous polypeptide (e.g., a deaminase) can be inserted into the napDNAbp at an amino acid residue selected from the group consisting of: 1002, 1003, 1025, 1052-1056, 1242-1247, 1061-1077, 943-947, 686-691, 569-578, 530-539, and 1060-1077, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue of another Cas9 polypeptide. The deaminase (e.g., an adenosine deaminase variant) can be inserted N-terminally or C-terminally to, or replace, the residue. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted at the C-terminus of the residue.

[0239] In some embodiments, an adenosine deaminase variant (e.g., a TadA variant) is inserted at an amino acid residue selected from the group consisting of: 1015, 1022, 1029, 1040, 1068, 1247, 1054, 1026, 768, 1067, 1248, 1052, and 1246 in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, an adenosine deaminase variant (e.g., a TadA variant) is inserted at residues 792-872, 792-906, or 2-791 in the above Cas9 reference sequence, or in place of the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the adenosine deaminase variant is inserted N-terminally to an amino acid selected from the group consisting of: 1015, 1022, 1029, 1040, 1068, 1247, 1054, 1026, 768, 1067, 1248, 1052, and 1246 in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the adenosine deaminase variant is inserted C-terminally to an amino acid selected from the group consisting of: 1015, 1022, 1029, 1040, 1068, 1247, 1054, 1026, 768, 1067, 1248, 1052, and 1246 in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the adenosine deaminase variant is inserted to replace an amino acid selected from the group consisting of: 1015, 1022, 1029, 1040, 1068, 1247, 1054, 1026, 768, 1067, 1248, 1052, and 1246 in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide.

[0240] In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted at amino acid residue 768, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted N-terminal to amino acid residue 768, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted C-terminal to amino acid residue 768, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted to replace amino acid residue 768, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide.

[0241] In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted at amino acid residue 791 or amino acid residue 792, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted N-terminally to amino acid residue 791, or N-terminally to amino acid residue 792, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted C-terminally to amino acid 791, or N-terminally to amino acid 792, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted to replace amino acid 791, or to replace amino acid 792, as numbered in the above Cas9 reference sequence, or to replace the corresponding amino acid residue in another Cas9 polypeptide.

[0242] In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted at amino acid residue 1016, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted N-terminal to amino acid residue 1016, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted C-terminal to amino acid residue 1016, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted to replace amino acid residue 1016, as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide.

[0243] In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted at amino acid residue 1022 or 1023, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted N-terminally to amino acid residue 1022 or 1023, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted C-terminally to amino acid residue 1022 or 1023, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted to replace amino acid residue 1022, or to replace amino acid residue 1023, as numbered in the above Cas9 reference sequence, or to replace the corresponding amino acid residue in another Cas9 polypeptide.

[0244] In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted at amino acid residue 1026 or at amino acid residue 1029, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted N-terminally to amino acid residue 1026 or at amino acid residue 1029, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted C-terminally to amino acid residue 1026 or at amino acid residue 1029, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted to replace amino acid residue 1026, or to replace amino acid residue 1029, as numbered in the above Cas9 reference sequence, or to replace the corresponding amino acid residue in another Cas9 polypeptide.

[0245] In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted at amino acid residue 1040 as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted N-terminal to amino acid residue 1040 as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted C-terminal to amino acid residue 1040 as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted to replace amino acid residue 1040 as numbered in the above Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide.

[0246] In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted at amino acid residue 1052 or 1054, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted N-terminally to amino acid residue 1052 or to amino acid residue 1054, as numbered in the Cas9 reference sequence above, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted C-terminally to amino acid residue 1052 or to amino acid residue 1054, as numbered in the Cas9 reference sequence above, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted at and replaces amino acid residue 1052 or to and replaces amino acid residue 1054, as numbered in the Cas9 reference sequence above, or at and replaces the corresponding amino acid residue in another Cas9 polypeptide.

[0247] In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted at amino acid residue 1067, or at amino acid residue 1068, or at amino acid residue 1069, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted N-terminal to amino acid residue 1067, or at amino acid residue 1068, or at amino acid residue 1069, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted C-terminal to amino acid residue 1067, or to amino acid residue 1068, or to amino acid residue 1069, as numbered in the above Cas9 reference sequence, or to the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted to replace amino acid residue 1067, or to replace amino acid residue 1068, or to replace amino acid residue 1069, as numbered in the above Cas9 reference sequence, or to the corresponding amino acid residue in another Cas9 polypeptide.

[0248] In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted at amino acid residue 1246, or at amino acid residue 1247, or at amino acid residue 1248, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted N-terminal to amino acid residue 1246, or at amino acid residue 1247, or at amino acid residue 1248, as numbered in the above Cas9 reference sequence, or at the corresponding amino acid residue in another Cas9 polypeptide. In some embodiments, the deaminase (e.g., an adenosine deaminase variant) is inserted C-terminal to amino acid residue 1246, or to amino acid residue 1247, or to amino acid residue 1248, as numbered in the above Cas9 reference sequence, or to the corresponding amino acid residue in another Cas9 polypeptide. In some embodime...

Claims

1. An adenosine deaminase variant having increased cytidine deaminase activity and / or increased cytidine deaminase specificity compared to a reference adenosine deaminase, wherein the adenosine deaminase variant comprises two or more amino acid modifications compared to the reference adenosine deaminase.

2. The adenosine deaminase variant comprising the modification has the following amino acid sequence: The adenosine deaminase variant of claim 1, having at least about 85% or more amino acid sequence identity to the adenosine deaminase variant of claim 1.

3. a) the two or more modifications are present at amino acid positions selected from the group consisting of 2, 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167 of an amino acid sequence having at least about 85% amino acid sequence identity to SEQ ID NO:1, or at corresponding amino acid positions in another adenosine deaminase; and / or b) the two or more modifications are selected from the group consisting of S2X, V4X, F6X, H8X, R13X, T17X, ​​R23X, E27X, P29X, V30X, R47X, A48X, I49X, G67X, Y76X, D77X, S82X, F84X, H96X, G100X, R107X, G112X, A114X, G115X, M118X, D119X, H122X, N127X, A142X, and A143X, which have an amino acid sequence identity of at least about 85% to SEQ ID NO:1; and R147X, Y147X, F149X, A158X, Q159X, A162X, S165X, T166X, and D167X, or a corresponding amino acid position in another adenosine deaminase, where X represents the presence of any amino acid, and preferably the two or more modifications are selected from the group consisting of S2H, V4K, V4S, V4T, V4Y, F6G, F6H, F6Y, H8Q, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R25, R36, R47, R5, R6, R7, R8, R15, R16, R27, R17, R28, R29, R30, R31, R32, R33, R34, R35, R36, R47, R49, R50, R51, R52, R53, R54, R55, R56, R57, R58, R59, R60, R61, R62, R63, R64, R65, R66, R67, R68, R69, R71, R72, R73, R74, R75, R76, R77, R78, R79, R79, R78, R79, R79, R71, R72, R75, R76, R77, R78, R79 ... 3G, T17A, T17W, R23Q, E27C, E27G, E27H, E27K, E27Q, E27S, E27G, P29A, P29G, P29K, V30F, V30I, V30L, R47G, R47S, A48G, I49K, I4 9M, I49N, I49Q, I49T, G67W, I76H, I76R, I76W, I76Y, Y76H, Y76I, Y76R, Y76W, D77G, S82T, F84A, F84L, F84M, H96N, G100A, G100K, R 107C, T111H, G112H, A114C, G115M, M118L, D119N, H122G, H122N, H122R, H122T, N127I, N127K, N127P, A142E, A143E, R147H, Y147D, F149Y, A158V, Q159S, A162C, A162N, A162Q, S165P, T166I and D167N, or the corresponding amino acid positions in another adenosine deaminase, and / or d) the two or more modifications are selected from those listed in any of Tables 1A-1F; 3. The adenosine deaminase variant according to claim 1 or 2.

4. a) the two or more modifications do not include modifications at amino acid positions selected from the group consisting of 30, 47-49, 82-84, 107-111, 139, 142, 143, 146-149, 151-161, 166, and 167; and / or b) the two or more modifications are V30I, V30L, V30, R47F, R47M, R47Q, R47W, P48A, P48D, P48E, P48H, P48K, P48L, P48R, P48S, P48T, I49V, V82G, V82S, V82T, L84F, L84I, R107A, R1 07C, R107H, R107K, R107N, R107P, D108A, D108E, D108F, D108G, D108I, D108K, D 108L, D108M, D108N, D108Q, D108S, D108V, D108W, D108Y, A109S, K110I, T111R, D139L, D139M, A142N, A143D, A143E, A143G, A143L, S146C, S146R, S146T, D147A , D147R, D147T, D147Y, F148A, F149A, F149N, F149Y, M151V, R152H, R152P, R153 C, Q154H, Q154L, Q154R, Q154S, E155D, E155G, E155V, I156D, I156F, I156Y, K157N, A158K, Q159L, K160E, K161T, T166I, T166R, and D167N, The adenosine deaminase variant of claim 1.

5. The two or more modifications are of an amino acid sequence having at least about 85% or more amino acid sequence identity to SEQ ID NO:1: a first modification at amino acid position 2 and one or more additional modifications at amino acid positions selected from the group consisting of 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167; a first modification at amino acid position 4 and one or more additional modifications at amino acid positions selected from the group consisting of 2, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167; a first modification at amino acid position 6 and one or more additional modifications at amino acid positions selected from the group consisting of 2, 4, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167; a first modification at amino acid position 13 and one or more additional modifications at amino acid positions selected from the group consisting of 2, 4, 6, 8, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167; a first modification at amino acid position 27 and one or more additional modifications at amino acid positions selected from the group consisting of 2, 4, 6, 8, 13, 17, 23, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167; a first modification at amino acid position 29 and one or more additional modifications at amino acid positions selected from the group consisting of 2, 4, 6, 8, 13, 17, 23, 27, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167; a first modification at amino acid position 100 and one or more additional modifications at amino acid positions selected from the group consisting of 2, 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167; a first modification at amino acid position 112 and one or more additional modifications at amino acid positions selected from the group consisting of 2, 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167; a first modification at amino acid position 114 and one or more additional modifications at amino acid positions selected from the group consisting of 2, 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167; a first modification at amino acid position 115 and one or more additional modifications at amino acid positions selected from the group consisting of 2, 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167; a first modification at amino acid position 162 and one or more additional modifications at amino acid positions selected from the group consisting of 2, 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 165, 166, and 167; or a first modification at amino acid position 165 and one or more additional modifications at amino acid positions selected from the group consisting of 2, 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 166, and 167; The adenosine deaminase variant according to any one of claims 1 to 4, wherein the amino acid is located at an amino acid position selected from the group consisting of:

6. a) the two or more modifications are an amino acid sequence having at least about 85% or more identity to SEQ ID NO:1 E27H, Y76I, and F84M, E27H, I49K, and Y76I, E27S, I49K, and Y76I, E27S, I49K, Y76I, and A162N, E27K and D119N, E27H and Y76I, E27S, I49K, and G67W, I49T, G67W, and H96N, E27C, Y76I, and D119N, R13G, E27Q, and N127K, T17A, E27H, I49M, Y76I, and M118L, I49Q, Y76I, and G115M, S2H, I49K, Y76I, and G112H, R47S and R107C, H8Q, I49Q, and Y76I, T17A, A48G, S82T, and A142E, E27G and I49N, E27G, D77G, and S165P, E27S, I49K, and S82T, E27S, I49K, S82T, and G115M, E27S, V30I, I49K, and S82T, E27S, V30F, I49K, S82T, F84A, R107C, and A142E, E27S, V30F, I49K, S82T, F84A, G112H, and A142E, E27S, V30F, I49K, S82T, F84A, G115M, and A142E, E27S, I49K, S82T, F84L, and R107C, E27S, I49K, S82T, F84L, and G112H, E27S, I49K, S82T, F84L, and G115M, E27S, I49K, S82T, F84L, R107C, and G112H, E27S, I49K, S82T, F84L, R107C, and G115M, E27S, I49K, S82T, F84L, R107C, and A142E, E27S, I49K, S82T, F84L, G112H, and A142E, E27S, I49K, S82T, F84L, G115M, and A142E, E27S, I49K, S82T, F84L, R107C, G112H, G115M, and A142E, E27S, V30I, I49K, S82T, and F84L, E27S, P29G, I49K, and S82T, E27S, P29G, I49K, S82T, and G115M, E27S, P29G, I49K, S82T, and A142E, P29G, I49K, and S82T, E27G, I49K, and S82T, E27G, I49K, S82T, R107C, and A142E, V4K, E27H, I49K, Y76I, and A114C, V4K, E27H, I49K, Y76I, and D77G, F6Y, E27H, I49K, Y76I, G100A, and H122R, V4T, E27H, I49K, Y76R, and H122G, F6Y, E27H, I49K, and Y76W, F6Y, E27H, I49K, Y76I, and D119N, F6Y, E27H, I49K, Y76I, and A114C, F6Y, E27H, I49K, and Y76I, V4K, E27H, I49K, Y76W, and H122T, F6G, E27H, I49K, Y76R, and G100K, F6H, E27H, I49K, Y76I, and H122N, E27H, I49K, Y76I, and A114C, F6Y, E27H, I49K, Y76H, H122R, and T166I, E27H, I49K, Y76I, and N127P, R23Q, E27H, I49K, and Y76R, E27H, I49K, Y76H, H122R, and A158V, F6Y, E27H, I49K, Y76I, and T111H, E27H, I49K, Y76I, and R147H, E27H, I49K, Y76I, and A143E, F6Y, E27H, I49K, and Y76R, T17W, E27H, I49K, Y76H, H122G, and A158V, V4S, E27H, I49K, A143E, and Q159S, E27H, I49K, Y76I, N127I, and A162Q, T17A, E27H, and A48G, T17A, E27K, and A48G, T17A, E27S, and A48G, T17A, E27S, A48G, and I49K, T17A, E27G, and A48G, T17A, A48G, and I49N, T17A, E27G, A48G, and I49N, T17A, E27Q, and A48G, E27S, I49K, S82T, and R107C, E27S, I49K, S82T, and G112H, E27S, I49K, S82T, and A142E, E27S, I49K, S82T, R107C, and G112H, E27S, I49K, S82T, R107C, and G115M, E27S, I49K, S82T, R107C, and A142E, E27S, I49K, S82T, G112H, and A142E, E27S, I49K, S82T, G115M, and A142E, E27S, I49K, S82T, R107C, G112H, G115M, and A142E, E27S, V30I, I49K, S82T, and R107C, E27S, V30I, I49K, S82T, and G112H, E27S, V30I, I49K, S82T, and G115M, E27S, V30I, I49K, S82T, and A142E, E27S, V30I, I49K, S82T, R107C, and G112H, E27S, V30I, I49K, S82T, R107C, and G115M, E27S, V30I, I49K, S82T, R107C, and A142E, E27S, V30I, I49K, S82T, G112H, and A142E, E27S, V30I, I49K, S82T, G115M, and A142E, E27S, V30I, I49K, S82T, R107C, G112H, G115M, and A142E, E27S, V30L, I49K, and S82T, E27S, V30L, I49K, S82T, and R107C, E27S, V30L, I49K, S82T, and G112H, E27S, V30L, I49K, S82T, and G115M, E27S, V30L, I49K, S82T, and A142E, E27S, V30L, I49K, S82T, R107C, and G112H, E27S, V30L, I49K, S82T, R107C, and G115M, E27S, V30L, I49K, S82T, R107C, and A142E, E27S, V30L, I49K, S82T, G112H, and A142E, E27S, V30L, I49K, S82T, G115M, and A142E, E27S, V30L, I49K, S82T, R107C, G112H, G115M, and A142E, E27S, V30F, I49K, S82T, and F84A, E27S, V30F, I49K, S82T, F84A, and R107C, E27S, V30F, I49K, S82T, F84A, and G112H, E27S, V30F, I49K, S82T, F84A, and G115M, E27S, V30F, I49K, S82T, F84A, and A142E, E27S, V30F, I49K, S82T, F84A, R107C, and G112H, E27S, V30F, I49K, S82T, F84A, R107C, and G115M, E27S, V30F, I49K, S82T, F84A, R107C, G112H, G115M, and A142E, E27S, I49K, S82T, and F84L, E27S, I49K, S82T, F84L, and A142E, E27S, V30I, I49K, S82T, F84L, and R107C, E27S, V30I, I49K, S82T, F84L, and G112H, E27S, V30I, I49K, S82T, F84L, and G115M, E27S, V30I, I49K, S82T, F84L, and A142E, E27S, V30I, I49K, S82T, F84L, R107C, and G112H, E27S, V30I, I49K, S82T, F84L, R107C, and G115M, E27S, V30I, I49K, S82T, F84L, R107C, and A142E, E27S, V30I, I49K, S82T, F84L, G112H, and A142E, E27S, V30I, I49K, S82T, F84L, G115M, and A142E, E27S, V30I, I49K, S82T, F84L, R107C, G112H, G115M, and A142E, E27S, P29G, I49K, S82T, and R107C, E27S, P29G, I49K, S82T, and G112H, E27S, P29G, I49K, S82T, R107C, and G112H, E27S, P29G, I49K, S82T, R107C, and G115M, E27S, P29G, I49K, S82T, R107C, and A142E, E27S, P29G, I49K, S82T, G112H, and A142E, E27S, P29G, I49K, S82T, G115M, and A142E, E27S, P29G, I49K, S82T, R107C, G112H, G115M, and A142E, P29G, I49K, S82T, and R107C, P29G, I49K, S82T, and G112H, P29G, I49K, S82T, and G115M, P29G, I49K, S82T, and A142E, P29G, I49K, S82T, R107C, and G112H, P29G, I49K, S82T, R107C, and G115M, P29G, I49K, S82T, R107C, and A142E, P29G, I49K, S82T, G112H, and A142E, P29G, I49K, S82T, G115M, and A142E, P29G, I49K, S82T, R107C, G112H, G115M, and A142E, P29K, I49K, and S82T, P29K, I49K, S82T, and R107C, P29K, I49K, S82T, and G112H, P29K, I49K, S82T, and G115M, P29K, I49K, S82T, and A142E, P29K, I49K, S82T, R107C, and G112H, P29K, I49K, S82T, R107C, and G115M, P29K, I49K, S82T, R107C, and A142E, P29K, I49K, S82T, G112H, and A142E, P29K, I49K, S82T, G115M, and A142E, P29K, I49K, S82T, R107C, G112H, G115M, and A142E, P29K, V30I, I49K, and S82T, P29K, V30I, I49K, S82T, and R107C, P29K, V30I, I49K, S82T, and G112H, P29K, V30I, I49K, S82T, and G115M, P29K, V30I, I49K, S82T, and A142E, P29K, V30I, I49K, S82T, R107C, and G112H, P29K, V30I, I49K, S82T, R107C, and G115M, P29K, V30I, I49K, S82T, R107C, and A142E, P29K, V30I, I49K, S82T, G112H, and A142E, P29K, V30I, I49K, S82T, G115M, and A142E, P29K, V30I, I49K, S82T, R107C, G112H, G115M, and A142E, P29K, I49K, S82T, and F84L, P29K, I49K, S82T, F84L, and R107C, P29K, I49K, S82T, F84L, and G112H, P29K, I49K, S82T, F84L, and G115M, P29K, I49K, S82T, F84L, and A142E, P29K, I49K, S82T, F84L, R107C, and G112H, P29K, I49K, S82T, F84L, R107C, and G115M, P29K, I49K, S82T, F84L, R107C, and A142E, P29K, I49K, S82T, F84L, G112H, and A142E, P29K, I49K, S82T, F84L, G115M, and A142E, P29K, I49K, S82T, F84L, R107C, G112H, G115M, and A142E, P29K, V30I, I49K, S82T, and F84L, P29K, V30I, I49K, S82T, F84L, and R107C, P29K, V30I, I49K, S82T, F84L, and G112H, P29K, V30I, I49K, S82T, F84L, and G115M, P29K, V30I, I49K, S82T, F84L, and A142E, P29K, V30I, I49K, S82T, F84L, R107C, and G112H, P29K, V30I, I49K, S82T, F84L, R107C, and G115M, P29K, V30I, I49K, S82T, F84L, R107C, and A142E, P29K, V30I, I49K, S82T, F84L, G112H, and A142E, P29K, V30I, I49K, S82T, F84L, G115M, and A142E, P29K, V30I, I49K, S82T, F84L, R107C, G112H, G115M, and A142E, E27G, I49K, S82T, and R107C, E27G, I49K, S82T, and G112H, E27G, I49K, S82T, and G115M, E27G, I49K, S82T, and A142E, E27G, I49K, S82T, R107C, and G112H, E27G, I49K, S82T, R107C, and G115M, E27G, I49K, S82T, G112H, and A142E, E27G, I49K, S82T, G115M, and A142E, E27G, I49K, S82T, R107C, G112H, G115M, and A142E, E27H, I49K, and S82T, E27H, I49K, S82T, and R107C, E27H, I49K, S82T, and G112H, E27H, I49K, S82T, and G115M, E27H, I49K, S82T, and A142E, E27H, I49K, S82T, R107C, and G112H, E27H, I49K, S82T, R107C, and G115M, E27H, I49K, S82T, R107C, and A142E, E27H, I49K, S82T, G112H, and A142E, E27H, I49K, S82T, G115M, and A142E, E27H, I49K, S82T, R107C, G112H, G115M, and A142E, E27S and S82T, E27S, S82T, and R107C, E27S, S82T, and G112H, E27S, S82T, and G115M, E27S, S82T, and A142E, E27S, S82T, R107C, and G112H, E27S, S82T, R107C, and G115M, E27S, S82T, R107C, and A142E, E27S, S82T, G112H, and A142E, E27S, S82T, G115M, and A142E, E27S, S82T, R107C, G112H, G115M, and A142E, P29A, and S82T, P29A, S82T, and R107C, P29A, S82T, and G112H, P29A, S82T, and G115M, P29A, S82T, and A142E, P29A, S82T, R107C, and G112H, P29A, S82T, R107C, and G115M, P29A, S82T, R107C, and A142E, P29A, S82T, G112H, and A142E, P29A, S82T, G115M, and A142E, P29A, S82T, R107C, G112H, G115M, and A142E, E27S, V30I, and S82T, E27S, V30I, S82T, and R107C, E27S, V30I, S82T, and G112H, E27S, V30I, S82T, and G115M, E27S, V30I, S82T, and A142E, E27S, V30I, S82T, R107C, and G112H, E27S, V30I, S82T, R107C, and G115M, E27S, V30I, S82T, R107C, and A142E, E27S, V30I, S82T, G112H, and A142E, E27S, V30I, S82T, G115M, and A142E, E27S, V30I, S82T, R107C, G112H, G115M, and A142E, P29A, V30I, S82T, and F84L, P29A, V30I, S82T, F84L, and R107C, P29A, V30I, S82T, F84L, and G112H, P29A, V30I, S82T, F84L, and G115M, P29A, V30I, S82T, F84L, and A142E, P29A, V30I, S82T, F84L, R107C, and G112H, P29A, V30I, S82T, F84L, R107C, and G115M, P29A, V30I, S82T, F84L, R107C, and A142E, P29A, V30I, S82T, F84L, G112H, and A142E, P29A, V30I, S82T, F84L, G115M, and A142E, P29A, V30I, S82T, F84L, R107C, G112H, G115M, and A142E, E27S, P29A, V30L, I49K, S82T, F84L, R107C, G112H, G115M, and A142E, V4K, and A114C, V4K, and D77G, F6Y, G100A, and H122R, V4T, I76R, and H122G, F6Y and I76W, F6Y and D119N, F6Y and A114C, V4K, I76W, and H122T, F6G, I76R, and G100K, F6H and H122N, F6Y, I76H, H122R, and T166I, R23Q, and I76R, I76H, H122R, and A158V, F6Y and T111H, T111H, H122G, and A162C, F6Y and I76R, T17W, I76H, H122G, and A158V, V4S, I76Y, A143E, and Q159S, N127I and A162Q, E27H, Y76I, F84M, and F149Y, E27H, I49K, Y76I, and F149Y, T17A, E27H, I49M, Y76I, M118L, and F149Y, T17A, A48G, S82T, A142E, and F149Y, E27G and F149Y, E27G, I49N, and F149Y, E27H, Y76I, F84M, Y147D, F149Y, T166I, and D167N, E27H, I49K, Y76I, Y147D, F149Y, T166I, D167N, T17A, E27H, I49M, Y76I, M118L, Y147D, F149Y, T166I, and D167N, T17A, A48G, S82T, A142E, Y147D, F149Y, T166I, and D167N, E27G, Y147D, F149Y, T166I, and D167N, E27G, I49N, Y147D, F149Y, T166I, and D167N, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, and A142E, F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, G115M, D119N, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, G115M, H122G, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, G115M, N127P, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, G115M, A142E, and A143E, F6Y, E27H, I49K, S82T, R107C, G112H, G115M, and A143E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, D119N, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, H122G, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, N127P, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, A142E, and A143E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, and A143E, F6Y, E27H, I49K, D77G, S82T, R107C, G112H, A114C, G115M, and A142E, F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, D119N, and A142E, F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, H122G, and A142E, F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, N127P, and A142E, F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, A142E, and A143E, F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, and A143E, F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, D119N, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, H122G, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, N127P, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, G115M, D119N, H122G, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, G115M, D119N, N127P, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, G115M, H122G, N127P, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, D119N, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, H122G, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, N127P, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, A142E, and A143E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, and A143E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, H122G, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, N127P, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, A142E, and A143E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, and A143E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, D119N, H122G, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, D119N, N127P, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, H122G, N127P, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, H122G, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, N127P, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, H122G, N127P, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, H122G, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, N127P, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, A142E, and A143E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, and A143E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, H122G, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, N127P, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, H122G, N127P, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, A142E, and A143E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, and A143E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, A142E, and A143E, and F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, and A143E, or the corresponding amino acid positions in another adenosine deaminase; and / or b) the two or more modifications are E27S, I49K, and S82T, E27S, V30I, I49K, S82T, and F84L, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, H122G, and A142E, and F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, H122G, N127P, and A142E and / or c) The adenosine deaminase variant according to any one of claims 1 to 5, wherein the two or more modifications comprise a combination of modifications selected from the modifications listed in any one of Tables 1A to 1F.

7. 1. An adenosine deaminase variant having increased cytidine deaminase activity and / or increased cytidine deaminase specificity compared to a reference adenosine deaminase, said adenosine deaminase variant being selected from the group consisting of: The adenosine deaminase variant comprises a modification in one or more of region A comprising amino acid residues 82-84, region B comprising amino acid residues 27-30 and 47-49, region C comprising residues 107-115, or the C-terminal helix comprising residues 139-167, compared to a) the modification in region A modifies the active site of the deaminase; or b) said modification in region B is present in one or more of loop 1 comprising residues 25-30, loop 3 comprising residues 46-47, or helix 2 comprising residues 48-51; or c) the alteration is in Loop 1, or d) the modification is present in loop 3, or e) the modification is in the C-terminal helix comprising residues 139-167; Optionally, the modification is associated with unwinding a helix; and optionally, the unwinding of the helix occurs between residues 145 and 155, or at about residue 150; or f) the adenosine deaminase variant further comprises an R at amino acid position 166 of SEQ ID NO: 1; or g) The adenosine deaminase variant of claim 7, wherein the adenosine deaminase variant does not contain an R amino acid at position 48 of SEQ ID NO:

1.

9. a) the adenosine deaminase variant exhibits at least about a 30-fold, 50-fold, or 70-fold increase in cytidine deaminase activity and / or cytidine deaminase specificity over a reference adenosine deaminase; and / or b) the adenosine deaminase variant retains at least about 30%, 50%, or 70% of the adenosine deaminase activity of the reference adenosine deaminase; and / or c) the adenosine deaminase variant has a ratio of cytidine to adenine deaminating activity of at least about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1; and / or d) the modification increases selectivity for deamination of cytidine compared to a reference adenosine deaminase; The adenosine deaminase variant according to any one of claims 1 to 8.

10. A fusion protein comprising a polynucleotide programmable DNA binding domain and an adenosine deaminase variant according to any one of claims 1 to 9, Optionally, the polynucleotide-programmable DNA binding domain is a Cas9, Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, or Cas12j / CasΦ domain.

11. A multimolecular complex comprising a polynucleotide-programmable DNA-binding protein, an adenosine deaminase variant according to any one of claims 1 to 9, and a guide RNA.

12. a) comprising an adenosine deaminase variant according to any one of claims 1 to 9, a polynucleotide-programmable DNA binding protein, and one or more guide polynucleotides; or b) a fusion protein according to claim 10 or 11, and one or more guide polynucleotides; 1. A base editing system, wherein the base editing system results in A to G and C to T edits in a target polynucleotide.

13. A polynucleotide encoding an adenosine deaminase variant described in any one of claims 1 to 9, a fusion protein described in claim 10, a multimolecular complex described in claim 11, or a base editing system described in claim 12.

14. A vector comprising the polynucleotide of claim 13. a) the vector is a mammalian expression vector, or b) The vector of claim 14, wherein the vector is a viral vector, and optionally the viral vector is selected from the group consisting of an adeno-associated virus (AAV), a retroviral vector, an adenoviral vector, a lentiviral vector, a Sendai virus vector, and a herpes virus vector.

16. A cell comprising the vector of claim 14 or 15.

17. A composition comprising an adenosine deaminase variant described in any one of claims 1 to 9, a fusion protein described in claim 10, a multimolecular complex described in claim 11, a base editing system described in claim 12, or a cell described in claim 16, optionally further comprising a pharma- ceutically acceptable excipient, diluent, or carrier.

18. 1. A method for in vitro or ex vivo editing of the genome of a cell, said method comprising:

14. The method of claim 13, comprising contacting a target polynucleotide sequence in a cell with the fusion protein of claim 10 and one or more guide polynucleotides, the multimolecular complex of claim 11, or the base editing system of claim 12, to generate one or more modifications in the genome of the cell, thereby editing the genome of the cell.

19. 16. A composition comprising the fusion protein of claim 10 and one or more guide polynucleotides, the multimolecular complex of claim 11, the base editing system of claim 12, or the vector of claim 14 or 15, for use in a method for treating a genetic disease or disorder in a subject, the method comprising: The composition comprises contacting a target polynucleotide in a cell of the subject with the fusion protein and one or more guide polynucleotides, the multimolecular complex, the base editing system, or the vector, to generate one or more modifications in the genome of the cell.

20. 13. A composition comprising the fusion protein of claim 10 and the multimolecular complex of claim 11, or the base editing system of claim 12, for use in a method for editing a C to T in a target polynucleotide, the method comprising contacting a target polynucleotide with the fusion protein and one or more guide polynucleotides, the multimolecular complex, or the base editing system, thereby editing the target polynucleotide.

21. 13. A composition comprising the fusion protein of claim 10, the multimolecular complex of claim 11, or the base editing system of claim 12, for use in a method for introducing an A to G and / or a C to T edit in the genome of a cell, the method comprising introducing into a cell the fusion protein and a guide polynucleotide, the multimolecular complex, or the base editing system to perform an A to G edit, a C to T edit, or a combination thereof, in the genome of the cell.

22. 13. A composition comprising the fusion protein of claim 10, the multimolecular complex of claim 11, or the base editing system of claim 12, for use in a method for correcting a single nucleotide polymorphism (SNP) in a polynucleotide, the method comprising contacting a target polynucleotide with the fusion protein and one or more guide polynucleotides, the multimolecular complex, or the base editing system, thereby editing the SNP by deaminating the SNP or its complementary nucleobase.

23. 13. A composition comprising the fusion protein of claim 10, the multimolecular complex of claim 11, or the base editing system of claim 12, for use in a method for editing a regulatory sequence present in the genome of a cell, the method comprising contacting a regulatory sequence with the fusion protein and one or more guide polynucleotides, the multimolecular complex, or the base editing system, thereby editing the regulatory sequence.

24. 1. A method for producing an adenosine deaminase variant having increased cytidine deaminase activity and / or cytidine deaminase specificity, said method comprising the step of: and generating one or more modifications in an amino acid sequence having at least 85% amino acid identity to S2H, V4K, V4S, V4T, V4Y, F6G, F6H, F6Y, H8Q, R13G, T17A, T17W, R23Q, E27C, E27G, E27H, E27K, E27Q, E27S, E27G, P29A, P29G, P29K, V30F, V30I, R47G, R47S, A48G, I49K, I49M, I49N, I49Q, I49T, G67W, I76H, I76R, I76W, Y76H, Y76R, Y76W, F84A, F84M, H96N, G100A, G100K, T111H, G112H, A114C, G115M, M118L, H122G, H122R, H122T, N127I, N127K, N127P, A142E, R147H, A158V, Q159S, A162C, A162N, A162Q and S165P, or the corresponding amino acid positions in another adenosine deaminase, preferably a) the one or more modifications are E27H, Y76I, and F84M, E27H, I49K, and Y76I, E27S, I49K, and Y76I, E27S, I49K, Y76I, and A162N, E27K and D119N, E27H and Y76I, E27S, I49K, and G67W, I49T, G67W, and H96N, E27C, Y76I, and D119N, R13G, E27Q, and N127K, T17A, E27H, I49M, Y76I, and M118L, I49Q, Y76I, and G115M, S2H, I49K, Y76I, and G112H, R47S and R107C, H8Q, I49Q, and Y76I, T17A, A48G, S82T, and A142E, E27G and I49N, E27G, D77G, and S165P, E27S, I49K, and S82T, E27S, I49K, S82T, and G115M, E27S, V30I, I49K, and S82T, E27S, V30F, I49K, S82T, F84A, R107C, and A142E, E27S, V30F, I49K, S82T, F84A, G112H, and A142E, E27S, V30F, I49K, S82T, F84A, G115M, and A142E, E27S, I49K, S82T, F84L, and R107C, E27S, I49K, S82T, F84L, and G112H, E27S, I49K, S82T, F84L, and G115M, E27S, I49K, S82T, F84L, R107C, and G112H, E27S, I49K, S82T, F84L, R107C, and G115M, E27S, I49K, S82T, F84L, R107C, and A142E, E27S, I49K, S82T, F84L, G112H, and A142E, E27S, I49K, S82T, F84L, G115M, and A142E, E27S, I49K, S82T, F84L, R107C, G112H, G115M, and A142E, E27S, V30I, I49K, S82T, and F84L, E27S, P29G, I49K, and S82T, E27S, P29G, I49K, S82T, and G115M, E27S, P29G, I49K, S82T, and A142E, P29G, I49K, and S82T, E27G, I49K, and S82T, E27G, I49K, S82T, R107C, and A142E, V4K, E27H, I49K, Y76I, and A114C, V4K, E27H, I49K, Y76I, and D77G, F6Y, E27H, I49K, Y76I, G100A, and H122R, V4T, E27H, I49K, Y76R, and H122G, F6Y, E27H, I49K, and Y76W, F6Y, E27H, I49K, Y76I, and D119N, F6Y, E27H, I49K, Y76I, and A114C, F6Y, E27H, I49K, and Y76I, V4K, E27H, I49K, Y76W, and H122T, F6G, E27H, I49K, Y76R, and G100K, F6H, E27H, I49K, Y76I, and H122N, E27H, I49K, Y76I, and A114C, F6Y, E27H, I49K, Y76H, H122R, and T166I, E27H, I49K, Y76I, and N127P, R23Q, E27H, I49K, and Y76R, E27H, I49K, Y76H, H122R, and A158V, F6Y, E27H, I49K, Y76I, and T111H, E27H, I49K, Y76I, and R147H, E27H, I49K, Y76I, and A143E, F6Y, E27H, I49K, and Y76R, T17W, E27H, I49K, Y76H, H122G, and A158V, V4S, E27H, I49K, A143E, and Q159S, E27H, I49K, Y76I, N127I, and A162Q, T17A, E27H, and A48G, T17A, E27K, and A48G, T17A, E27S, and A48G, T17A, E27S, A48G, and I49K, T17A, E27G, and A48G, T17A, A48G, and I49N, T17A, E27G, A48G, and I49N, T17A, E27Q, and A48G, E27S, I49K, S82T, and R107C, E27S, I49K, S82T, and G112H, E27S, I49K, S82T, and A142E, E27S, I49K, S82T, R107C, and G112H, E27S, I49K, S82T, R107C, and G115M, E27S, I49K, S82T, R107C, and A142E, E27S, I49K, S82T, G112H, and A142E, E27S, I49K, S82T, G115M, and A142E, E27S, I49K, S82T, R107C, G112H, G115M, and A142E, E27S, V30I, I49K, S82T, and R107C, E27S, V30I, I49K, S82T, and G112H, E27S, V30I, I49K, S82T, and G115M, E27S, V30I, I49K, S82T, and A142E, E27S, V30I, I49K, S82T, R107C, and G112H, E27S, V30I, I49K, S82T, R107C, and G115M, E27S, V30I, I49K, S82T, R107C, and A142E, E27S, V30I, I49K, S82T, G112H, and A142E, E27S, V30I, I49K, S82T, G115M, and A142E, E27S, V30I, I49K, S82T, R107C, G112H, G115M, and A142E, E27S, V30L, I49K, and S82T, E27S, V30L, I49K, S82T, and R107C, E27S, V30L, I49K, S82T, and G112H, E27S, V30L, I49K, S82T, and G115M, E27S, V30L, I49K, S82T, and A142E, E27S, V30L, I49K, S82T, R107C, and G112H, E27S, V30L, I49K, S82T, R107C, and G115M, E27S, V30L, I49K, S82T, R107C, and A142E, E27S, V30L, I49K, S82T, G112H, and A142E, E27S, V30L, I49K, S82T, G115M, and A142E, E27S, V30L, I49K, S82T, R107C, G112H, G115M, and A142E, E27S, V30F, I49K, S82T, and F84A, E27S, V30F, I49K, S82T, F84A, and R107C, E27S, V30F, I49K, S82T, F84A, and G112H, E27S, V30F, I49K, S82T, F84A, and G115M, E27S, V30F, I49K, S82T, F84A, and A142E, E27S, V30F, I49K, S82T, F84A, R107C, and G112H, E27S, V30F, I49K, S82T, F84A, R107C, and G115M, E27S, V30F, I49K, S82T, F84A, R107C, G112H, G115M, and A142E, E27S, I49K, S82T, and F84L, E27S, I49K, S82T, F84L, and A142E, E27S, V30I, I49K, S82T, F84L, and R107C, E27S, V30I, I49K, S82T, F84L, and G112H, E27S, V30I, I49K, S82T, F84L, and G115M, E27S, V30I, I49K, S82T, F84L, and A142E, E27S, V30I, I49K, S82T, F84L, R107C, and G112H, E27S, V30I, I49K, S82T, F84L, R107C, and G115M, E27S, V30I, I49K, S82T, F84L, R107C, and A142E, E27S, V30I, I49K, S82T, F84L, G112H, and A142E, E27S, V30I, I49K, S82T, F84L, G115M, and A142E, E27S, V30I, I49K, S82T, F84L, R107C, G112H, G115M, and A142E, E27S, P29G, I49K, S82T, and R107C, E27S, P29G, I49K, S82T, and G112H, E27S, P29G, I49K, S82T, R107C, and G112H, E27S, P29G, I49K, S82T, R107C, and G115M, E27S, P29G, I49K, S82T, R107C, and A142E, E27S, P29G, I49K, S82T, G112H, and A142E, E27S, P29G, I49K, S82T, G115M, and A142E, E27S, P29G, I49K, S82T, R107C, G112H, G115M, and A142E, P29G, I49K, S82T, and R107C, P29G, I49K, S82T, and G112H, P29G, I49K, S82T, and G115M, P29G, I49K, S82T, and A142E, P29G, I49K, S82T, R107C, and G112H, P29G, I49K, S82T, R107C, and G115M, P29G, I49K, S82T, R107C, and A142E, P29G, I49K, S82T, G112H, and A142E, P29G, I49K, S82T, G115M, and A142E, P29G, I49K, S82T, R107C, G112H, G115M, and A142E, P29K, I49K, and S82T, P29K, I49K, S82T, and R107C, P29K, I49K, S82T, and G112H, P29K, I49K, S82T, and G115M, P29K, I49K, S82T, and A142E, P29K, I49K, S82T, R107C, and G112H, P29K, I49K, S82T, R107C, and G115M, P29K, I49K, S82T, R107C, and A142E, P29K, I49K, S82T, G112H, and A142E, P29K, I49K, S82T, G115M, and A142E, P29K, I49K, S82T, R107C, G112H, G115M, and A142E, P29K, V30I, I49K, and S82T, P29K, V30I, I49K, S82T, and R107C, P29K, V30I, I49K, S82T, and G112H, P29K, V30I, I49K, S82T, and G115M, P29K, V30I, I49K, S82T, and A142E, P29K, V30I, I49K, S82T, R107C, and G112H, P29K, V30I, I49K, S82T, R107C, and G115M, P29K, V30I, I49K, S82T, R107C, and A142E, P29K, V30I, I49K, S82T, G112H, and A142E, P29K, V30I, I49K, S82T, G115M, and A142E, P29K, V30I, I49K, S82T, R107C, G112H, G115M, and A142E, P29K, I49K, S82T, and F84L, P29K, I49K, S82T, F84L, and R107C, P29K, I49K, S82T, F84L, and G112H, P29K, I49K, S82T, F84L, and G115M, P29K, I49K, S82T, F84L, and A142E, P29K, I49K, S82T, F84L, R107C, and G112H, P29K, I49K, S82T, F84L, R107C, and G115M, P29K, I49K, S82T, F84L, R107C, and A142E, P29K, I49K, S82T, F84L, G112H, and A142E, P29K, I49K, S82T, F84L, G115M, and A142E, P29K, I49K, S82T, F84L, R107C, G112H, G115M, and A142E, P29K, V30I, I49K, S82T, and F84L, P29K, V30I, I49K, S82T, F84L, and R107C, P29K, V30I, I49K, S82T, F84L, and G112H, P29K, V30I, I49K, S82T, F84L, and G115M, P29K, V30I, I49K, S82T, F84L, and A142E, P29K, V30I, I49K, S82T, F84L, R107C, and G112H, P29K, V30I, I49K, S82T, F84L, R107C, and G115M, P29K, V30I, I49K, S82T, F84L, R107C, and A142E, P29K, V30I, I49K, S82T, F84L, G112H, and A142E, P29K, V30I, I49K, S82T, F84L, G115M, and A142E, P29K, V30I, I49K, S82T, F84L, R107C, G112H, G115M, and A142E, E27G, I49K, S82T, and R107C, E27G, I49K, S82T, and G112H, E27G, I49K, S82T, and G115M, E27G, I49K, S82T, and A142E, E27G, I49K, S82T, R107C, and G112H, E27G, I49K, S82T, R107C, and G115M, E27G, I49K, S82T, G112H, and A142E, E27G, I49K, S82T, G115M, and A142E, E27G, I49K, S82T, R107C, G112H, G115M, and A142E, E27H, I49K, and S82T, E27H, I49K, S82T, and R107C, E27H, I49K, S82T, and G112H, E27H, I49K, S82T, and G115M, E27H, I49K, S82T, and A142E, E27H, I49K, S82T, R107C, and G112H, E27H, I49K, S82T, R107C, and G115M, E27H, I49K, S82T, R107C, and A142E, E27H, I49K, S82T, G112H, and A142E, E27H, I49K, S82T, G115M, and A142E, E27H, I49K, S82T, R107C, G112H, G115M, and A142E, E27S and S82T, E27S, S82T, and R107C, E27S, S82T, and G112H, E27S, S82T, and G115M, E27S, S82T, and A142E, E27S, S82T, R107C, and G112H, E27S, S82T, R107C, and G115M, E27S, S82T, R107C, and A142E, E27S, S82T, G112H, and A142E, E27S, S82T, G115M, and A142E, E27S, S82T, R107C, G112H, G115M, and A142E, P29A, and S82T, P29A, S82T, and R107C, P29A, S82T, and G112H, P29A, S82T, and G115M, P29A, S82T, and A142E, P29A, S82T, R107C, and G112H, P29A, S82T, R107C, and G115M, P29A, S82T, R107C, and A142E, P29A, S82T, G112H, and A142E, P29A, S82T, G115M, and A142E, P29A, S82T, R107C, G112H, G115M, and A142E, E27S, V30I, and S82T, E27S, V30I, S82T, and R107C, E27S, V30I, S82T, and G112H, E27S, V30I, S82T, and G115M, E27S, V30I, S82T, and A142E, E27S, V30I, S82T, R107C, and G112H, E27S, V30I, S82T, R107C, and G115M, E27S, V30I, S82T, R107C, and A142E, E27S, V30I, S82T, G112H, and A142E, E27S, V30I, S82T, G115M, and A142E, E27S, V30I, S82T, R107C, G112H, G115M, and A142E, P29A, V30I, S82T, and F84L, P29A, V30I, S82T, F84L, and R107C, P29A, V30I, S82T, F84L, and G112H, P29A, V30I, S82T, F84L, and G115M, P29A, V30I, S82T, F84L, and A142E, P29A, V30I, S82T, F84L, R107C, and G112H, P29A, V30I, S82T, F84L, R107C, and G115M, P29A, V30I, S82T, F84L, R107C, and A142E, P29A, V30I, S82T, F84L, G112H, and A142E, P29A, V30I, S82T, F84L, G115M, and A142E, P29A, V30I, S82T, F84L, R107C, G112H, G115M, and A142E, E27S, P29A, V30L, I49K, S82T, F84L, R107C, G112H, G115M, and A142E, V4K, and A114C, V4K, and D77G, F6Y, G100A, and H122R, V4T, I76R, and H122G, F6Y and I76W, F6Y and D119N, F6Y and A114C, V4K, I76W, and H122T, F6G, I76R, and G100K, F6H and H122N, F6Y, I76H, H122R, and T166I, R23Q, and I76R, I76H, H122R, and A158V, F6Y and T111H, T111H, H122G, and A162C, F6Y and I76R, T17W, I76H, H122G, and A158V, V4S, I76Y, A143E, and Q159S, N127I and A162Q, E27H, Y76I, F84M, and F149Y, E27H, I49K, Y76I, and F149Y, T17A, E27H, I49M, Y76I, M118L, and F149Y, T17A, A48G, S82T, A142E, and F149Y, E27G and F149Y, E27G, I49N, and F149Y, E27H, Y76I, F84M, Y147D, F149Y, T166I, and D167N, E27H, I49K, Y76I, Y147D, F149Y, T166I, D167N, T17A, E27H, I49M, Y76I, M118L, Y147D, F149Y, T166I, and D167N, T17A, A48G, S82T, A142E, Y147D, F149Y, T166I, and D167N, E27G, Y147D, F149Y, T166I, and D167N, E27G, I49N, Y147D, F149Y, T166I, and D167N, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, and A142E, F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, G115M, D119N, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, G115M, H122G, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, G115M, N127P, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, G115M, A142E, and A143E, F6Y, E27H, I49K, S82T, R107C, G112H, G115M, and A143E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, D119N, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, H122G, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, N127P, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, A142E, and A143E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, and A143E, F6Y, E27H, I49K, D77G, S82T, R107C, G112H, A114C, G115M, and A142E, F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, D119N, and A142E, F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, H122G, and A142E, F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, N127P, and A142E, F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, A142E, and A143E, F6Y, E27H, I49K, D77G, S82T, R107C, G112H, G115M, and A143E, F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, D119N, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, H122G, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, A114C, G115M, N127P, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, G115M, D119N, H122G, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, G115M, D119N, N127P, and A142E, F6Y, E27H, I49K, S82T, R107C, G112H, G115M, H122G, N127P, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, D119N, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, H122G, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, N127P, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, A142E, and A143E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, G115M, and A143E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, H122G, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, N127P, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, A142E, and A143E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, and A143E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, D119N, H122G, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, D119N, N127P, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, H122G, N127P, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, H122G, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, N127P, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, H122G, N127P, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, H122G, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, N127P, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, A142E, and A143E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, and A143E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, H122G, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, N127P, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, H122G, N127P, and A142E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, A142E, and A143E, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, and A143E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, A142E, and A143E, and F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, D119N, H122G, N127P, and A143E, or at corresponding amino acid positions in another adenosine deaminase; and / or b) the one or more modifications are E27S, I49K, and S82T, E27S, V30I, I49K, S82T, and F84L, F6Y, E27H, I49K, Y76W, S82T, R107C, G112H, G115M, and A142E, F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, H122G, and A142E, and F6Y, E27H, I49K, Y76W, D77G, S82T, R107C, G112H, A114C, G115M, H122G, N127P, and A142E and / or c) the one or more modifications include a combination of modifications selected from those listed in any of Tables 1A-1F; method.

25. 25. An adenosine deaminase variant produced by the method of claim 24.

26. A kit comprising a fusion protein described in claim 10, a multimolecular complex described in claim 11, or a base editing system described in claim 12, a polynucleotide described in claim 13, a vector described in claim 14 or 15, a cell described in claim 16, or a composition described in claim 17, and instructions for its use in base editing.

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