RNA-guided transcriptional regulation

• US 9,267,135 B2
• Filed: 06/30/2014
• Issued: 02/23/2016
• 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): Marvich, Maria
• Application Number:
  US14/319,289
• Publication Number:
  US 20140356959A1
• Time in Patent Office: 603 Days
• US Class Current: 1/1
• 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 gene in a cell in vitro comprising

introducing into the cell a nucleic acid encoding a guide RNA fused to a target of an RNA-binding domain wherein the nucleic acid is operably linked to an expression control sequence, wherein the guide RNA is complementary to a target nucleic acid sequence comprising the gene,

introducing into the cell a second nucleic acid encoding a transcriptional regulator protein or domain that modulates the target nucleic acid expression, and which is fused to an RNA-binding domain wherein the second nucleic acid is operably linked to an expression control sequence,

introducing into the cell a third nucleic acid encoding a nuclease null Cas9 protein of a Type II CRISPR system wherein the third nucleic acid is operably linked to an expression control sequence, wherein the nuclease null Cas9 protein interacts with the guide RNA which binds to the target nucleic acid sequence in a site specific manner,

wherein the cell produces the guide RNA fused to the target of the RNA-binding domain, the Cas9 protein and the transcriptional regulator protein or domain fused to the RNA-binding domain,

wherein the guide RNA and the Cas9 protein co-localize to the target nucleic acid sequence and wherein the transcriptional regulator protein or domain modulates expression of the gene.

2. The method of claim 1 wherein the cell is a eukaryotic cell.

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

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

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

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

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

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

9. The method of claim 1 wherein the transcriptional regulator protein or domain is a transcriptional activator.

10. A method of modulating expression of a viral gene in a cell in vitro comprising

introducing into the cell a nucleic acid encoding a guide RNA fused to a target of an RNA-binding domain wherein the nucleic acid is operably linked to an expression control sequence, wherein the guide RNA is complementary to a viral target nucleic acid sequence comprising the viral gene,

introducing into the cell a second nucleic acid encoding a transcriptional regulator protein or domain that modulates the target nucleic acid expression, and which is fused to an RNA-binding domain wherein the second nucleic acid is operably linked to an expression control sequence,

introducing into the cell a third nucleic acid encoding a nuclease null Cas9 protein of a Type II CRISPR system, wherein the third nucleic acid is operably linked to an expression control sequence, wherein the nuclease null Cas9 protein interacts with the guide RNA which binds to the viral target nucleic acid sequence in a site specific manner,

wherein the cell produces the guide RNA fused to the target of the RNA-binding domain, the Cas9 protein and the transcriptional regulator protein or domain fused to the RNA-binding domain,

wherein the guide RNA and the Cas9 protein co-localize to the viral target nucleic acid sequence and wherein the transcriptional regulator protein or domain modulates expression of the viral gene.

11. The method of claim 10 wherein the cell is a eukaryotic cell.

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

13. The method or claim 10 wherein the cell is a human cell.

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

15. The method of claim 10 wherein the guide RNA is between about 20 to about 100 nucleotides.

16. The method of claim 10 wherein the guide RNA is between about 100 to about 250 nucleotides.

17. The method of claim 10 wherein the transcriptional regulator protein or domain is a transcriptional activator.