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 guiding RNA to target the endonuclease to a specific nucleotide sequence for cleavage. 
     
     
         2 . The endonuclease of  claim 1 , wherein the endonuclease is derived from a Cas9 protein and comprises a RuvC-like nuclease domain and a HNH-like nuclease domain. 
     
     
         3 . The endonuclease of  claim 1 , wherein the endonuclease acid has a sequence consisting of SEQ ID NO:2. 
     
     
         4 . An isolated nucleic acid encoding the endonuclease of  claim 1 . 
     
     
         5 . The nucleic acid of  claim 5 , wherein the nucleic acid is RNA. 
     
     
         6 . The nucleic acid of  claim 5 , wherein the nucleic acid is DNA. The nucleic acid of  claim 6 , wherein the DNA is codon optimized for translation in mammalian cells. 
     
     
         8 . The nucleic acid of  claim 6 , wherein the DNA is codon optimized for translation in human cells. 
     
     
         9 . The nucleic acid of  claim 8 , wherein the nucleic acid has a sequence consisting of SEQ ID NO:3. 
     
     
         10 . The nucleic acid of  claim 6 , which is operably linked to a promoter control sequence. 
     
     
         11 . A vector comprising the nucleic acid of  claim 10 . 
     
     
         12 . A method for modifying a chromosomal sequence in a eukaryotic cell, the method comprising:
 a) introducing into the eukaryotic cell (i) an RNA-guided endonuclease or a nucleic acid encoding the RNA-guided endonuclease, wherein the RNA-guided endonuclease comprises a nuclear localization signal, (ii) at least one guiding RNA or at least one DNA molecule encoding a guiding RNA, wherein each guiding RNA guides the RNA-guided endonuclease to a targeted site in the chromosomal sequence, and optionally, (iii) at least one donor polynucleotide comprising a donor sequence; and   b) culturing the cell such that the RNA-guided endonuclease introduces a double-stranded break in the targeted site in the chromosomal sequence and the double-stranded break is repaired by a DNA repair process such that the chromosomal sequence is modified by a deletion of at least one nucleotide, an insertion of at least one nucleotide, a substitution of at least one nucleotide, or a combination thereof.   
     
     
         13 . The method of  claim 12 , wherein the RNA-guided endonuclease is derived from a Cas9 protein and comprises a RuvC-like nuclease domain and a HNH-like nuclease domain. 
     
     
         14 . The method of  claim 12 , wherein the nucleic acid encoding the RNA-guided endonuclease is RNA or DNA. 
     
     
         15 . The method of  claim 12 , wherein the guiding RNA comprises a first 5′ region that is complementary to the target site, a second internal region that forms a secondary structure, and a third 3′ region that remains essentially single-stranded. 
     
     
         16 . The method of  claim 12 , wherein the DNA molecule encoding the guiding RNA comprises the guiding RNA coding sequence that is operably linked to a mammalian U6 or mammalian H1 promoter control sequence. 
     
     
         17 . The method of  claim 12 , wherein no donor polynucleotide is introduced into the cell, and the double-stranded break is repaired by a non-homologous end-joining repair process such that the chromosomal sequence is inactivated. 
     
     
         18 . The method of  claim 12 , wherein the donor polynucleotide is introduced into the cell, the donor sequence of the donor polynucleotide is flanked by an upstream sequence and a downstream sequence, wherein the upstream and downstream sequences share substantial sequence identity with sequences upstream and downstream, respectively, of the target site, and the double-stranded break is repaired by homology-directed repair process such that the donor sequence is integrated into the chromosomal sequence. 
     
     
         19 . The method of  claim 12 , wherein the donor polynucleotide is introduced into the cell, the donor polynucleotide is a linear molecule comprising the donor sequence that is optionally flanked by short overhangs, and the double-stranded break is repaired by a non-homologous end-joining repair process such that the donor sequence is integrated into the chromosomal sequence. 
     
     
         20 . The method of  claim 12 , wherein the donor polynucleotide is introduced into the cell, the donor polynucleotide comprises the target site recognized by the RNA-guided endonuclease, and the double-stranded break is repaired by a non-homologous end-joining repair process such that the donor sequence is integrated into the chromosomal sequence. 
     
     
         21 . The method of  claim 12 , wherein the cell is a human cell, a non-human mammalian cell, a non-mammalian vertebrate cell, an invertebrate cell, a plant cell, or a single cell eukaryotic organism.