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 . An isolated endonuclease comprising at least one nuclear localization signal, at least one nuclease domain, and at least one domain that interacts with a guide RNA to target the endonuclease to a specific nucleotide sequence for cleavage. 
     
     
         2 . The isolated endonuclease of  claim 1 , wherein the endonuclease is derived from a Cas9 protein. 
     
     
         3 . The isolated endonuclease of  claim 1 , wherein the endonuclease is modified to lack at least one functional nuclease domain. 
     
     
         4 . The isolated endonuclease of  claim 1 , further comprising a cell-penetrating domain, a marker domain, or both. 
     
     
         5 . The isolated endonuclease of  claim 1 , which is part of a protein-RNA complex comprising the guide RNA. 
     
     
         6 . The isolated endonuclease of  claim 5 , wherein the guide RNA is a single molecule comprising a 5′ region that is complementary to a target site. 
     
     
         7 . An isolated nucleic acid encoding the endonuclease of  claim 1 . 
     
     
         8 . The isolated nucleic acid of  claim 7 , wherein the nucleic acid is codon optimized for translation in mammalian cells. 
     
     
         9 . The isolated nucleic acid of  claim 7 , wherein the nucleic acid is codon optimized for translation in human cells. 
     
     
         10 . The isolated nucleic acid of  claim 7 , which is operably linked to a promoter control sequence. 
     
     
         11 . A vector comprising the isolated nucleic acid of  claim 10 . 
     
     
         12 . The vector of  claim 11 , further comprising a sequence encoding a guide RNA that is operably linked to a promoter control sequence. 
     
     
         13 . A method for modifying a chromosomal sequence in a eukaryotic cell, the method comprising:
 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 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.   
     
     
         14 . The method of  claim 13 , wherein the at least one RNA-guided endonuclease is derived from a Cas9 protein. 
     
     
         15 . The method of  claim 13 , wherein the nucleic acid encoding the at least one RNA-guided endonuclease is codon optimized for expression in the eukaryotic cell. 
     
     
         16 . The method of  claim 15 , wherein the nucleic acid encoding the at least one RNA-guided endonuclease is mRNA. 
     
     
         17 . The method of  claim 15 , wherein the nucleic acid encoding the at least one RNA-guided endonuclease is DNA. 
     
     
         18 . The method of  claim 17 , wherein the DNA is part of a vector that further comprises a sequence encoding the at least one guide RNA. 
     
     
         19 . The method of  claim 13 , wherein the eukaryotic cell is a human cell, a non-human mammalian cell, a one cell animal embryo, a non-mammalian vertebrate cell, an invertebrate cell, a plant cell, or a single cell eukaryotic organism.