CRISPR-BASED GENOME MODIFICATION AND REGULATION
First Claim
1. A method for modifying a chromosomal sequence in a eukaryotic cell, the method comprising:
- a) introducing into the eukaryotic cell at least two RNA-guided nickase systems or nucleic acid encoding said systems, and, optionally, at least one donor polynucleotide, wherein each RNA-guided nickase system comprises (i) a RNA-guided endonuclease that is modified to cleave one strand of a double-stranded sequence and (ii) a guide RNA comprising a first region having complementarity to a target site in the chromosomal sequence and a second region that interacts with the RNA-guided endonuclease, and wherein the target sites of the two RNA-guided endonuclease are in close proximity but on opposite strands of the chromosomal sequence; and
b) culturing the eukaryotic cell such the two RNA-guided endonucleases together introduce a double-stranded break in the chromosomal sequence, and repair of the double-stranded break by a DNA repair process leads to modification of the chromosomal sequence.
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Abstract
The present invention provides RNA-guided endonucleases, which are engineered for expression in eukaryotic cells or embryos, and methods of using the RNA-guided endonuclease for targeted genome modification in in eukaryotic cells or embryos. Also provided are fusion proteins, wherein each fusion protein comprises a CRISPR/Cas-like protein or fragment thereof and an effector domain. The effector domain can be a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. Also provided are methods for using the fusion proteins to modify a chromosomal sequence or regulate expression of a chromosomal sequence.
84 Citations
21 Claims
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1. A method for modifying a chromosomal sequence in a eukaryotic cell, the method comprising:
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a) introducing into the eukaryotic cell at least two RNA-guided nickase systems or nucleic acid encoding said systems, and, optionally, at least one donor polynucleotide, wherein each RNA-guided nickase system comprises (i) a RNA-guided endonuclease that is modified to cleave one strand of a double-stranded sequence and (ii) a guide RNA comprising a first region having complementarity to a target site in the chromosomal sequence and a second region that interacts with the RNA-guided endonuclease, and wherein the target sites of the two RNA-guided endonuclease are in close proximity but on opposite strands of the chromosomal sequence; and b) culturing the eukaryotic cell such the two RNA-guided endonucleases together introduce a double-stranded break in the chromosomal sequence, and repair of the double-stranded break by a DNA repair process leads to modification of the chromosomal sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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Specification