Crispr-based genome modification and regulation

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.

What is claimed is: 
     
         1 . A method for integrating an exogenous sequence into a chromosomal sequence of 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 (iii) at least one donor polynucleotide comprising the exogenous sequence; 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 integration of the exogenous sequence into the chromosomal sequence.   
     
     
         2 . The method of  claim 1 , wherein the RNA-guided endonuclease is derived from a clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated (Cas) (CRISPR/Cas) type II system protein. 
     
     
         3 . The method of  claim 2 , wherein the CRISPR/Cas type II system protein is a Cas9 protein. 
     
     
         4 . The method of  claim 1 , wherein the target site is a Rosa26 locus, a HPRT locus, or an AAVS 1 locus. 
     
     
         5 . The method of  claim 1 , wherein the target site in the chromosomal sequence is immediately followed by a protospacer adjacent motif (PAM). 
     
     
         6 . The method of  claim 1 , wherein the guide RNA comprises a first region that is complementary to the target site in the chromosomal sequence. 
     
     
         7 . The method of  claim 1 , wherein the guide RNA comprises a second region that interacts with the RNA-guided endonuclease. 
     
     
         8 . The method of  claim 1 , wherein the exogenous sequence in the donor polynucleotide is flanked by sequences having substantial sequence identity to sequences on either side of the target site in the chromosomal sequence. 
     
     
         9 . The method of  claim 1 , wherein the exogenous sequence in the donor polynucleotide is flanked by targeted cleavage sites that are recognized by the RNAguided endonuclease. 
     
     
         10 . The method of  claim 1 , wherein the nucleic acid encoding the RNA-guided endonuclease is m RNA 
     
     
         11 . The method of  claim 1 , wherein the nucleic acid encoding the RNA-guided endonuclease is DNA 
     
     
         12 . The method of  claim 11 , wherein the DNA is part of a vector that further comprises sequence encoding the guide RNA 
     
     
         13 . The method of  claim 1 , wherein the eukaryotic cell is a human cell, a nonhuman mammalian cell, a non-human mammalian embryo, or a plant cell.