Genome engineering
Methods are provided for the use of Cas9 in genome engineering of stem cells. Methods include introducing into the stem cell a first foreign nucleic acid encoding a guide RNA complementary to a target DNA and which guides a Cas9 enzyme to the target DNA, wherein the RNA and the enzyme are members of a co-localization complex for the target DNA, introducing into the stem cell a second foreign nucleic acid encoding a donor nucleic acid sequence, wherein the guide RNA and the donor nucleic acid sequences are expressed, wherein the guide RNA and the Cas 9 enzyme co-localize to the target DNA, the Cas 9 enzyme cleaves the target DNA and the donor nucleic acid is inserted into the target DNA to produce altered DNA in the stem cell.
1. A method of altering target DNA in a human induced pluripotent stem cell in vitro comprising providing to the stem cell a Cas 9 enzyme that forms a co-localization complex with a guide RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner,
introducing into the stem cell a guide RNA complementary to the target DNA and which guides the enzyme to the target DNA, wherein the guide RNA and the enzyme are members of a co-localization complex for the target DNA,
introducing into the stem cell a donor nucleic acid sequence, wherein the guide RNA and the donor nucleic acid sequence are linked,
wherein the guide RNA and the Cas 9 enzyme co-localize to the target DNA, the Cas 9 enzyme cleaves the target DNA and the donor nucleic acid sequence is inserted into the target DNA to produce altered DNA in the stem cell.
2. The method of claim 1 wherein the guide RNA is between about 10 to about 500 nucleotides.
3. The method of claim 1 wherein the guide RNA is between about 20 to about 100 nucleotides.
4. The method of claim 1 wherein the guide RNA is a crRNA-tracrRNA fusion.
5. The method of claim 1 wherein the donor nucleic acid sequence is inserted by homologous recombination.
6. The method of claim 1 wherein the donor nucleic acid sequence is inserted by nonhomologous end joining.
7. The method of claim 1 wherein the guide RNA is introduced into the stem cell as a first foreign nucleic acid which is expressed by the stem cell.
8. The method of claim 1 further comprising introducing multiple donor nucleic acid sequences and multiple guide RNAs to produce multiple alterations to the DNA in the cell.
9. The method of claim 1 wherein the Cas9 enzyme is S. pyogenes Cas9.
10. The method of claim 1 wherein the target DNA is genomic DNA.
11. The method of claim 1 wherein the guide RNA is about 100 nucleotides.