COMPOSITIONS AND METHODS FOR EPIGENOME EDITING

• US 20180023064A1
• Filed: 02/09/2016
• Published: 01/25/2018
• Est. Priority Date: 02/09/2015
• Status: Active Grant

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: GERSBACH, Charles A., HILTON, Isaac
• Granted Patent: US 10,676,726 B2
• Time in Patent Office: Days
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 203/01048 Histone acetyltransferase (2.3.1.48)

Abstract

Disclosed herein are CRISPR/Cas9-based gene activation systems that include a fusion protein of a Cas9 protein and a protein having histone acetyltransferase activity, and methods of using said systems.

Claims

1. A fusion protein comprising two heterologous polypeptide domains, wherein the first polypeptide domain comprises a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein and the second polypeptide domain comprises a peptide having histone acetyltransferase activity.

2. The fusion protein of claim 1, wherein the fusion protein activates transcription of a target gene.

3. The fusion protein of claim 1 or 2, wherein the Cas protein comprises Cas9.

4. The fusion protein of claim 3, wherein the Cas9 comprises at least one amino acid mutation which knocks out nuclease activity of Cas9.

5. The fusion protein of claim 4, wherein the Cas protein comprises SEQ ID NO:

1 or SEQ ID NO;

10.

6. The fusion protein of any one of claims 1-5, wherein the second polypeptide domain comprises a histone acetyltransferase effector domain.

7. The fusion protein of claim 6, wherein the histone acetyltransferase effector domain is a p300 histone acetyltransferase effector domain.

8. The fusion protein of any one of claims 1-7, wherein the second polypeptide domain comprises SEQ ID NO:

2 or SEQ ID NO;

3.

9. The fusion protein of any one of claims 1-8, wherein the first polypeptide domain comprises SEQ ID NO:

1 or SEQ ID NO;

10 and the second polypeptide domain comprises SEQ ID NO;

2 or SEQ ID NO. 3.

10. The fusion protein of any one of claims 1-9, wherein the first polypeptide domain comprises SEQ ID NO:

1 and the second polypeptide domain comprises SEQ ID NO. 3, or the first polypeptide domain comprises SEQ ID NO;

10 and the second polypeptide domain comprises SEQ ID NO. 3.

11. The fusion protein of any one of claims 1-10, further comprising a linker connecting the first polypeptide domain to the second polypeptide domain.

12. The fusion protein of any one of claims 1-11, wherein the fusion protein comprises an amino acid sequence of SEQ ID NO:

140, 141, or 149.

13. A DNA targeting system comprising the fusion protein of any one of claims 1-12 and at least one guide RNA (gRNA).

14. The DNA targeting system of claim 13, wherein the at least one gRNA comprises a 12-22 base pair complementary polynucleotide sequence of the target DNA sequence followed by a protospacer-adjacent motif.

15. The DNA targeting system of claim 13 or 14, wherein the at least one gRNA targets a target region, the target region comprises a target enhancer, target regulatory element, a cis-regulatory region of a target gene, or a trans-regulatory region of a target gene.

16. The DNA targeting system of claim 15, wherein the target region is a distal or proximal cis-regulatory region of the target gene.

17. The DNA targeting system of claim 15 or 16, wherein the target region is an enhancer region or a promoter region of the target gene.

18. The DNA targeting system of any one of claims 15-17, wherein the target gene is an endogenous gene or a transgene.

19. The DNA targeting system of claim 15, wherein the target region comprises a target enhancer or a target regulatory element.

20. The DNA targeting system of claim 19, wherein the target enhancer or target regulatory element control the gene expression of more than one target gene.

21. The DNA targeting system of any one of claims 15-20, wherein the DNA targeting system comprises between one and ten different gRNAs.

22. The DNA targeting system of any one of claims 15-21, wherein the DNA targeting system comprises one gRNA.

23. The DNA targeting system of any one of claims 15-22, wherein the target region is located on the same chromosome as the target gene.

24. The DNA targeting system of claim 23, wherein the target region is located about 1 base pair to about 100,000 base pairs upstream of a transcription start site of the target gene.

25. The DNA targeting system of claim 24, wherein the target region is located about 1000 base pairs to about 50,000 base pairs upstream of the transcription start site of the target gene.

26. The DNA targeting system of any one of claims 15-22, wherein the target region is located on a different chromosome as the target gene.

27. The DNA targeting system of any one of claims 15-28, wherein the different gRNAs bind to different target regions.

28. The DNA targeting system of claim 27, wherein the different gRNAs bind to target regions of different target genes.

29. The DNA targeting system of claim 27, wherein the expression of two or more target genes are activated.

30. The DNA targeting system of any one of claims 15-29, wherein the target gene is selected from the group consisting of IL1RN, MYOD1, OCT4, HBE, HBG, HBD, HBB, MYOCD, PAX7, FGF1A, FGF1B, and FGF1C.

31. The DNA targeting system of claim 30, wherein the target region is at least one of HS2 enhancer of the human β

-globin locus, distal regulatory region (DRR) of the MYOD gene, core enhancer (CE) of the MYOD gene, proximal (PE) enhancer region of the OCT4 gene, or distal (DE) enhancer region of the OCT4 gene.

32. The DNA targeting system of any one of claims 13-31, wherein the gRNA comprises at least one of SEQ ID NOs:

23-73, 188-223, or 224-254.

33. An isolated polynucleotide encoding the fusion protein of any one of claims 1-12 or the DNA targeting system of any one of claims 13-32.

34. A vector comprising the isolated polynucleotide of claim 33.

35. A cell comprising the isolated polynucleotide of claim 33 or the vector of claim 34.

36. A kit comprising the fusion protein of any one of claims 1-12, the DNA targeting system of claims 13-32, the isolated polynucleotide of claim 33, the vector of claim 34, or the cell of claim 35.

37. A method of activating gene expression of a target gene in a cell, the method comprising contacting the cell with the fusion protein of any one of claims 1-12, the DNA targeting system of claims 13-32, the isolated polynucleotide of claim 33, or the vector of claim 34.

38. A method of activating gene expression of a target gene in a cell, the method comprising contacting the cell with a polynucleotide encoding a DNA targeting system, wherein the DNA targeting system comprises the fusion protein of any one of claims 1-12 and at least one guide RNA (gRNA).

39. The method of claim 38, wherein the at least one gRNA comprises a 12-22 base pair complementary polynucleotide sequence of the target DNA sequence followed by a protospacer-adjacent motif.

40. The method of claim 38 or 39, wherein the at least one gRNA targets a target region, the target region is a cis-regulatory region or a trans-regulatory region of a target gene.

41. The method of claim 40, wherein the target region is a distal or proximal cis-regulatory region of the target gene.

42. The method of claim 40 or 41, wherein the target region is an enhancer region or a promoter region of the target gene.

43. The method of claim 40-42, wherein the target gene is an endogenous gene or a transgene.

44. The method of claim 43, wherein the DNA targeting system comprises between one and ten different gRNAs.

45. The method of claim 43, wherein the DNA targeting system comprises one gRNA.

46. The method of claim 40-45, wherein the target region is located on the same chromosome as the target gene.

47. The method of claim 46, wherein the target region is located about 1 base pair to about 100,000 base pairs upstream of a transcription start site of the target gene.

48. The method of claim 46, wherein the target region is located about 1000 base pairs to about 50,000 base pairs upstream of the transcription start site of the target gene.

49. The method of claim 40-45, wherein the target region is located on a different chromosome as the target gene.

50. The method of claim 40-45, wherein the different gRNAs bind to different target regions.

51. The method of claim 50, wherein the different gRNAs bind to target regions of different target genes.

52. The method of claim 51, wherein the expression of two or more target genes are activated.

53. The method of claim 40-52, wherein the target gene is selected from the group consisting of IL1RN, MYOD1, OCT4, HBE, HBG, HBD, HBB, MYOCD, PAX7, FGF1A, FGF1B, and FGF1C.

54. The method of claim 53, wherein the target region is at least one of HS2 enhancer of the human β

-globin locus, distal regulatory region (DRR) of the MYOD gene, core enhancer (CE) of the MYOD gene, proximal (PE) enhancer region of the OCT4 gene, or distal (DE) enhancer region of the OCT4 gene.

55. The method of claim 37-54, wherein the gRNA comprises at least one of SEQ ID NOs:

23-73, 188-223, or 224-254.

56. The method of any one of claims 37-55, wherein the DNA targeting system is delivered to the cell virally or non-virally.

57. The method of any one of claims 37-56, wherein the cell is a mammalian cell.