CRISPR-BASED GENOME MODIFICATION AND REGULATION
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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 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.
175 Citations
48 Claims
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1-12. -12. (canceled)
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13. A method for modifying a chromosomal sequence in a eukaryotic cell or embryo, the method comprising:
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a) introducing into the eukaryotic cell or embryo (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 or embryo such that each guide RNA directs an RNA-guided endonuclease to a targeted site in the chromosomal sequence where the RNA-guided endonuclease introduces a double-stranded break in the targeted site, and the double-stranded break is repaired by a DNA repair process such that the chromosomal sequence is modified. - View Dependent Claims (14, 15, 16, 17, 18, 19)
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- 20. A fusion protein comprising a CRISPR/Cas-like protein or fragment thereof and an effector domain.
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24-28. -28. (canceled)
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31. (canceled)
- 32. A method for modifying a chromosomal sequence or regulating expression of a chromosomal sequence in a cell or embryo, the method comprising introducing into the cell or embryo (a) at least one fusion protein or nucleic acid encoding at least one fusion protein, wherein the fusion protein comprises a CRISPR/Cas-like protein or a fragment thereof and an effector domain, and (b) at least one guide RNA or DNA encoding at least one guide RNA, wherein the one guide RNA guides the CRISPR/Cas-like protein of the fusion protein to a targeted site in the chromosomal sequence and the effector domain of the fusion protein modifies the chromosomal sequence or regulates expression of the chromosomal sequence.
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37. (canceled)
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42-43. -43. (canceled)
Specification