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 . A method of modifying a target DNA molecule, the method comprising:
 allowing a CRISPR complex to target a target sequence of the target DNA molecule, said CRISPR complex comprising:   (a) a Cas9; and   (b) RNA comprising:
 (i) a guide sequence linked to a tracr mate sequence, wherein the guide sequence is capable of hybridizing to the target sequence, and 
 (ii) a tracr sequence that hybridizes to the tracr mate sequence to form a duplex structure, 
   wherein said allowing a CRISPR complex to target a target sequence of the target DNA molecule takes place in vitro or in a human or non-human animal or plant, or in a cell or tissue thereof,   resulting in modification of the target DNA molecule.   
     
     
         2 . The method of  claim 1 , wherein the tracr mate and tracr sequences have at least 50% sequence complementarity along the length of the shorter of the two. 
     
     
         3 . The method of  claim 1 , wherein the tracr mate and tracr sequences have at least 50% sequence complementarity along the length of the shorter of the two, wherein the shorter of the two has a length comprising about 12 to about 27 nucleotides. 
     
     
         4 . The method of  claim 1 , wherein the tracr mate and tracr sequences have at least 50% sequence complementarity along the length of the shorter of the two, wherein the shorter of the two has a length comprising about 15 to about 27 nucleotides. 
     
     
         5 . The method of  claim 1 , wherein the duplex structure has a total of 10, 11, 17, or 21 complementary pairs. 
     
     
         6 . The method of  claim 1 , wherein the duplex structure has a total of 11 or 17 complementary pairs. 
     
     
         7 . A method of modifying a target DNA molecule, the method comprising:
 allowing a CRISPR complex to target a target sequence of the target DNA molecule, said CRISPR complex comprising:   (a) a Cas9; and   (b) RNA comprising:
 (i) a guide sequence linked to a tracr mate sequence, wherein the guide sequence is capable of hybridizing to the target sequence, and 
 (ii) a tracr sequence that hybridizes to the tracr mate sequence to form a duplex structure, 
   wherein the duplex structure has a total number of Watson-Crick base pairs in the range of 9-19,   wherein said allowing a CRISPR complex to target a target sequence of the target DNA molecule takes place in vitro or in a human or non-human animal or plant, or in a cell or tissue thereof,   resulting in modification of the target DNA molecule.   
     
     
         8 . The method of  claim 7 , wherein the duplex structure has a total number of Watson-Crick base pairs in the range of 9-16. 
     
     
         9 . The method of  claim 7 , wherein the duplex structure has a total of 9, 16, or 19 Watson-Crick base pairs. 
     
     
         10 . The method of  claim 7 , wherein the duplex structure has a total of 9 or 16 Watson-Crick base pairs. 
     
     
         11 . The method of  claim 1  wherein said modification of the target DNA molecule is cleavage of the target DNA molecule. 
     
     
         12 . The method of  claim 1 , wherein the target sequence is at least 15 nucleotides (nt) to 20 nt long. 
     
     
         13 . The method of  claim 1 , wherein the target sequence is at least 15 nucleotides (nt) to 25 nt long. 
     
     
         14 . The method of  claim 1 , wherein the target DNA is chromosomal DNA. 
     
     
         15 . The method of  claim 1 , wherein the tracr sequence comprises UAGCAAGUUAAAAUAAGGCUAGUCCGUUUU. 
     
     
         16 . The method of  claim 1 , wherein the RNA comprises one or more modified nucleotides. 
     
     
         17 . The method of  claim 1 , wherein the guide sequence and/or the tracr sequence is conjugated to an avidin/biotin complex, an acridine, fluorescent label or fluorcoumarin. 
     
     
         18 . The method of  claim 1 , wherein the tracr mate and tracr sequences have at least 50% sequence complementarity along the length of the shorter of the two when optimally aligned. 
     
     
         19 . A method for cleaving or editing a target DNA molecule having a target sequence or modulating transcription of at least one gene target having the target sequence with an engineered Type II CRISPR complex, the method comprising allowing the CRISPR complex to target the target DNA molecule or the gene target,
 wherein the CRISPR complex comprises:   a) RNA comprising (i) an engineered guide sequence linked to a tracr mate sequence wherein the guide sequence is capable of hybridizing with the target sequence and (ii) a tracr sequence that hybridizes to the tracr mate sequence to form a duplex structure, and   b) a Cas9,   
       wherein the RNA forms the complex with the Cas9 and targets the Cas9 of the complex to the target DNA molecule or the gene target, whereby said target DNA molecule is cleaved or edited or transcription of the gene target is modulated. 
     
     
         20 . The method of  claim 19 , wherein each of (i) and (ii) are separate molecules and wherein at least a portion of the tracr mate sequence in (i) hybridizes to at least a portion of (ii). 
     
     
         21 . The method of  claim 19 , wherein the tracr mate sequence in (i) and the tracr sequence in (ii) hybridizes to form a duplex structure. 
     
     
         22 . The method of  claim 21 , wherein the tracr mate and tracr sequences have at least 50% sequence complementarity along the length of the shorter of the two. 
     
     
         23 . The method of  claim 21 , wherein the tracr mate and tracr sequences have at least 50% sequence complementarity along the length of the shorter of the two, wherein the shorter of the two has a length comprising about 12 to about 27 nucleotides. 
     
     
         24 . The method of  claim 21 , wherein the tracr mate and tracr sequences have at least 50% sequence complementarity along the length of the shorter of the two, wherein the shorter of the two has a length comprising about 15 to about 27 nucleotides. 
     
     
         25 . The method of  claim 21 , wherein the duplex structure has a total number of Watson-Crick base pairs in the range of 9-19. 
     
     
         26 . The method of  claim 21 , wherein the duplex structure has a total number of Watson-Crick base pairs in the range of 9-16. 
     
     
         27 . The method of  claim 21 , wherein the duplex structure has a total of 9, 16, or 19 Watson-Crick base pairs. 
     
     
         28 . The method of  claim 21 , wherein the duplex structure has a total of 10, 11, 17, or 21 complementary pairs.