METHODS OF EDITING SINGLE NUCLEOTIDE POLYMORPHISM USING PROGRAMMABLE BASE EDITOR SYSTEMS

1 . A method of editing a methyl-CpG-binding protein 2 (MECP2) polynucleotide comprising a single nucleotide polymorphism (SNP) associated with Rett Syndrome (RTT), the method comprising contacting the MECP2 polynucleotide with a base editor in complex with one or more guide polynucleotides, wherein the base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain, and wherein one or more of said guide polynucleotides target said base editor to effect an A•T to G•C alteration of the SNP associated with RTT.

2 . The method of claim 1 , wherein the contacting is in a cell, a eukaryotic cell, a mammalian cell, or human cell.

3 . The method of claim 1 , wherein the cell is in vivo or ex vivo.

4 . (canceled)

5 . The method of claim 1 , wherein the alteration is one or more of R106W, R168*, R133C, T158M, R255*, R270*, and R306C.

6 . The method of claim 1 , wherein the A•T to G•C alteration at the SNP associated with RTT changes a cysteine to an arginine, methionine to a threonine, or stop codon to arginine in the methyl CpG binding protein 2 (MECP2) polypeptide; or

wherein the SNP associated with RTT results in expression of an MECP2 polypeptide comprising an arginine at amino acid position 168, 133, 255, 270, or 306; or a threonine at position 158.

7 . (canceled)

8 . The method of claim 1 , wherein the polynucleotide programmable DNA binding domain is a Streptococcus pyogenes Cas9 (SpCas9) or variant thereof.

9 . The method of claim 1 , wherein the polynucleotide programmable DNA binding domain comprises a modified SpCas9 having an altered protospacer-adjacent motif (PAM) specificity.

10 . (canceled)

11 . The method of claim 9 , wherein the modified SpCas9 comprises the amino acid substitutions L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, T1337R and one or more of L1111, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, R1335Q, T1337, T1337L, T1337Q, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337H, T1337Q, and T1337M, or corresponding amino acid substitutions thereof; or

wherein the modified SpCas9 comprises the amino acid substitutions D1135L, S1136R, G1218S, E1219V, A1322R, R1335Q, and T1337, and one or more of L1111R, G1218R, E1219F, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, T1337L, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, and T1337M, or corresponding amino acid substitutions thereof.

12 . (canceled)

13 . The method of claim 1 , wherein the polynucleotide programmable DNA binding domain is a nuclease inactive or nickase variant.

14 - 15 . (canceled)

16 . The method of claim 1 , wherein the adenosine deaminase domain is a TadA deaminase domain.

17 . The method of claim 16 , wherein the TadA deaminase domain is TadA*7.10 domain.

18 . The method of claim 1 , wherein the one or more guide RNAs comprises a CRISPR RNA (crRNA) and a trans-encoded small RNA (tracrRNA), wherein the crRNA comprises a nucleic acid sequence complementary to a Mecp2 nucleic acid sequence comprising the SNP associated with RTT; or

wherein the base editor is in complex with a single guide RNA (sgRNA) comprising a nucleic acid sequence complementary to an MECP2 nucleic acid sequence comprising the SNP associated with RTT.

19 . (canceled)

20 . A cell produced by introducing into the cell, or a progenitor thereof:

a base editor, or a polynucleotide encoding said base editor, to said cell, wherein said base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain; and

one or more guide polynucleotides that target the base editor to effect an A•T to G•C alteration of a single nucleotide polymorphism (SNP) associated with Rett Syndrome (RTT).

21 . The cell of claim 20 , wherein the cell is a neuron.

22 . The cell of claim 21 , wherein the neuron expresses a methyl-CpG-binding protein 2 (Mecp2) polypeptide.

23 . The cell of claim 20 , wherein the cell is from a subject having RTT.

24 . The cell of claim 20 , wherein the cell is a mammalian cell or a human cell.

25 - 41 . (canceled)

42 . A method of treating Rett Syndrome (RTT) in a subject comprising administering to said subject a cell of claim 20 ; or

a base editor, or a polynucleotide encoding said base editor, to said subject, wherein said base editor comprises a polynucleotide programmable DNA binding domain and an adenosine deaminase domain; and

one or more guide polynucleotides that target the base editor to effect an A•T to G•C alteration of a single nucleotide polymorphism (SNP) associated with RTT.

43 . The method of claim 42 , wherein the subject is a mammal or a human.

44 . The method of claim 42 , comprising delivering the base editor, or polynucleotide encoding said base editor, and said one or more guide polynucleotides to a cell of the subject.

45 . The method of claim 44 , wherein the cell is a neuron.

46 - 60 . (canceled)

61 . A base editor comprising:

(i) a modified Streptococcus pyogenes Cas9 (SpCas9) comprising the amino acid substitutions L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, T1337R and one or more of L1111, D1135L, 51136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, R1335Q, T1337, T1337L, T1337Q, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337H, T1337Q, and T1337M, or corresponding amino acid substitutions thereof; and

(ii) an adenosine deaminase; or

(i) a modified SpCas9 comprising the amino acid substitutions D1135L, S1136R, G1218S, E1219V, A1322R, R1335Q, and T1337, and one or more of L1111R, G1218R, E1219F, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, T1337L, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, and T1337M, or corresponding amino acid substitutions thereof; and

(ii) an adenosine deaminase.

62 . (canceled)