RNA-guided transcriptional regulation

• US 10,640,789 B2
• Filed: 02/22/2016
• Issued: 05/05/2020
• Est. Priority Date: 06/04/2013
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Church, George M., Mali, Prashant G., Esvelt, Kevin M.
• Primary Examiner(s): Wu, Julie
• Application Number:
  US15/049,451
• Publication Number:
  US 20160237456A1
• Time in Patent Office: 1,534 Days
• Field of Search: None
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• 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 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/635 Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/00 Hydrolases acting on ester bonds (3.1)

Abstract

Methods of modulating expression of a target nucleic acid in a cell are provided including introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to DNA, wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a nuclease-null Cas9 protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the nuclease-null Cas9 protein, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the nuclease-null Cas9 protein and the transcriptional regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid.

Claims

1. A method of modulating expression of a target nucleic acid in a eukaryotic cell comprising

providing to the cell a nucleic acid encoding a guide RNA complementary to the target nucleic acid sequence and an aptamer comprising a target of an RNA binding domain, wherein the aptamer is attached to the 3′

end or the 5′

end of the guide RNA, wherein the guide RNA is a tracrRNA-crRNA fusion,

providing to the cell a nucleic acid encoding a transcriptional activator or repressor and an RNA binding domain, wherein the RNA binding domain binds to the target of the RNA binding domain,

providing to the cell a nucleic acid encoding a nuclease null Cas9 protein that interacts with the guide RNA, and

wherein the cell expresses the guide RNA having the aptamer attached to the 3′

end or the 5′

end of the guide RNA, the transcriptional activator or repressor having the RNA binding domain and the Cas9 protein, and

wherein the guide RNA including the transcriptional activator or repressor connected thereto and the Cas9 protein co-localize to the target nucleic acid sequence and wherein the transcriptional activator or repressor modulates expression of the target nucleic acid.

2. The method of claim 1 wherein the cell is a yeast cell, a plant cell or a mammalian cell.

3. The method or claim 1 wherein the cell is a human cell.

4. The method of claim 1 wherein the guide RNA is between about 10 to about 250 nucleotides.

5. The method of claim 1 wherein the guide RNA is between about 20 to about 100 nucleotides.

6. The method of claim 1 wherein the guide RNA is between about 100 to about 250 nucleotides.

7. The method of claim 1 wherein the target nucleic acid is genomic DNA, mitochondrial DNA, viral DNA or exogenous DNA.

15. The method of claim 1 wherein the aptamer comprises two copies of MS2 bacteriophage coat-protein binding RNA stem-loop.

16. The method of claim 1 wherein the RNA binding domain comprises MS2 bacteriophage coat-protein.

29. The method of claim 1 wherein multiple guide RNAs are introduced to the cell with each guide RNA being complementary to a different target nucleic acid sequence and having an aptamer comprising a target of an RNA binding domain, wherein the aptamer is attached to the 3′

end or the 5′

end of the guide RNA, wherein each guide RNA is a tracrRNA-crRNA fusion, and wherein multiple guide RNAs having the transcriptional activator or repressor connected thereto modulate expression of the target nucleic acid.

30. The method of claim 1 wherein multiple guide RNAs are introduced to the cell with each guide RNA being complementary to a different target nucleic acid sequence and having an aptamer comprising a target of an RNA binding domain, wherein the aptamer is attached to the 3′

end or the 5′

end of the guide RNA, wherein each guide RNA is a tracrRNA-crRNA fusion, and wherein multiple guide RNAs having the transcriptional activator or repressor connected thereto modulate expression of different target nucleic acids.

8. A method of modulating expression of a target nucleic acid in a eukaryotic cell comprising

providing to the cell a nucleic acid encoding a transcriptional activator or repressor and an RNA binding domain,

providing to the cell a nucleic acid encoding a guide RNA having a spacer sequence fully complementary to viral DNA including the target nucleic acid sequence and an aptamer comprising a target of an RNA binding domain attached to the 3′

end or the 5′

end of the guide RNA, wherein the guide RNA is a tracrRNA-crRNA fusion, and wherein the RNA binding domain binds to the target of the RNA binding domain, wherein the cell expresses the guide RNA having the aptamer attached to the 3′

end or the 5′

end of the guide RNA and the transcriptional activator or repressor having the RNA binding domain,

providing to the cell a nuclease null Cas9 protein that interacts with the guide RNA,

wherein the guide RNA including the transcriptional activator or repressor connected thereto and the Cas9 protein co-localize to the target nucleic acid sequence and wherein the transcriptional activator or repressor modulates expression of the target nucleic acid.

9. The method of claim 8

wherein the Cas9 protein is provided to the cell by introducing to the cell a nucleic acid encoding the Cas9 protein, and

wherein the cell expresses the Cas9 protein.

10. The method of claim 8 wherein the cell is a yeast cell, a plant cell or a mammalian cell.

11. The method or claim 8 wherein the cell is a human cell.

12. The method of claim 8 wherein the guide RNA is between about 10 to about 250 nucleotides.

13. The method of claim 8 wherein the guide RNA is between about 20 to about 100 nucleotides.

14. The method of claim 8 wherein the guide RNA is between about 100 to about 250 nucleotides.

17. The method of claim 9 wherein the aptamer comprises two copies of MS2 bacteriophage coat-protein binding RNA stem-loop.

18. The method of claim 9 wherein the RNA binding domain comprises MS2 bacteriophage coat-protein.

31. The method of claim 8 wherein multiple guide RNAs are introduced to the cell with each guide RNA being complementary to a different target nucleic acid sequence and having an aptamer comprising a target of an RNA binding domain, wherein the aptamer is attached to the 3′

end or the 5′

end of the guide RNA, wherein each guide RNA is a tracrRNA-crRNA fusion, and wherein multiple guide RNAs having the transcriptional activator or repressor connected thereto modulate expression of the target nucleic acid.

32. The method of claim 8 wherein multiple guide RNAs are introduced to the cell with each guide RNA being complementary to a different target nucleic acid sequence and having an aptamer comprising a target of an RNA binding domain, wherein the aptamer is attached to the 3′

end or the 5′

end of the guide RNA, wherein each guide RNA is a tracrRNA-crRNA fusion, and wherein multiple guide RNAs having the transcriptional activator or repressor connected thereto modulate expression of different target nucleic acids.

19. A method of modulating expression of a target nucleic acid in a eukaryotic cell comprising

providing to the cell a guide RNA complementary to the target nucleic acid sequence, and an aptamer comprising a target of an RNA binding domain, wherein the aptamer is attached to the 3′

end or the 5′

end of the guide RNA, wherein the guide RNA is a tracrRNA-crRNA fusion,

providing to the cell a transcriptional activator or repressor and an RNA binding domain attached thereto, wherein the RNA binding domain binds to the target of the RNA binding domain,

providing to the cell a nuclease null Cas9 protein that interacts with the guide RNA,

wherein the guide RNA including the transcriptional activator or repressor connected thereto and the Cas9 protein co-localize to the target nucleic acid sequence and wherein the transcriptional activator or repressor modulates expression of the target nucleic acid.

20. The method of claim 19 wherein the cell is a yeast cell, a plant cell or a mammalian cell.

21. The method or claim 19 wherein the cell is a human cell.

22. The method of claim 19 wherein the guide RNA is between about 10 to about 250 nucleotides.

23. The method of claim 19 wherein the guide RNA is between about 20 to about 100 nucleotides.

24. The method of claim 19 wherein the guide RNA is between about 100 to about 250 nucleotides.

25. The method of claim 19 wherein the aptamer comprises two copies of MS2 bacteriophage coat-protein binding RNA stem-loop.

26. The method of claim 19 wherein the RNA binding domain comprises MS2 bacteriophage coat-protein.

27. The method of claim 19 wherein multiple guide RNAs are introduced to the cell with each guide RNA being complementary to a different target nucleic acid sequence and having an aptamer comprising a target of an RNA binding domain, wherein the aptamer is attached to the 3′

end or the 5′

end of the guide RNA, wherein each guide RNA is a tracrRNA-crRNA fusion, and wherein multiple guide RNAs having the transcriptional activator or repressor connected thereto modulate expression of the target nucleic acid.

28. The method of claim 19 wherein multiple guide RNAs are introduced to the cell with each guide RNA being complementary to a different target nucleic acid sequence and having an aptamer comprising a target of an RNA binding domain, wherein the aptamer is attached to the 3′

end or the 5′

end of the guide RNA, wherein each guide RNA is a tracrRNA-crRNA fusion, and wherein multiple guide RNAs having the transcriptional activator or repressor connected thereto modulate expression of different target nucleic acids.