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 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 modifying a chromosomal sequence in a eukaryotic cell by integrating a donor sequence, the method comprising introducing into the eukaryotic cell:
 (i) a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) (CRISPR/Cas) type II protein linked to at least one nuclear localization signal (NLS) or a nucleic acid encoding the CRISPR/Cas type II protein linked to at least one NLS, wherein the CRISPR/Cas type II protein is a Cas9 protein, and the nucleic acid encoding the CRISPR/Cas type II protein is codon optimized for expression in the eukaryotic cell;   (ii) a guide RNA or DNA encoding the guide RNA, wherein the guide RNA comprises a first region that is complementary to a target site in the chromosomal sequence that is immediately followed by a protospacer adjacent motif, and a second region that interacts with the CRISPR/Cas type II protein; and   (iii) a donor polynucleotide comprising the donor sequence;
 wherein the guide RNA guides the CRISPR/Cas type II protein to the target site in the chromosomal sequence, the CRISPR/Cas type II protein introduces a double-stranded break at the target site, and repair of the double-stranded break by a DNA repair process leads to integration or exchange of the donor sequence into the chromosomal sequence. 
   
     
     
         2 . The method of  claim 1 , wherein the CRISPR/Cas type II protein is linked to at least one fluorescent protein marker. 
     
     
         3 . The method of  claim 1 , wherein the guide RNA is a single molecule. 
     
     
         4 . The method of  claim 3 , wherein the guide RNA is enzymatically synthesized. 
     
     
         5 . The method of  claim 1 , wherein the guide RNA comprises a crRNA and a tracrRNA. 
     
     
         6 . The method of  claim 5 , wherein the crRNA is chemically synthesized and the tracrRNA is enzymatically synthesized, or both are enzymatically synthesized. 
     
     
         7 . The method of  claim 1 , wherein DNA encoding the guide RNA is operably linked to a promoter sequence for expression in the eukaryotic cell and is part of a vector. 
     
     
         8 . The method of  claim 1 , wherein the nucleic acid encoding the CRISPR/Cas type II protein is mRNA. 
     
     
         9 . The method of  claim 1 , wherein the nucleic acid encoding the CRISPR/Cas type II protein is DNA. 
     
     
         10 . The method of  claim 1 , wherein the nucleic acid encoding the CRISPR/Cas type II protein is part of a vector and the nucleic acid encoding the CRISPR/Cas type II protein is operably linked to a promoter sequence for expression in the eukaryotic cell. 
     
     
         11 . The method of  claim 10 , wherein the vector further comprises sequence encoding the guide RNA that is operably linked to a promoter sequence for expression in the eukaryotic cell. 
     
     
         12 . The method of  claim 1 , wherein the donor sequence comprises at least one nucleotide change relative to the target site in the chromosomal sequence. 
     
     
         13 . The method of  claim 1 , wherein the donor 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. 
     
     
         14 . The method of  claim 1 , wherein the eukaryotic cell is a human cell, a non-human mammalian cell, a non-mammalian vertebrate cell, an invertebrate cell, a plant cell, or single cell eukaryotic organism. 
     
     
         15 . A method for modifying a chromosomal sequence in a eukaryotic cell by integrating a donor sequence, the method comprising introducing into the eukaryotic cell:
 (i) a nucleic acid encoding a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) (CRISPR/Cas) type II protein linked to at least one nuclear localization signal (NLS), wherein the CRISPR/Cas type II protein is a Cas9 protein, and the nucleic acid encoding the CRISPR/Cas type II protein is codon optimized for expression in the eukaryotic cell;   (ii) a guide RNA or DNA encoding the guide RNA, wherein the guide RNA comprises a first region that is complementary to a target site in the chromosomal sequence that is immediately followed by a protospacer adjacent motif, and a second region that interacts with the CRISPR/Cas type II protein; and   (iii) a donor polynucleotide comprising the donor sequence;
 wherein the nucleic acid encoding the CRISPR/Cas type II protein is RNA, the nucleic acid encoding the CRISPR/Cas type II protein is DNA, or the nucleic acid encoding the CRISPR/Cas type II protein is DNA that is part of a vector that further comprises DNA encoding the guide RNA; and 
 wherein the guide RNA guides the CRISPR/Cas type II protein to the target site in the chromosomal sequence, the CRISPR/Cas type II protein introduces a double-stranded break at the target site, and repair of the double-stranded break by a DNA repair process leads to integration or exchange of the donor sequence into the chromosomal sequence. 
   
     
     
         16 . The method of  claim 15 , wherein the guide RNA is a single molecule. 
     
     
         17 . The method of  claim 16 , wherein the guide RNA is enzymatically synthesized. 
     
     
         18 . The method of  claim 15 , wherein the guide RNA comprises a crRNA and a tracrRNA. 
     
     
         19 . The method of  claim 18 , wherein the crRNA is chemically synthesized and the tracrRNA is enzymatically synthesized, or both are enzymatically synthesized. 
     
     
         20 . The method of  claim 15 , wherein the vector further comprises operably linked promoter control sequences. 
     
     
         21 . The method of  claim 15 , wherein the donor sequence comprises at least one nucleotide change relative to the target site in the chromosomal sequence. 
     
     
         22 . The method of  claim 15 , wherein the donor 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. 
     
     
         23 . The method of  claim 15 , wherein the eukaryotic cell is a human cell, a non-human mammalian cell, a non-mammalian vertebrate cell, an invertebrate cell, a plant cell, or single cell eukaryotic organism.