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 non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising
 I. a first regulatory element operably linked to a polynucleotide sequence encoding a CRISPR-Cas system chimeric RNA (chiRNA, wherein the chiRNA comprises   (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell and directing sequence-specific binding of a CRISPR complex to the target sequence,   (b) a tracr mate sequence capable of hybridizing to a tracr sequence, and   (c) a tracr sequence, wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation, and the chiRNA comprises two or more hairpins, and   II. a second regulatory element operably linked to an enzyme-coding sequence encoding a Cas9 comprising one or more nuclear localization sequences (NLSs) in the proximity of a terminus of the Cas9,   wherein components I and II are located on the same or different vectors of the system.   
     
     
         2 . The composition of  claim 1 , wherein the one or more vectors encode multiple chiRNAs to provide a multiplexed system. 
     
     
         3 . The composition of  claim 1 , wherein the first regulatory element is a polymerase III promoter. 
     
     
         4 . The composition of  claim 1 , wherein the second regulatory element is a polymerase II promoter. 
     
     
         5 . The composition of  claim 1 , wherein the Cas9 comprises one or more NLSs of sufficient strength to drive accumulation of said Cas9 in a detectable amount in the nucleus of a eukaryotic cell. 
     
     
         6 . The composition of  claim 1 , wherein the tracr sequence exhibits at least 50% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. 
     
     
         7 . The composition of  claim 1 , wherein the Cas9 is a g  pyogenes.    
     
     
         8 . The composition of  claim 1 , wherein the Cas9 is a  S. thermophilus  Cas9. 
     
     
         9 . The composition of  claim 1 , wherein the enzyme-coding sequence encoding the Cas9 is codon-optimized for expression in a eukaryotic cell. 
     
     
         10 . The composition of  claim 1 , wherein the guide sequence is at least 15 nucleotides in length. 
     
     
         11 . The composition of  claim 1 , wherein the chimeric RNA comprises two, three, four or five hairpins. 
     
     
         12 . A non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising
 I. a first regulatory element operably linked to a polynucleotide sequence encoding a CRISPR-Cas system RNA comprising   (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell and directing sequence-specific binding of a CRISPR complex to the target sequence, and   (b) a tracr mate sequence capable of hybridizing to a tracr sequence,   II. a second regulatory element operably linked to an enzyme-coding sequence encoding a Cas9 comprising one or more nuclear localization sequences (NLSs) in the proximity of a terminus of the Cas9, and   III. a third regulatory element operably linked to a polynucleotide sequence encoding a tracrRNA comprising a tracr sequence,   wherein components I, II and III are located on the same or different vectors of the system.   
     
     
         13 . The composition of  claim 12 , wherein the one or more vectors encode multiple guide sequences and a single tracr sequence to provide a multiplexed system. 
     
     
         14 . The composition of  claim 12 , wherein the first regulatory element is a polymerase III promoter. 
     
     
         15 . The composition of  claim 12 , wherein the second regulatory element is a polymerase II promoter. 
     
     
         16 . The composition of  claim 12 , wherein the third regulatory element is a polymerase III promoter. 
     
     
         17 . The composition of  claim 12 , wherein the Cas9 comprises one or more NLSs of sufficient strength to drive accumulation of said Cas9 in a detectable amount in the nucleus of a eukaryotic cell. 
     
     
         18 . The composition of  claim 12 , wherein the tracr sequence exhibits at least 50% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. 
     
     
         19 . The composition of  claim 12 , wherein the Cas9 is a  S. pyogenes  s9. 
     
     
         20 . The composition of  claim 12 , wherein the Cas9 is a  S. thermophilus  Cas9. 
     
     
         21 . The composition of  claim 12 , wherein the enzyme-coding sequence encoding the Cas9 is codon-optimized for expression in a eukaryotic cell. 
     
     
         22 . The composition of  claim 12 , wherein the guide sequence is at least 15 nucleotides in length. 
     
     
         23 . A eukaryotic cell comprising the composition of  claim 1 . 
     
     
         24 . An organism comprising the eukaryotic cell of  claim 23 . 
     
     
         25 . A non-human organism comprising the eukaryotic cell of  claim 23 . 
     
     
         26 . A kit comprising the composition of  claim 1  and instructions for using said kit. 
     
     
         27 . A method of altering the expression of a genomic locus of interest in a eukaryotic cell comprising contacting the genomic locus with the composition of  claim 1 , and determining if the expression of the genomic locus has been altered. 
     
     
         28 . The method of  claim 27  wherein the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence based on the presence of a CRISPR motif sequence. 
     
     
         29 . The method of  claim 28 , wherein the CRISPR motif sequence is NAG.