Compositions and methods for gene editing

1. A fusion protein comprising, from N-terminus to C-terminus:

(i) a DNA methyltransferase domain, a nuclease-deficient RNA-guided DNA endonuclease enzyme, and a Krüppel associated box domain; or

(ii) a Krüppel associated box domain, a nuclease-deficient RNA-guided DNA endonuclease enzyme, and a DNA methyltransferase domain.

2. The fusion protein of claim 1 , wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is a nuclease-deficient Class II CRISPR endonuclease.

3. The fusion protein of claim 1 , wherein the DNA methyltransferase domain comprises a Dnmt3A protein and a Dnmt3L protein (a Dnmt3A-3L domain).

4. The fusion protein of claim 1 , wherein the fusion protein comprises, from N-terminus to C-terminus, the DNA methyltransferase domain, the nuclease-deficient RNA-guided DNA endonuclease enzyme, and the Krüppel associated box domain.

5. The fusion protein of claim 4 , wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9 and the DNA methyltransferase domain comprises a Dnmt3A protein and a Dnmt3L protein (a Dnmt3A-3L domain); wherein the dCas9 is covalently linked to the Dnmt3A-3L domain via an XTEN linker.

6. The fusion protein of claim 1 , wherein the fusion protein comprises, from N-terminus to C-terminus, the Krüppel associated box, the nuclease-deficient RNA-guided DNA endonuclease enzyme, an XTEN linker, and the DNA methyltransferase domain.

7. The fusion protein of claim 1 , wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9 and the DNA methyltransferase domain comprises a Dnmt3A protein and a Dnmt3L protein (a Dnmt3A-3L domain).

8. The fusion protein of claim 7 , wherein the dCas9 is covalently linked to the Dnmt3A-3L domain via a XTEN linker and wherein the Krüppel associated box domain is covalently linked to the dCas9 via a peptide linker.

9. The fusion protein of claim 1 , wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is covalently linked to the Krüppel associated box domain via a peptide linker.

10. The fusion protein of claim 1 , wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is covalently linked to the DNA methyltransferase domain via a peptide linker.

11. The fusion protein of claim 1 , wherein the Krüppel associated box domain in (ii) is covalently linked to the DNA methyltransferase domain via an XTEN linker.

12. The fusion protein of claim 5 , wherein the XTEN linker comprises about 16 to 80 amino acid residues.

13. The fusion protein of claim 1 , further comprising a nuclear localization signal peptide.

14. The fusion protein of claim 1 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 12, 13, 14, or 15.

15. A nucleic acid sequence encoding the fusion protein of claim 1 .

16. A complex comprising: (i) a fusion protein of claim 1 ; and (ii) a polynucleotide comprising: (a) a DNA-targeting sequence that is complementary to a target polynucleotide sequence; and (b) a binding sequence for the nuclease-deficient RNA-guided DNA endonuclease enzyme, wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is bound to the polynucleotide via the binding sequence.

17. A vector comprising a nucleic acid sequence encoding the fusion protein of claim 1 .

18. A cell comprising the fusion protein of claim 1 .

19. A method of silencing a target nucleic acid sequence in a cell, comprising: (i) delivering a first polynucleotide encoding a fusion protein of claim 1 to a cell containing the target nucleic acid; and (ii) delivering to the cell a second polynucleotide comprising: (a) a DNA-targeting sequence that is complementary to the target nucleic acid sequence; and (b) a binding sequence for the nuclease-deficient RNA-guide DNA endonuclease enzyme.

20. A method of silencing a target nucleic acid sequence in a cell, the method comprising delivering a complex to a cell containing the target nucleic acid; wherein the complex comprises: (i) a fusion protein of claim 1 ; and (ii) a polynucleotide comprising: (a) a DNA-targeting sequence that is complementary to a target polynucleotide sequence; and (b) a binding sequence for the nuclease-deficient RNA-guided DNA endonuclease enzyme, wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is bound to the polynucleotide via the binding sequence.

21. The fusion protein of claim 1 , wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9 which comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:23; ddCas12a which comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:34; dLbCfp1 which comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:35; or dFnCfp1 which comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:36.

22. The fusion protein of claim 1 , wherein the K{umlaut over (r)}uppel associated box domain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:16.

23. The fusion protein of claim 3 , wherein the Dnmt3A protein comprises an amino acid sequence with at least 85% sequence identity to SEQ ID NO:26, and the Dnmt3L protein comprises an amino acid sequence with at least 85% sequence identity to SEQ ID NO:28.

24. The fusion protein of claim 1 , wherein the fusion protein comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:15.

25. The fusion protein of claim 1 , wherein the nuclease-deficient RNA-guided DNA endonuclease enzyme is selected from the group consisting of dCas9, ddCpf1, ddCas12a, ddLbCpf1 and ddFnCpf1.

26. A fusion protein comprising, a DNA methyltransferase domain, a nuclease-deficient RNA-guided DNA endonuclease enzyme, and a Krüppel associated box domain; wherein

(1) the nuclease-deficient RNA-guided DNA endonuclease enzyme is dCas9 which comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:23; ddCas12a which comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:34; dLbCfp1 which comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:35; or dFnCfp1 which comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:36;

(2) the Krüppel associated box domain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:16; and

(3) the DNA methyltransferase domain comprises a Dnmt3A protein, a Dnmt3L protein, or a Dnmt3A protein and a Dnmt3L protein; wherein the Dnmt3A protein comprises an amino acid sequence with at least 85% sequence identity to SEQ ID NO:26, and the Dnmt3L protein comprises an amino acid sequence with at least 85% sequence identity to SEQ ID NO:28.

27. The fusion protein of claim 26 , wherein the Krüppel associated box domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:16.

28. The fusion protein of claim 27 , wherein the Krüppel associated box domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:16.

29. The fusion protein of claim 28 , wherein the Krüppel associated box domain comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:16.

30. The fusion protein of claim 26 , wherein the Dnmt3A protein comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:26.

31. The fusion protein of claim 30 , wherein the Dnmt3A protein comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO:26.

32. The fusion protein of claim 31 , wherein the Dnmt3A protein comprises an amino acid sequence with at least 99% sequence identity to SEQ ID NO:26.

33. The fusion protein of claim 26 , wherein the Dnmt3L protein comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:28.

34. The fusion protein of claim 33 , wherein the Dnmt3L protein comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO:28.

35. The fusion protein of claim 34 , wherein the Dnmt3L protein comprises an amino acid sequence with at least 99% sequence identity to SEQ ID NO:28.

36. The fusion protein of claim 26 , wherein the dCas9 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:23.

37. The fusion protein of claim 36 , wherein the dCas9 comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:23.

38. The fusion protein of claim 37 , wherein the dCas9 comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:23.

39. The fusion protein according to claim 25 , wherein the Krüppel associated box domain comprises an amino acid sequence of SEQ ID NO: 16, the Dnmt3A protein comprises an amino acid sequence of SEQ ID NO: 26, the Dnmt3L protein comprises an amino acid sequence of SEQ ID NO: 28 and the dCas9 comprises an amino acid sequence of SEQ ID NO: 23.

40. The fusion protein according to claim 39 , wherein the fusion protein comprises, from N-terminus to C-terminus, a DNA methyltransferase domain, a nuclease-deficient RNA-guided DNA endonuclease enzyme, and a Krüppel associated box domain.

41. The fusion protein according to claim 39 , wherein the fusion protein comprises, from N-terminus to C-terminus, a DNA methyltransferase domain, a nuclease-deficient RNA-guided DNA endonuclease enzyme, and a Krüppel associated box domain.