RNA-Guided Human Genome Engineering
First Claim
1. A method of altering a eukaryotic cell comprisingproviding to the eukaryotic cell a guide RNA sequence complementary to a target nucleic acid sequence,providing to the eukaryotic cell a Cas9 enzyme that interacts with the guide RNA sequence and cleaves the target nucleic acid sequence in a site specific manner,wherein the guide RNA sequence binds to the complementary target nucleic acid sequence and the Cas9 enzyme cleaves the target nucleic acid sequence in a site specific manner;
- wherein the guide RNA sequence includes a guide sequence of GN19 complementary to the target nucleic acid sequence and a scaffold sequence and wherein the guide RNA sequence is between about 100 to about 250 nucleotides.
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Abstract
A method of altering a eukaryotic cell is provided including transfecting the eukaryotic cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner.
242 Citations
25 Claims
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1. A method of altering a eukaryotic cell comprising
providing to the eukaryotic cell a guide RNA sequence complementary to a target nucleic acid sequence, providing to the eukaryotic cell a Cas9 enzyme that interacts with the guide RNA sequence and cleaves the target nucleic acid sequence in a site specific manner, wherein the guide RNA sequence binds to the complementary target nucleic acid sequence and the Cas9 enzyme cleaves the target nucleic acid sequence in a site specific manner; wherein the guide RNA sequence includes a guide sequence of GN19 complementary to the target nucleic acid sequence and a scaffold sequence and wherein the guide RNA sequence is between about 100 to about 250 nucleotides. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A eukaryotic cell including
a nucleic acid encoding a guide RNA sequence complementary to a target nucleic acid sequence, a nucleic acid encoding a Cas9 enzyme that interacts with the guide RNA sequence and cleaves the target nucleic acid sequence in a site specific manner, wherein the eukaryotic cell expresses the guide RNA and the Cas9 enzyme, the guide RNA binds to the complementary target nucleic acid and the Cas9 enzyme cleaves the target nucleic acid in a site specific manner, wherein the guide RNA sequence includes a guide sequence of GN19 complementary to the target nucleic acid sequence and a scaffold sequence and wherein the guide RNA sequence is between about 100 to about 250 nucleotides.
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14. A method of altering a eukaryotic cell comprising
providing to the eukaryotic cell a guide RNA complementary to a target nucleic acid sequence, providing to the eukaryotic cell a Cas9 enzyme that interacts with the guide RNA and cleaves the target nucleic acid sequence in a site specific manner, wherein the guide RNA binds to the complementary target nucleic acid sequence and the Cas9 enzyme cleaves the target nucleic acid sequence in a site specific manner; wherein the guide RNA includes a guide sequence complementary to the target nucleic acid sequence and a scaffold sequence connected to the guide sequence and having the following nucleic acid sequence and structure; - View Dependent Claims (15, 16, 17, 18)
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19. A method of altering expression of a target nucleic acid sequence in a eukaryotic cell comprising
providing to the eukaryotic cell a guide RNA sequence complementary to the target nucleic acid sequence, providing to the eukaryotic cell a Cas9 enzyme that interacts with the guide RNA sequence and cleaves the target nucleic acid sequence in a site specific manner, wherein the guide RNA sequence binds to the complementary target nucleic acid sequence and the Cas9 enzyme cleaves the target nucleic acid sequence in a site specific manner whereby expression of the target nucleic acid sequence is altered; wherein the guide RNA sequence includes a guide sequence of GN19 complementary to the target nucleic acid sequence and a scaffold sequence and wherein the guide RNA sequence is between about 100 to about 250 nucleotides. - View Dependent Claims (20, 21, 22, 23, 24, 25)
Specification