RNA-Guided Transcriptional Regulation

• US 20140356956A1
• Filed: 06/30/2014
• Published: 12/04/2014
• Est. Priority Date: 06/04/2013
• Status: Active Application

• 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.
• Application Number:
  US14/319,530
• Publication Number:
  US 20140356956A1
• Time in Patent Office: Days
• US Class Current: 435/441
• 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 altering a target nucleic acid in a cell comprising

providing to the cell two or more RNAs with each RNA being complementary to an adjacent site in the target nucleic acid,

providing to the cell a Cas9 protein nickase and being guided by the two or more RNAs, and

wherein the two or more RNAs and the Cas9 protein nickase co-localize to the DNA target nucleic acid and nick the target nucleic acid resulting in two or more adjacent nicks.

2. The method of claim 1

wherein the two or more RNAs are provided to the cell by introducing into the cell a first foreign nucleic acid encoding the two or more RNAs,

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

wherein the two or more RNAs and the Cas9 protein nickase are expressed and wherein the Cas9 protein nickase co-localizes with the two or more RNAs to the target nucleic acid and nicks the DNA target nucleic acid resulting in two or more adjacent nicks.

3. The method of claim 1 wherein the two or more adjacent nicks are on the same strand of the double stranded DNA.

4. The method of claim 1 wherein the two or more adjacent nicks are on the same strand of the double stranded DNA and result in homologous recombination.

5. The method of claim 1 wherein the two or more adjacent nicks are on different strands of the double stranded DNA.

6. The method of claim 1 wherein the two or more adjacent nicks are on different strands of the double stranded DNA and create double stranded breaks.

7. The method of claim 1 wherein the two or more adjacent nicks are on different strands of the double stranded DNA and create double stranded breaks resulting in nonhomologous end joining.

8. The method of claim 1 wherein the two or more adjacent nicks are on different strands of the double stranded DNA and are offset with respect to one another.

9. The method of claim 1 wherein the two or more adjacent nicks are on different strands of the double stranded DNA and are offset with respect to one another and create double stranded breaks.

10. The method of claim 1 wherein the two or more adjacent nicks are on different strands of the double stranded DNA and are offset with respect to one another and create double stranded breaks resulting in nonhomologous end joining.

11. The method of claim 1 further including introducing into the cell a third foreign nucleic acid encoding a donor nucleic acid sequence wherein the two or more nicks results in homologous recombination of the target nucleic acid with the donor nucleic acid sequence.

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

24. The method of claim 2 further including introducing into the cell a third foreign nucleic acid encoding a donor nucleic acid sequence wherein the two or more nicks results in homologous recombination of the target nucleic acid with the donor nucleic acid sequence.

13. A method of altering a viral DNA target nucleic acid in a cell comprising

providing to the cell two or more RNAs with each RNA being complementary to an adjacent site in the viral DNA target nucleic acid,

providing to the cell a Cas9 protein nickase and being guided by the two or more RNAs, and

wherein the two or more RNAs and the Cas9 protein nickase co-localize to the viral DNA target nucleic acid and nick the viral DNA target nucleic acid resulting in two or more adjacent nicks.

14. The method of claim 13

wherein the two or more RNAs are provided to the cell by introducing into the cell a first foreign nucleic acid encoding the two or more RNAs,

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

wherein the two or more RNAs and the Cas9 protein nickase are expressed and wherein the Cas9 protein nickase co-localizes with the two or more RNAs to the viral DNA target nucleic acid and nicks the viral DNA target nucleic acid resulting in two or more adjacent nicks.

15. The method of claim 13 wherein the two or more adjacent nicks are on the same strand of the double stranded DNA.

16. The method of claim 13 wherein the two or more adjacent nicks are on the same strand of the double stranded DNA and result in homologous recombination.

17. The method of claim 13 wherein the two or more adjacent nicks are on different strands of the double stranded DNA.

18. The method of claim 13 wherein the two or more adjacent nicks are on different strands of the double stranded DNA and create double stranded breaks.

19. The method of claim 13 wherein the two or more adjacent nicks are on different strands of the double stranded DNA and create double stranded breaks resulting in nonhomologous end joining.

20. The method of claim 13 wherein the two or more adjacent nicks are on different strands of the double stranded DNA and are offset with respect to one another.

21. The method of claim 13 wherein the two or more adjacent nicks are on different strands of the double stranded DNA and are offset with respect to one another and create double stranded breaks.

22. The method of claim 13 wherein the two or more adjacent nicks are on different strands of the double stranded DNA and are offset with respect to one another and create double stranded breaks resulting in nonhomologous end joining.

23. The method of claim 13 further including providing to the cell a donor nucleic acid sequence wherein the two or more nicks results in homologous recombination of the target nucleic acid with the donor nucleic acid sequence.