RNA-Guided Transcriptional Regulation

• US 20140356959A1
• Filed: 06/30/2014
• Published: 12/04/2014
• 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.
• Granted Patent: US 9,267,135 B2
• Time in Patent Office: Days
• US Class Current: 435/455
• 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 cell comprising

providing to the cell a guide RNA complementary to the target nucleic acid sequence including a transcriptional activator or repressor domain as a fusion protein for modulating target nucleic acid expression in vivo,

providing to the cell a nuclease null Cas9 protein that interacts with the guide RNA and binds to the target nucleic acid sequence in a site specific manner,

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

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

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

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

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

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

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

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

2. The method of claim 2

wherein the guide RNA including the transcriptional activator or repressor domain as a fusion protein is provided to the cell by introducing to the cell a nucleic acid encoding the guide RNA including the transcriptional activator or repressor domain as a fusion protein,

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 guide RNA including the transcriptional activator or repressor domain as a fusion protein and the Cas9 protein.

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

providing to the cell a guide RNA complementary to viral DNA including the target nucleic acid sequence, wherein the guide RNA includes a transcriptional activator or repressor domain as a fusion protein for modulating target nucleic acid expression in vivo,

providing to the cell a nuclease null Cas9 protein that interacts with the guide RNA and binds to the target nucleic acid sequence in a site specific manner,

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

11. The method of claim 10

wherein the guide RNA including the transcriptional activator or repressor domain as a fusion protein is provided to the cell by introducing to the cell a nucleic acid encoding the guide RNA including the transcriptional activator or repressor domain as a fusion protein,

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 guide RNA including the transcriptional activator or repressor domain as a fusion protein and the Cas9 protein.

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

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

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

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

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

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