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 inserting a donor nucleic acid sequence into a cell using homologous recombination comprising
providing to the cell two guide RNAs with each guide RNA having a spacer sequence, a tracr mate sequence and a tracr sequence, and with a portion of the tracr sequence being hybridized to the tracr mate sequence and with the tracr mate sequence and the tracr sequence being linked by a linker nucleic acid sequence and with each spacer sequence being complementary to an adjacent site in a DNA target nucleic acid,
providing to the cell a donor nucleic acid sequence,
providing to the cell a Cas9 protein nickase, and
wherein each of the two guide RNAs co-localize with the Cas9 protein nickase to the DNA target nucleic acid resulting in an offset nick with 5′-overhangs, and
wherein the donor nucleic acid sequence is inserted into the target nucleic acid at the offset nick using homologous recombination.
2. The method of claim 1
wherein the two guide RNAs are provided to the cell by introducing into the cell a first foreign nucleic acid encoding the two guide 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 guide RNAs and the Cas9 protein nickase are expressed.
3. The method of claim 1 wherein the target nucleic acid is genomic DNA, mitochondrial DNA, viral DNA or exogenous DNA.
4. The method of claim 1 wherein the tracr sequence is between 90 to 500 nucleotides.
5. The method of claim 1 wherein the tracr sequence is between 90 to 200 nucleotides.
6. The method of claim 1 wherein the tracr sequence is between 100 to 200 nucleotides.