Crispr-cas component systems, methods and compositions for sequence manipulation

Abstract

The invention provides for systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells and methods for selecting specific cells by introducing precise mutations utilizing the CRISPR/Cas system.

What is claimed is: 
     
         1 . An ex vivo human cell comprising:
 (i) a Cas9,   (ii) a first RNA sequence comprising a sequence complementary to a target DNA sequence, wherein the first RNA sequence comprises a tracr-mate sequence, and   (iii) a second RNA sequence comprising a sequence complementary to the tracr-mate sequence,
 wherein the first RNA sequence and the second RNA sequence are hybridized as hybridized RNA sequences, 
 wherein the Cas9 is complexed with the hybridized RNA sequences, and 
 wherein the hybridized RNA sequences and the Cas9 do not occur naturally together. 
   
     
     
         2 . The ex vivo human cell of  claim 1 , wherein the Cas9 comprises one or more SV40 large T-antigen nuclear localization signals. 
     
     
         3 . The ex vivo human cell of  claim 2 , wherein at least one of the SV40 large T-antigen nuclear localization signals is at the C-terminus or the N-terminus of the Cas9. 
     
     
         4 . The ex vivo human cell of  claim 2 , wherein at least one of the SV40 large T-antigen nuclear localization signals is at the C-terminus and at least one of the SV40 large T-antigen nuclear localization signals is at the N-terminus of the Cas9. 
     
     
         5 . The ex vivo human cell of  claim 1 , wherein the Cas9 is flanked on each end by one or more nuclear localization signals. 
     
     
         6 . The ex vivo human cell of  claim 2 , wherein the SV40 large T-antigen nuclear localization signal comprises the sequence PKKKRKV (SEQ ID NO: 1). 
     
     
         7 . The ex vivo human cell of  claim 1 , wherein the sequence complementary to a target DNA sequence is between 15-25 nucleotides in length. 
     
     
         8 . The ex vivo human cell of  claim 1 , wherein the tracr-mate sequence comprises the sequence GUUUUAGAGCUA. 
     
     
         9 . The ex vivo human cell of  claim 1 , wherein the first RNA sequence and/or the second RNA sequence comprises one or more methylated nucleotides. 
     
     
         10 . The ex vivo human cell of  claim 1 , wherein the target sequence comprises a non-coding sequence. 
     
     
         11 . The ex vivo human cell of  claim 10 , wherein the non-coding sequence comprises a regulatory element. 
     
     
         12 . An ex vivo human cell comprising an  S. pyogenes  Cas9 linked to one or more nuclear localization signals, or a codon optimized polynucleotide for expression of the  S. pyogenes  Cas9, in the ex vivo human cell. 
     
     
         13 . An ex vivo human cell comprising:
 (i) an  S. pyogenes  Cas9,   (ii) a first RNA sequence comprising a sequence complementary to a target DNA sequence, in the human cell wherein the first RNA sequence comprises a tracr-mate sequence, and   (iii) a second RNA sequence comprising a sequence complementary to the tracr-mate sequence,
 wherein the first RNA sequence and the second RNA sequence are hybridized as hybridized RNA sequences, 
 wherein the  S. pyogenes  Cas9 is complexed with the hybridized RNA sequences, and 
 wherein the hybridized RNA sequences and the  S. pyogenes  Cas9 do not occur naturally together. 
   
     
     
         14 . The ex vivo human cell of  claim 13 , wherein the  S. pyogenes  Cas9 comprises one or more SV40 large T-antigen nuclear localization signals. 
     
     
         15 . The ex vivo human cell of  claim 14 , wherein the one or more SV40 large T-antigen nuclear localization signals is at either the C-terminus or the N-terminus of the  S. pyogenes  Cas9. 
     
     
         16 . The ex vivo human cell of  claim 14 , wherein the one or more SV40 large T-antigen nuclear localization signals are at the C-terminus and the N-terminus of the  S. pyogenes  Cas9. 
     
     
         17 . The ex vivo human cell of  claim 13 , wherein the  S. pyogenes  Cas9 is flanked on each end by one or more nuclear localization signals. 
     
     
         18 . The ex vivo human cell of  claim 14 , wherein the SV40 large T-antigen nuclear localization signal comprises the sequence PKKKRKV (SEQ ID NO: 1). 
     
     
         19 . The ex vivo human cell of  claim 13 , wherein the sequence complementary to a target DNA sequence is between 15-25 nucleotides in length. 
     
     
         20 . The ex vivo human cell of  claim 13 , wherein the tracr-mate sequence comprises the sequence GUUUUAGAGCUA. 
     
     
         21 . The ex vivo human cell of  claim 13 , wherein the first RNA sequence and/or the second RNA sequence comprises one or more methylated nucleotides. 
     
     
         22 . The ex vivo human cell of  claim 13 , wherein the target sequence comprises a non-coding sequence. 
     
     
         23 . The ex vivo human cell of  claim 21 , wherein the non-coding sequence comprises a regulatory element. 
     
     
         24 . An ex vivo human cell comprising:
 (i) an  S. pyogenes  Cas9,   (ii) a first RNA sequence comprising a sequence that is complementary to a target DNA sequence in the human cell and a tracr-mate sequence, wherein the tracr-mate sequence comprises the sequence GUUUUAGAGCUA,
 wherein the first RNA sequence comprises one or more methylated nucleotides, and 
   (iii) a second RNA sequence comprising a sequence complementary to the tracr-mate sequence,
 wherein the second RNA sequence comprises one or more methylated nucleotides, 
 wherein the first RNA sequence and the second RNA sequence are hybridized as hybridized RNA sequences, 
 wherein the  S. pyogenes  Cas9 is complexed with the hybridized RNA sequences, and 
 wherein the hybridized RNA sequences and the  S. pyogenes  Cas9 do not occur naturally together. 
   
     
     
         25 . The ex vivo human cell of  claim 24 , wherein the  S. pyogenes  Cas9 comprises two SV40 large T-antigen nuclear localization signals. 
     
     
         26 . The ex vivo human cell of  claim 25 , wherein the two SV40 large T-antigen nuclear localization signals are at either the C-terminus or the N-terminus of the  S. pyogenes  Cas9. 
     
     
         27 . The ex vivo human cell of  claim 25 , wherein the two SV40 large T-antigen nuclear localization signals are at the C-terminus and the N-terminus of the  S. pyogenes  Cas9. 
     
     
         28 . The ex vivo human cell of  claim 25 , wherein the SV40 large T-antigen nuclear localization signal comprises the sequence PKKKRKV (SEQ ID NO: 1). 
     
     
         29 . The ex vivo human cell of  claim 24 , wherein the target sequence comprises a non-coding sequence. 
     
     
         30 . The ex vivo human cell of  claim 24 , wherein the first RNA sequence comprises a sequence that is hybridized to a target DNA sequence and that is between 15-25 nucleotides in length.