RNA-guided transcriptional regulation
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.
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.