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 eukaryotic cell comprising a target chromosomal sequence; and
 (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, wherein the at least one RNA-guided endonuclease is a clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR associated (Cas) (CRISPR-Cas) type II system protein, wherein the nucleic acid encoding the CRISPR-Cas9 type II protein is codon optimized for expression in the eukaryotic cell, and wherein the CRISPR-Cas type II system protein is a Cas9 protein, and   (ii) at least one engineered guide RNA or DNA encoding at least one engineered guide RNA, each guide RNA comprising
 (1) a first region at the 5′ end that is capable of base pairing with a target site in the chromosomal sequence, and 
 (2) a second region that forms a secondary structure which interacts with the at least one RNA-guided endonuclease; and 
   
       whereby base pairing of the first region to the target site is capable of targeting the Cas9 protein to the target chromosomal sequence. 
     
     
         2 . The eukaryotic cell of  claim 1 , wherein the genome of said eukaryotic cell comprises the target chromosomal sequence. 
     
     
         3 . The eukaryotic cell of  claim 1 , wherein the eukaryotic cell is a mammalian cell or a human cell. 
     
     
         4 . The eukaryotic cell of  claim 3 , wherein (A) the guide RNA comprises a single molecule such that the first region and the second region on present on the same RNA molecule or (B) the guide RNA comprises a double-molecule such that the first region and the second region are present on different RNA molecules. 
     
     
         5 . The eukaryotic cell of  claim 4 , comprising two or more first regions, or one or more nucleic acids comprising two or more nucleotide sequences encoding different first regions. 
     
     
         6 . The eukaryotic cell of  claim 1 , wherein the system comprises a nucleic acid molecule comprising a nucleotide sequence encoding the second region, and wherein said nucleic acid molecule comprising a nucleotide sequence encoding the second region does not comprise a nucleotide sequence encoding said Cas9 protein. 
     
     
         7 . The eukaryotic cell of  claim 1 , wherein the system comprises a double stranded DNA molecule comprising nucleotide sequences encoding both the first region of the guide RNA and the second region of the guide RNA, wherein the nucleotide sequences encoding the first region and the second region are on the same strand. 
     
     
         8 . The eukaryotic cell of  claim 1 , wherein one or more of a) the nucleic acid comprising the nucleotide sequence encoding said Cas9 protein and b) the one or more nucleic acids comprising one or more nucleotide sequences encoding said guide RNA, are one or more vectors, wherein the one or more vectors are selected from the group consisting of plasmid vectors, phagemids, cosmids, artificial/mini-chromosomes, transposons, and viral vectors; and wherein the nucleotide sequence encoding said Cas9 protein and/or the one or more nucleotide sequences encoding said guide RNA is operably linked to a control element operable in said eukaryotic cell. 
     
     
         9 . The eukaryotic cell of  claim 1 , wherein the Cas9 protein comprises one or more Nuclear Localization Signals (NLSs). 
     
     
         10 . The eukaryotic cell of  claim 9 , wherein the one or more NLSs comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO: 2. 
     
     
         11 . The eukaryotic cell of  claim 1 , wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain. 
     
     
         12 . The eukaryotic cell of  claim 1 , comprising two or more different guide RNAs, or one or more nucleic acids comprising two or more nucleotide sequences encoding different guide RNAs. 
     
     
         13 . The eukaryotic cell of  claim 1 , wherein the system comprises a donor polynucleotide and the system is capable of editing the target chromosomal sequence by inserting a sequence of the donor polynucleotide into a cleaved strand of the target chromosomal sequence. 
     
     
         14 . The eukaryotic cell of  claim 1 , wherein the nucleotide sequence encoding said Cas9 protein comprises a nucleotide sequence modification that replaces one or more codons of a wild-type Cas9-encoding nucleotide sequence with one or more different codons encoding the same amino acid. 
     
     
         15 . The eukaryotic cell of  claim 1 , wherein said Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain and is capable of cleaving only one strand of DNA. 
     
     
         16 . The eukaryotic cell of  claim 15 , wherein the nucleotide sequence encoding said Cas9 protein comprises a nucleotide sequence modification that replaces one or more codons of a wild-type Cas9-encoding nucleotide sequence with one or more different codons encoding the same amino acid. 
     
     
         17 . The eukaryotic cell of  claim 15 , comprising two or more different guide RNAs, or one or more nucleic acids comprising two or more nucleotide sequences encoding different guide RNAs. 
     
     
         18 . The eukaryotic cell of  claim 15 , wherein (A) the guide RNA comprises a single molecule such that the first region and the second region on present on the same RNA molecule or (B) the guide RNA comprises a double-molecule such that the first region and the second region are present on different RNA molecules. 
     
     
         19 . The eukaryotic cell of  claim 15 , wherein the Cas9 protein comprises one or more Nuclear Localization Signals (NLSs). 
     
     
         20 . The eukaryotic cell of  claim 19 , wherein the one or more NLSs comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO: 2. 
     
     
         21 . A eukaryotic cell comprising a target chromosomal sequence and
 an engineered and/or non-naturally occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising   a) a Cas9 protein, or a nucleic acid comprising a nucleotide sequence encoding said Cas9 protein; and   b) at least one engineered guide RNA or DNA encoding at least one engineered guide RNA, each guide RNA comprising
 i) a first region at the 5′ end that is capable of base pairing with a target site in the chromosomal sequence, and 
 ii) a second region that forms a secondary structure which interacts with the at least one RNA-guided endonuclease; and; 
   
       wherein (I) the guide RNA comprises a nucleotide sequence that is modified relative to naturally occurring guide RNAs, and/or (II) the Cas9 protein comprises an amino acid sequence that is modified relative to naturally occurring Cas9 proteins,
 wherein the guide RNA is capable of forming a complex with the Cas9 protein, thereby targeting the Cas9 protein to the target chromosomal sequence, 
 whereby said system is capable of cleaving or editing the target chromosomal sequence. 
 
     
     
         22 . The eukaryotic cell of  claim 21 , wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain. 
     
     
         23 . The eukaryotic cell of  claim 21 , wherein one or more of the nucleic acids of a) and b) are one or more vectors, wherein the one or more vectors are selected from the group consisting of plasmid vectors, phagemids, cosmids, artificial/mini-chromosomes, transposons, and viral vectors; wherein the nucleotide sequence encoding said Cas9 protein and/or the one or more nucleotide sequence encoding said DNA-targeting RNA are operably linked to control elements operable in said eukaryotic cell. 
     
     
         24 . The eukaryotic cell of  claim 21 , wherein the Cas9 protein comprises one or more Nuclear Localization Signals (NLSs). 
     
     
         25 . The eukaryotic cell of  claim 24 , wherein said Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain and is capable of cleaving only one strand of DNA. 
     
     
         26 . The eukaryotic cell of  claim 25 , wherein the one or more NLSs comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO: 2. 
     
     
         27 . A non-human plant or animal comprising the eukaryotic cell of  claim 1 .