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 localizing an effector domain to a target nucleic acid sequence 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 the effector domain 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 or nickase 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 effector domain fused to the RNA binding domain and the Cas9 protein, and
wherein the guide RNA including the effector domain connected thereto and the Cas9 protein co-localize to the target nucleic acid sequence.
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.
8. The method of claim 1 wherein the aptamer comprises two copies of MS2 bacteriophage coat-protein binding RNA stem-loop.
9. The method of claim 1 wherein the RNA binding domain comprises MS2 bacteriophage coat-protein.
10. 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 have the effector domain connected thereto.
11. A method of editing a target gene in a eukaryotic cell comprising
providing to the cell a nucleic acid encoding a guide RNA complementary to a target DNA sequence within or adjacent to the target gene 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 an effector domain 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 nickase 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 effector domain fused to the RNA binding domain and the Cas9 protein, and
wherein the guide RNA including the effector domain connected thereto and the Cas9 protein co-localize to the target DNA sequence and thereby edit the target gene.
12. The method of claim 11 wherein the cell is a yeast cell, a plant cell or a mammalian cell.
13. The method of claim 11 wherein the cell is a human cell.
14. The method of claim 11 wherein the guide RNA is between about 10 to about 250 nucleotides.
15. The method of claim 11 wherein the guide RNA is between about 20 to about 100 nucleotides.
16. The method of claim 11 wherein the guide RNA is between about 100 to about 250 nucleotides.
17. The method of claim 11 wherein the target nucleic acid is genomic DNA, mitochondrial DNA, viral DNA or exogenous DNA.
18. The method of claim 11 wherein the aptamer comprises two copies of MS2 bacteriophage coat-protein binding RNA stem-loop.
19. The method of claim 11 wherein the RNA binding domain comprises MS2 bacteriophage coat-protein.
20. The method of claim 11 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 have the effector domain connected thereto.