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.

1 - 34 . (canceled) 
     
     
         35 . A method of selecting one or more prokaryotic cell(s) by introducing one or more mutations in one or more prokaryotic cell(s), the method comprising:
 introducing one or more vectors into the prokaryotic cell(s);   wherein the one or more vectors drive expression of one or more of: a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template;   and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, and an editing template are produced in the prokaryotic cell(s);   introducing the editing template into a target polynucleotide through recombination, wherein the editing template comprises the one or more mutations that abolish CRISPR enzyme cleavage;   allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of the target polynucleotide;   wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to a tracr sequence;   wherein binding of the CRISPR complex to the target polynucleotide induces cell death;   thereby allowing one or more prokaryotic cell(s) in which one or more mutations have been introduced to be selected.   
     
     
         36 . The method of  claim 35 , wherein the CRISPR enzyme is a type II CRISPR system enzyme. 
     
     
         37 . The method of  claim 35 , wherein the CRISPR enzyme is a Cas9. 
     
     
         38 . The method of  claim 37 , wherein the Cas9 is  S. pyogenes  Cas9. 
     
     
         39 . The method of  claim 35  wherein the CRISPR enzyme is not endogenous to the prokaryote(s). 
     
     
         40 . The method of  claim 35  wherein the prokaryote(s) is  S. pneumoniae  or  E. coli.   
     
     
         41 . The method of  claim 35  wherein the one or more vectors are plasmids. 
     
     
         42 . A method of inducing cell death of one or more prokaryotic cell(s) comprising:
 introducing one or more vectors into the prokaryotic cell(s);   wherein the one or more vectors drive expression of one or more of: a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence;   and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, are produced in the prokaryotic cell(s);   wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within a target polynucleotide, and (2) the tracr mate sequence that is hybridized to the tracr sequence;   wherein binding of the CRISPR complex to the target polynucleotide results in Cas9-directed cleavage at a targeted site in the prokaryote(s) and induces cell death.   
     
     
         43 . The method of  claim 42 , wherein the CRISPR enzyme is a type II CRISPR system enzyme. 
     
     
         44 . The method of  claim 42 , wherein the CRISPR enzyme is a Cas9. 
     
     
         45 . The method of  claim 44 , wherein the Cas9 is  S. pyogenes  Cas9. 
     
     
         46 . The method of  claim 42 , wherein the CRISPR enzyme is not endogenous to the prokaryote(s). 
     
     
         47 . The method of  claim 42  wherein the prokaryote(s) is  S. pneumoniae  or  E. coli.   
     
     
         48 . The method of  claim 42  wherein the one or more vectors are plasmids. 
     
     
         49 . A method of generating one mutation or multiple mutations in one or more prokaryotic cell(s) by introducing the one mutation or multiple mutations in one or more prokaryotic cell(s) comprising:
 A. generating one mutation in one or more prokaryotic cell(s) by introducing the mutation in one or more prokaryotic cell(s), by a method comprising:   introducing one or more vectors into the prokaryotic cell(s);   wherein the one or more vectors drive expression of one or more of: a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template;   and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, and an editing template are produced in the prokaryotic cell(s);   allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide whereby the editing template comprising the mutation is introduced through recombination in the one or more prokaryote(s); and   wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to a tracr sequence;   thereby generating one or more prokaryotic cell(s) in which the mutation has been introduced;
 or 
   B. generating multiple mutations in one or more prokaryotic cell(s) by introducing the multiple mutations in one or more prokaryotic cell(s), by a method comprising:   introducing one or more vectors into the prokaryotic cell(s);   wherein the one or more vectors drive expression of one or more of: a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template;   and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, and an editing template are produced in the prokaryotic cell(s);   allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide whereby the editing template comprising the multiple mutations is introduced through recombination in the one or more prokaryote(s); and   wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to a tracr sequence;   thereby generating one or more prokaryotic cell(s) in which multiple mutations have been introduced;
 or 
   C. generating multiple mutations in one or more prokaryotic cell(s) by introducing the multiple mutations in one or more prokaryotic cell(s), by a method comprising:   introducing one or more vectors into the prokaryotic cell(s);   wherein the one or more vectors drive expression of one or more of: a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template;   and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, and an editing template are produced in the prokaryotic cell(s);   allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide whereby the editing template comprising a first mutation is introduced through recombination in the one or more prokaryote(s); and   wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to a tracr sequence;   thereby generating one or more prokaryotic cell(s) in which the first mutation has been introduced;   introducing one or more vectors into the prokaryotic cell(s) having the first mutation;   wherein the one or more vectors drive expression of one or more of: a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template;   and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence, and an editing template are produced in the prokaryotic cell(s);   allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide whereby the editing template comprising a second mutation is introduced through recombination in the one or more prokaryote(s); and   wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to a tracr sequence;   thereby generating one or more prokaryotic cell(s) in which the first and the second mutation has been introduced.   
     
     
         50 . The method of  claim 49 , wherein the CRISPR enzyme is a type II CRISPR system enzyme. 
     
     
         51 . The method of  claim 49 , wherein the CRISPR enzyme is Cas9. 
     
     
         52 . The method of  claim 51 , wherein the Cas9 is  S. pyogenes  Cas9. 
     
     
         53 . The method of  claim 49 , wherein the CRISPR enzyme is not endogenous to the prokaryote(s). 
     
     
         54 . The method of  claim 49  wherein the prokaryote(s) is  S. pneumoniae  or  E. coli.   
     
     
         55 . The method of  claim 49  wherein the one or more vectors are plasmids. 
     
     
         56 . A method for introducing mutation(s) into a prokaryote comprising introducing the mutation(s) by recombineering, and selecting for prokaryote(s) having the mutation(s)
 by introducing one or more vectors into the prokaryotic cell(s);   wherein the one or more vectors drive expression of one or more of: a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence and an editing oligonucleotide;   and all of a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template are produced in the prokaryotic cell(s);   introducing the editing oligonucleotide comprising the mutation(s) into a target polynucleotide by recombineering,   allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of the target polynucleotide, wherein the target sequence is in prokaryote(s) not having the mutation(s) from recombineering; and   wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to a target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to a tracr sequence;   wherein binding of the CRISPR complex to the target polynucleotide results in Cas9-directed cleavage at a targeted site in the prokaryote(s) not having the mutation(s) and induces cell death.   
     
     
         57 . The method of  claim 56 , wherein the CRISPR enzyme is a type II CRISPR system enzyme. 
     
     
         58 . The method of  claim 56 , wherein the CRISPR enzyme is Cas9. 
     
     
         59 . The method of  claim 56 , wherein the Cas9 is  S. pyogenes  Cas9. 
     
     
         60 . The method of  claim 56 , wherein the CRISPR enzyme is not endogenous to the prokaryote(s). 
     
     
         61 . The method of  claim 56  wherein the prokaryote(s) is  E. coli.