Genome Engineering
AssigneePresident and Fellows of Harvard College
InventorsGeorge M. CHURCH, Luhan YANG, Marc Guell CARGOL, Joyce Lichi YANG
Life of this patent
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Abstract
Methods of genome engineering in cells using a TALEN lacking repeat sequences or Cas9 is provided.
Claims18
A method of altering target DNA in a stem cell expressing 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 comprising
- (a) introducing into the stem cell a first foreign nucleic acid encoding the guide RNA complementary to the target DNA and which guides the 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.
The method of claim 104 wherein the guide RNA is between about 10 to about 500 nucleotides.
The method of claim 104 wherein the guide RNA is between about 20 to about 100 nucleotides.
The method of claim 104 wherein the guide RNA is a tracrRNA-crRNA fusion.
The method of claim 104 wherein the donor nucleic acid sequence is inserted by homologous recombination.
The method of claim 104 wherein the donor nucleic acid sequence is inserted by nonhomologous end joining.
The method of claim 104 wherein the RNA and the donor nucleic acid sequences are present on one or more plasmids.
The method of claim 104 further comprising repeating step (a) multiple times to produce multiple alterations to the DNA in the cell.
The method of claim 104 wherein after producing altered DNA in a stem cell, a nucleic acid encoding the Cas 9 enzyme is removed from the stem cell genome.
The method of claim 104 wherein the RNA and the donor nucleic acid sequences are expressed as a bound nucleic acid sequence.
A stem cell including a first foreign nucleic acid encoding for a Cas 9 enzyme that forms a co-localization complex with RNA complementary to target DNA and that cleaves the target DNA in a site specific manner.
The stem cell of claim 120 further including a second foreign nucleic acid encoding for a guide RNA complementary to the target DNA and which guides the enzyme to the target DNA, wherein the RNA and the enzyme are members of a co-localization complex for the target DNA.
The stem cell of claim 121 further including a third foreign nucleic acid encoding a donor nucleic acid sequence.
The stem cell of claim 120 further including an inducible promoter for promoting expression of the enzyme.
The stem cell of claim 120 wherein the first foreign nucleic acid is removable from genomic DNA of the cell using a transposase.
The stem cell of claim 120 wherein the guide RNA is between about 10 to about 500 nucleotides.
The stem cell of claim 120 wherein the guide RNA is between about 20 to about 100 nucleotides.
The stem cell of claim 120 wherein the guide RNA is a tracrRNA-crRNA fusion
Dependencies are inferred from the claim text.