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 (i) at least one RNA-guided endonuclease comprising at least one nuclear localization signal or nucleic acid encoding at least one RNA-guided endonuclease comprising at least one nuclear localization signal, (ii) at least one guide RNA or DNA encoding at least one guide RNA, and, optionally, (iii) at least one donor polynucleotide; and
b) culturing the eukaryotic cell such that the guide RNA guides the RNA-guided endonuclease to a target site in the chromosomal sequence where the RNA-guided endonuclease introduces a double-stranded break, 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.
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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 (i) at least one RNA-guided endonuclease comprising at least one nuclear localization signal or nucleic acid encoding at least one RNA-guided endonuclease comprising at least one nuclear localization signal, (ii) at least one guide RNA or DNA encoding at least one guide RNA, and, optionally, (iii) at least one donor polynucleotide; and b) culturing the eukaryotic cell such that the guide RNA guides the RNA-guided endonuclease to a target site in the chromosomal sequence where the RNA-guided endonuclease introduces a double-stranded break, 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