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