COMPOSITIONS AND METHODS FOR TREATING GLYCOGEN STORAGE DISEASE TYPE 1A

1 . An adenosine deaminase variant comprising a glycine (G) at amino acid position 82, a threonine (T) or an aspartic acid (D) at amino acid position 147, a serine (S) at amino acid position 154, and one or more of a histidine (H) at amino acid position 36, a tyrosine at amino acid position 76, a tyrosine at amino acid position 149, a lysine (K) at amino acid position 157, and an asparagine (N) at amino acid position 167 of the following amino acid sequence, wherein the adenosine deaminase has at least about 85% identity to said amino acid sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1), or corresponding alterations in another adenosine deaminase.

2 . An adenosine deaminase variant comprising any of the following combinations of alterations

a) I76Y+V82G+Y147T+Q154S;

b) L36H+V82G+Y147T+Q154S+N157K;

c) V82G+Y147D+F149Y+Q154S+D167N;

d) L36H+V82G+Y147D+F149Y+Q154S+N157K+D167N;

e) L36H+I76Y+V82G+Y147T+Q154S+N157K;

f) I76Y+V82G+Y147D+F149Y+Q154S+D167N;

g) Y147D+F149Y+D167N;

h) L36H; I76Y; V82G; Q154S; and N157K;

i) I76Y; V82G; Q154S; or

j) L36H+I76Y+V82G+Y147D+F149Y+Q154S+N157K+D167N with reference to SEQ ID NO: 1: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1), or corresponding combinations of alterations in another adenosine deaminase.

3 . The adenosine deaminase variant of claim 1 , comprising the following combination of alterations I76Y+V82G+Y147D+F149Y+Q154S+D167N of SEQ ID NO: 1, or corresponding alterations in another adenosine deaminase.

4 . The adenosine deaminase variant of claim 1 , wherein the adenosine deaminase has at least about 90% identity to SEQ ID NO: 1.

5 - 6 . (canceled)

7 . A fusion protein or complex comprising a polynucleotide programmable DNA binding domain and at least one adenosine deaminase variant domain, wherein the adenosine deaminase variant domain comprises a glycine (G) at amino acid position 82, a threonine (T) or an aspartic acid (D) at amino acid position 147, a serine (S) at amino acid position 154, and one or more of a histidine (H) at amino acid position 36, a tyrosine at amino acid position 76, a tyrosine at amino acid position 149, a lysine (K) at amino acid position 157, and an asparagine (N) at amino acid position 167 of the following amino acid sequence, wherein the adenosine deaminase has at least about 85% identity to said amino acid sequence MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1), or corresponding alterations in another adenosine deaminase.

8 - 10 . (canceled)

11 . The fusion protein or complex of claim 1 comprising a polynucleotide programmable DNA binding domain and at least one adenosine deaminase variant domain, wherein the adenosine deaminase variant domain comprises any of the following combinations of alterations

a) I76Y+V82G+Y147T+Q154S;

b) L36H+V82G+Y147T+Q154S+N157K;

c) V82G+Y147D+F149Y+Q154S+D167N;

d) L36H+V82G+Y147D+F149Y+Q154S+N157K+D167N;

e) L36H+I76Y+V82G+Y147T+Q154S+N157K;

f) I76Y+V82G+Y147D+F149Y+Q154S+D167N;

g) Y147D+F149Y+D167N;

h) L36H; I76Y; V82G; Q154S; and N157K;

i) I76Y; V82G; Q154S; or

j) L36H+I76Y+V82G+Y147D+F149Y+Q154S+N157K+D167N

with reference to SEQ ID NO: 1: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1), or corresponding combinations of alterations in another adenosine deaminase.

12 - 14 . (canceled)

15 . The fusion protein or complex of claim 7 , wherein the fusion protein comprises a TadA*7.10 adenosine deaminase domain and an adenosine deaminase variant domain; or wherein the polynucleotide programmable DNA binding domain is a Cas9 domain.

16 - 18 . (canceled)

19 . The fusion protein or complex of claim 7 , wherein the polynucleotide programmable DNA binding domain comprises a modified SaCas9 having an altered protospacer-adjacent motif (PAM) specificity;

wherein the SaCas9 has protospacer-adjacent motif (PAM) specificity for the nucleic acid sequence 5′-NNGRRT-3′ or 5′-GAGAAT-3′, and wherein the SaCas9 is a nuclease active SaCas9, a nuclease inactive SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n);

wherein the linker comprises the amino acid sequence: SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 359); and

further comprises one or more nuclear localization signals.

20 - 30 . (canceled)

31 . A base editor system comprising the fusion protein or complex of claim 7 , and one or more guide polynucleotides.

32 - 41 . (canceled)

42 . The base editor system of claim 31 , wherein the guide polynucleotide comprises or consists essentially of one of the following sequences:

(SEQ ID NO: 409)

mCsmAsmCsCAGUAUGGACACUGUCCAAAGUUUUAGUACUCUGUAAUGAA

AAUUACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAA

CUUGUUGGCGAGAmUsmUsmUsU

or

(SEQ ID NO: 410)

mCsmCsmASCCAGUAUGGACACUGUCCAAAGUUUUAGUACUCUGUAAUGA

AAAUUACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCA

ACUUGUUGGCGAGAmUsmUsmUSU,

wherein “m” denotes a 2′-O-methyl and “s” denotes a phosphorothioate; or

wherein the guide polynucleotide comprises a nucleic acid sequence, in 5′ to 3′ orientation, selected from CCACCAGUAUGGACACUGUC (SEQ ID NO: 371); CACCAGUAUGGACACUGUCC (SEQ ID NO: 372); ACCAGUAUGGACACUGUCCA (SEQ ID NO: 373); CCAGUAUGGACACUGUCCAA (SEQ ID NO: 374); CAGUAUGGACACUGUCCAAA (SEQ ID NO: 370); AGUAUGGACACUGUCCAAAG (SEQ ID NO: 375); GUAUGGACACUGUCCAAAGA (SEQ ID NO: 376); or UAUGGACACUGUCCAAAGAG (SEQ ID NO: 377).

43 - 44 . (canceled)

45 . A polynucleotide encoding the adenosine deaminase variant of claim 1 .

46 - 48 . (canceled)

49 . A cell comprising the polynucleotide of claim 45 .

50 . A cell comprising the adenosine deaminase variant of claim 1 .

51 - 56 . (canceled)

57 . A method of treating a genetic disease in a subject in need thereof, the method comprising administering to a cell of the subject the base editor system of claim 31 or a polynucleotide encoding the base editor system.

58 . A method of treating a genetic disease in a subject in need thereof, the method comprising administering to the subject the cell of claim 49 .

59 . The method of claim 57 , wherein after treatment the cell expresses a G6PC polypeptide capable of catalyzing the hydrolysis of D-glucose 6-phosphate to D-glucose and orthophosphate.

60 . (canceled)

61 . A method for correcting a single nucleotide polymorphism (SNP) in a polynucleotide, the method comprising:

contacting a target nucleotide sequence, at least a portion of which is located in the polynucleotide or its reverse complement, with the base editor system of claim 31 and editing the SNP by deaminating the SNP or its complement nucleobase upon targeting of the base editor to the target nucleotide sequence, wherein deaminating the SNP or its complement nucleobase corrects the SNP.

62 . The method of claim 61 , wherein the SNP is associated with Glycogen Storage Disease Type 1a (GSD1a).

63 . (canceled)

64 . A method of editing a glucose-6-phosphatase (G6PC) polynucleotide comprising a single nucleotide polymorphism (SNP) associated with Glycogen Storage Disease Type 1a (GSD1a), the method comprising contacting the G6PC polynucleotide with a fusion protein or complex of claim 7 in a complex with one or more guide polynucleotides, wherein one or more of the guide polynucleotides targets the base editor to effect an A·T to G·C alteration of the SNP associated with GSD1a.

65 . (canceled)

66 . The method of claim 57 , wherein the SNP changes a glutamine (Q) to a non-glutamine (X) amino acid or changes an arginine (R) to a non-arginine (X) in a G6PC polypeptide; wherein the SNP results in expression of an G6PC polypeptide having a non-glutamine (X) amino acid at position 347 or a non-arginine (X) amino acid at position 83; wherein the base editor correction replaces the non-glutamine amino acid (X) at position 347 with a glutamine or the non-arginine amino acid (X) at position 83 with an arginine; wherein the SNP results in expression of a G6PC polypeptide that prematurely terminates at amino acid position 347 or at a cysteine at position 83; wherein the SNP encodes one or more of Q347X and/or R83C.

67 - 72 . (canceled)

73 . The method of claim 64 , wherein the guide polynucleotide comprises a nucleic acid sequence, from 5′-3′, selected from the group consisting of CAGUAUGGACACUGUCCAAA (SEQ ID NO: 370); CCACCAGUAUGGACACUGUC (SEQ ID NO: 371); CACCAGUAUGGACACUGUCC (SEQ ID NO: 372); ACCAGUAUGGACACUGUCCA (SEQ ID NO: 373); CCAGUAUGGACACUGUCCAA (SEQ ID NO: 374); AGUAUGGACACUGUCCAAAG (SEQ ID NO: 375); GUAUGGACACUGUCCAAAGA (SEQ ID NO: 376); and UAUGGACACUGUCCAAAGAG (SEQ ID NO: 377).

74 . (canceled)

75 . A vector comprising the polynucleotide of claim 45 .

76 - 77 . (canceled)

78 . A composition comprising the fusion protein or complex of claim 7 .

79 - 81 . (canceled)

82 . A composition comprising the polynucleotide of claim 45 .

83 . (canceled)

84 . A composition comprising the cell of claim 49 .

85 - 87 . (canceled)

88 . A kit comprising the fusion protein or complex of claim 7 .

89 - 92 . (canceled)

93 . A modified guide RNA (gRNA) comprising modified nucleotides, wherein the guide comprises from 5′ to 3′ a polynucleotide sequence selected from the group consisting of:

(SEQ ID NO: 404)

CAGUAUGGACACUGUCCAAAGUUUUAGUACUCUGUAAUGAAAAUUACAGA

AUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGC

GAGAUUUU;

(SEQ ID NO: 405)

UUUCAGUAUGGACACUGUCCAAAGUUUUAGUACUCUGUAAUGAAAAUUAC

AGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUU

GGCGAGAUUUU;

(SEQ ID NO: 406)

CAGUAUGGACACUGUCCAAAGUUUUAGUACUCUGGAAACAGAAUCUACUA

AAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUU

U;

(SEQ ID NO: 407)

CCAGUAUGGACACUGUCCAAAGUUUUAGUACUCUGUAAUGAAAAUUACAG

AAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGG

CGAGAUUUU;

(SEQ ID NO: 408)

ACCAGUAUGGACACUGUCCAAAGUUUUAGUACUCUGUAAUGAAAAUUACA

GAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUG

GCGAGAUUUU;

(SEQ ID NO: 409)

CACCAGUAUGGACACUGUCCAAAGUUUUAGUACUCUGUAAUGAAAAUUAC

AGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUU

GGCGAGAUUUU;

(SEQ ID NO: 410)

CCACCAGUAUGGACACUGUCCAAAGUUUUAGUACUCUGUAAUGAAAAUUA

CAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGU

UGGCGAGAUUUU;

(SEQ ID NO: 411)

CAGUAUGGACACUGUCCAAAGUUUUAGUACUCUGUAGAAAUACAGAAUCU

ACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGA

UUUU;

(SEQ ID NO: 412)

CAGUAUGGACACUGUCCAAAGUUUUAGUACUCUGCGGAAACGCAGAAUCU

ACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGA

UUUU;

(SEQ ID NO: 413)

CAGUAUGGACACUGUCCAAAGUUUUAGUACUCGAAAGAAUCUACUAAAAC

AAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU;

(SEQ ID NO: 414)

CAGUAUGGACACUGUCCAAAGUUUUAGUACCCGAAAGCAUCUACUAAAAC

AAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU;

and

(SEQ ID NO: 415)

CAGUAUGGACACUGUCCAAAGUUUUAGUACUCUGUAAUGAAAAUUACAGA

AUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGC

GAGAUUUU.

94 - 112 . (canceled)

113 . A modified guide RNA (gRNA) comprising a nucleic acid sequence, from 5′ to 3′, selected from

(SEQ ID NO: 404)

mCsmAsmGsmUAUmGmGmACAmCUGUCCAAAmGUUUUmAmGmUACUCmUG

mUmAmAmUGmAAAmAmUmUmACmAGAAUCUACmUmAAAACAAGGCAAmAAUGm

CCmGUGUmUmUmAmUmCmUmCmGmUmCmAmAmCmUmUmGmUmUmGmGmCmGm

AmGmAmUsmUsmUsmU;

(SEQ ID NO: 405)

mUsmUsmUsCAGmUAUmGmGmACAmCUGUCCAAAmGUUUUmAmGmUACUC

mUGmUmAmAmUGmAAAmAmUmUmACmAGAAUCUACmUmAAAACAAGGCAAmAA

UGmCCmGUGUmUmUmAmUmCmUmCmGmUmCmAmAmCmUmUmGmUmUmGmGmC

mGmAmGmAmUsmUsmUsmU;

(SEQ ID NO: 404)

mCsmAsGsmUAUmGmGmAmCAmCmUGUCCAAmAmGUmUUmUmAmGmUACU

mCmUmGmUmAmAmUGmAmAmAmAmUmUmACmAmGAAmUCUACmUmAmAAACA

AGmGCAAmAAUGmCmCmGUGmUmUmUmAmUmCmUmCmGmUmCmAmAmCmUmU

mGmUmUmGmGmCmGmAmGmAmUsmUsmUsmU;

(SEQ ID NO: 404)

mCsmAsGsmUmAUmGmGmAmCAmCUGUCCAAmAmGUUUmUAmGmUACUmC

mUmGmUmAmAmUmGmAmAmAmAmUmUmAmCmAmGmAAmUCUACUmAmAAACA

AmGmGmCmAmAmAAUmGmCmCGUGmUmUmUmAmUmCmUmCmGmUmCmAmAmC

mUmUmGmUmUmGmGmCmGmAmGmAmUsmUsmUsmU;

or

(SEQ ID NO: 404)

mCsmAsGsmUmAUmGmGmAmCAmCmUGUCmCmAAmAmGmUmUmUmUmAmG

mUAmCmUmCmUmGmUmAmAmUmGmAmAmAmAmUmUmAmCmAmGmAmAmUCUA

CmUmAmAmAAmCAmAmGmGmCmAmAmAAUmGmCmCmGmUGmUmUmUmAmUmC

mUmCmGmUmCmAmAmCmUmUmGmUmUmGmGmCmGmAmGmAmUsmUsmUsmU,

wherein “m” denotes a 2′-O-methyl and “s” denotes a phosphorothioate; or

selected from

(SEQ ID NO: 404)

mCsmAsmGsUAUGGACACUGUCCAAAGUUUUAGUACmUmCmUmGmUmAmAmUmG

mAmAmAmAmUmUmAmCmAmGmAAUCUACUAAAACAAGGCAAAAUGCCGUGUUU

AUCUCGUCAACUUGUUGGCGAGAUsmUsmUsmU;

(SEQ ID NO: 404)

mCsmAsmGsUAUGGACACUGUCCAAAGUUUUAGUACmUmCmUmGmUmAmA

mUmGmAmAmAmAmUmUmAmCmAmGmAAUCUACUAAAACAAGGCAAAAUGCCGU

GUmUmUmAmUmCmUmCmGmUmCmAmAmCmUmUmGmUmUmGmGmCmGmAmGmA

mUsmUsmUsmU;

(SEQ ID NO: 404)

mCsmAsmGsmUAUmGmGmACAmCUGUCCAAAmGUUUUAGUACmUmCmUmG

mUmAmAmUmGmAmAmAmAmUmUmAmCmAmGmAAUCUACUAAAACAAGGCAAAA

UGCCmGUGUmUmUAmUmCmUmCmGmUmCmAmAmCmUmUmGmUmUmGmGmCmG

mAmGmAUsmUsmUsmU;

(SEQ ID NO: 406)

mCsmAsmGsUAUGGACACUGUCCAAAGUUUUAGUACmUmCmUmGGmAmAm

AmCmAmGmAAUCUACUAAAACAAGGCAAAAUGCCGUGUmUmUmAmUmCmUmCm

GmUmCmAmAmCmUmUmGmUmUmGmGmCmGmAmGmAmUsmUsmUsmU;

(SEQ ID NO: 406)

mCsmAsmGsmUAUmGmGmACAmCUGUCCAAAmGUUUUAGUACmUmCmUmG

mGmAmAmAmCmAmGmAAUCUACUAAAACAAGGCAAAAUGCCmGUGUmUmUAmU

mCmUmCmGmUmCmAmAmCmUmUmGmUmUmGmGmCmGmAmGmAUsmUsmUsmU,

wherein “m” denotes a 2′-O-methyl and “s” denotes a phosphorothioate;

selected from

(SEQ ID NO: 407)

mCsmCsmAsGUAUGGACACUGUCCAAAGUUUUAGUACUCUGUAAUGAAAAU

UACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUG

GCGAGAmUsmUsmUsU;

(SEQ ID NO: 408)

mAsmCsmCsAGUAUGGACACUGUCCAAAGUUUUAGUACUCUGUAAUGAAAA

UUACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUU

GGCGAGAmUsmUsmUsU;

(SEQ ID NO: 409)

mCsmAsmCsCAGUAUGGACACUGUCCAAAGUUUUAGUACUCUGUAAUGAAA

AUUACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGU

UGGCGAGAmUsmUsmUsU;

or

(SEQ ID NO: 410)

mCsmCsmAsCCAGUAUGGACACUGUCCAAAGUUUUAGUACUCUGUAAUGAA

AAUUACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUG

UUGGCGAGAmUsmUsmUsU,  wherein “m” denotes a 2′-O-methyl and “s”

denotes a phosphorothioate; selected from

(SEQ ID NO: 411)

mCsmAsmGsUAUGGACACUGUCCAAAGUUUUAGUACUCUGUAGAAAUACAG

AAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAG

AUsmUsmUsmU;

(SEQ ID NO: 412)

mCsmAsmGsUAUGGACACUGUCCAAAGUUUUAGUACUCUGCG GAAA

CGCAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUG

GCGAGAUsmUsmUsmU;

(SEQ ID NO: 406)

mCsmAsmGsUAUGGACACUGUCCAAAGUUUUAGUACUCUGGAAACAGAAUC

UACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUsmU

smUsmU;

(SEQ ID NO: 413)

mCsmAsmGsUAUGGACACUGUCCAAAGUUUUAGUACUCGAAAGAAUCUACU

AAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUsmUsmUs

mU;

(SEQ ID NO: 414)

mCsmAsmGsUAUGGACACUGUCCAAAGUUUUAGUACCCGAAAGCAUCUACU

AAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUsmUsmUs

mU [[ ] ] ,  wherein “m” denotes a 2′-O-methyl and “s” denotes a

phosphorothioate; or selected from

(SEQ ID NO: 415)

mCsmAsmGsUAUGGACACUGUCCAAAGUUUUAGUACUCUGUAAUGAAAAUU

ACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGG

CGAGAUsmUsmUsmU;

or

(SEQ ID NO: 415)

mCsmAsmGsUAUGGACACUGUCCAAAGUUUUAGUACUCUGUAAUGAAAAUU

ACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGG

CGAGAmUsmUsmUsU,  wherein “m” denotes a 2′-O-methyl and “s”

denotes a phosphorothioate.

wherein “m” denotes a 2′-O-methyl and “s” denotes a phosphorothioate.

114 - 117 . (canceled)

118 . A formulation comprising a lipid nanoparticle comprising an mRNA expressing a base editor and a gRNA, wherein the base editor comprises a Cas9 domain and at least one adenosine deaminase variant comprising V82G, Y147T/D, Q154S, and one or more of L36H, I76Y, F149Y, N157K, and D167N with reference to SEQ ID NO: 1: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1), or corresponding alterations in another adenosine deaminase; and the gRNA comprises

(SEQ ID NO: 370)

CAGUAUGGACACUGUCCAAA.

119 . A formulation comprising a lipid nanoparticle comprising an mRNA expressing a base editor, wherein the base editor comprises a Cas9 domain and at least one adenosine deaminase variant comprising V82G, Y147T/D, Q154S, and one or more of L36H, I76Y, F149Y, N157K, and D167N with reference to SEQ ID NO: 1: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALR QGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNH RVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1), or corresponding alterations in another adenosine deaminase; and a gRNA comprising

(SEQ ID NO: 370)

CAGUAUGGACACUGUCCAAA.

120 - 124 . (canceled)