Delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications

Abstract

The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are delivery systems and tissues or organ which are targeted as sites for delivery. Also 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 to ensure enhanced specificity for target recognition and avoidance of toxicity and to edit or modify a target site in a genomic locus of interest to alter or improve the status of a disease or a condition.

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,   (ii) a first RNA sequence with a sequence complementary to a target sequence within a BCL11A gene, wherein the first RNA sequence also comprises a tracr-mate sequence,   (ii) a second RNA sequence comprising a sequence complementary to the tracr-mate sequence,
 wherein the first RNA sequence and the second RNA sequence are in a single 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 the one or more SV40 large T-antigen nuclear localization signals are at the C-terminus of the Cas9. 
     
     
         3 . The ex vivo human cell of  claim 1 , wherein the Cas9 is flanked on each end by at least one nuclear localization signal. 
     
     
         4 . 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). 
     
     
         5 . The ex vivo human cell of  claim 1 , wherein the Cas9 is an  S. pyogenes  Cas9. 
     
     
         6 . The ex vivo human cell of  claim 1 , wherein the sequence complementary to the target sequence is between 15-25 nucleotides in length. 
     
     
         7 . The ex vivo human cell of  claim 1 , wherein the tracr-mate sequence comprises the sequence GUUUUAGAGCUA. 
     
     
         8 . The ex vivo human cell of  claim 1 , wherein the first RNA sequence comprises one or more methylated nucleotides. 
     
     
         9 . The ex vivo human cell of  claim 1 , wherein 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 . The ex vivo human cell of  claim 11 , wherein the regulatory element comprises an enhancer. 
     
     
         13 . An ex vivo human cell comprising:
 (i) an  S. pyogenes  Cas9 comprising one or more SV40 large T-antigen nuclear localization signals,   (ii) a first RNA sequence comprising a sequence complementary to a target sequence within a BCL11Agene, wherein the first RNA sequence also comprises a tracr-mate sequence,   (ii) a second RNA sequence comprising a sequence complementary to the tracr-mate sequence,
 wherein the first RNA sequence and the second RNA sequence are in a single 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 the one or more SV40 large T-antigen nuclear localization signals is at the C-terminus of the  S. pyogenes  Cas9 and/or at the N-terminus of the  S. pyogenes  Cas9. 
     
     
         15 . 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). 
     
     
         16 . The ex vivo human cell of  claim 13 , wherein the  S. pyogenes  Cas9 is flanked on each end by at least one nuclear localization signal. 
     
     
         17 . The ex vivo human cell of  claim 13 , wherein the sequence complementary to the target sequence is between 15-25 nucleotides in length. 
     
     
         18 . The ex vivo human cell of  claim 13 , wherein the tracr-mate sequence comprises the sequence GUUUUAGAGCUA. 
     
     
         19 . The ex vivo human cell of  claim 13 , wherein the first RNA sequence comprises one or more methylated nucleotides. 
     
     
         20 . The ex vivo human cell of  claim 13 , wherein the second RNA sequence comprises one or more methylated nucleotides. 
     
     
         21 . The ex vivo human cell of  claim 13 , wherein the target sequence comprises a non-coding sequence. 
     
     
         22 . The ex vivo human cell of  claim 21 , wherein the non-coding sequence comprises a regulatory element. 
     
     
         23 . An ex vivo human cell comprising:
 (i) an  S. pyogenes  Cas9 comprising one or more SV40 large T-antigen nuclear localization signals,   (ii) a first RNA sequence comprising a sequence complementary to a target sequence within a BCL11A gene,
 wherein the first RNA sequence comprises a tracr-mate sequence that comprises the sequence GUUUUAGAGCUA, 
 wherein the first RNA sequence comprises one or more methylated nucleotides, and 
   (ii) 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 in a single RNA molecule, 
 wherein the Cas9 is complexed with the RNA molecule, and 
 wherein the RNA molecule and the Cas9 do not occur naturally together. 
   
     
     
         24 . The ex vivo human cell of  claim 23 , wherein the target sequence comprises a non-coding sequence. 
     
     
         25 . The ex vivo human cell of  claim 23 , wherein the non-coding sequence comprises a regulatory element. 
     
     
         26 . The ex vivo human cell of  claim 23 , wherein the  S. pyogenes  Cas9 comprises two SV40 large T-antigen nuclear localization signals. 
     
     
         27 . The ex vivo human cell of  claim 23 , wherein the one or more SV40 large T-antigen nuclear localization signals is at the C-terminus of the  S. pyogenes  Cas9 and/or at the N-terminus of the  S. pyogenes  Cas9. 
     
     
         28 . The ex vivo human cell of  claim 23 , wherein the SV40 large T-antigen nuclear localization signals comprise the sequence PKKKRKV (SEQ ID NO: 1). 
     
     
         29 . The ex vivo human cell of  claim 23 , wherein the  S. pyogenes  Cas9 is flanked on each end by at least one nuclear localization signal. 
     
     
         30 . An ex vivo human cell comprising:
 (i) a Cas9 comprising one or more SV40 large T-antigen nuclear localization signals,   (ii) an RNA molecule comprising:
 (a) a sequence complementary to a target sequence within a BCL 11A gene, 
 (b) a tracr-mate sequence, and 
 (c) a sequence complementary to the tracr-mate sequence, 
   wherein the Cas9 is complexed with the RNA molecule, and   wherein the RNA molecule and the Cas9 do not occur naturally together.