RNA-Guided Human Genome Engineering
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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.
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Citations
41 Claims
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1-14. -14. (canceled)
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15. A method of altering a eukaryotic cell comprising
providing the eukaryotic cell with a guide RNA having a spacer sequence complementary to genomic DNA of the eukaryotic cell and a scaffold sequence, providing the eukaryotic cell with a codon optimized Cas9 of a Type II CRISPR system that interacts with the guide RNA and cleaves the genomic DNA in a site specific manner, wherein the guide RNA binds to complementary genomic DNA and the Cas enzyme of a Type II CRISPR system cleaves the genomic DNA in a site specific manner, and wherein the codon optimized Cas9 is a human codon optimized Cas9 encoded by a nucleic acid comprising SEQ ID NO:
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37. A method of altering a eukaryotic cell comprising
providing the eukaryotic cell with a guide RNA having a spacer sequence complementary to genomic DNA of the eukaryotic cell and a scaffold sequence, providing the eukaryotic cell with a codon optimized Cas9 of a Type II CRISPR system that interacts with the guide RNA and cleaves the genomic DNA in a site specific manner, wherein the guide RNA binds to complementary genomic DNA and the Cas enzyme of a Type II CRISPR system cleaves the genomic DNA in a site specific manner, wherein the guide RNA includes between 100 and 250 nucleotides, wherein the codon optimized Cas9 is a human codon optimized Cas9 encoded by a nucleic acid comprising SEQ ID NO: - 22.
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38. An RNA-guided genome editing system comprising
(1) a guide RNA sequence or a first nucleic acid sequence encoding the guide RNA sequence and (2) a Cas enzyme that forms a complex with the guide RNA sequence, or a second nucleic acid sequence encoding the Cas enzyme, wherein the guide RNA sequence comprises a spacer sequence complementary to a target nucleic acid sequence and which binds to the target nucleic acid sequence, and wherein the guide RNA sequence is a crRNA-tracrRNA fusion transcript having a secondary structure comprising a first hairpin connected to the spacer sequence and a second 3′ - hairpin.
- View Dependent Claims (39, 40, 41)
Specification