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 CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises   (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell,   (b) a tracr mate sequence, and   (c) a tracr sequence, and   II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences (NLSs) in the proximity of a terminus of the CRISPR enzyme,   wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,   wherein components I and II are located on the same or different vectors of the system,   wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence,   wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, and   wherein the chimeric RNA polynucleotide sequence comprises two or more hairpins.   
     
     
         2 . A multiplexed CRISPR enzyme system, wherein the system comprises a vector system comprising one or more vectors comprising
 I. a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises   (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell,   (h) a tracr mate sequence, and   (c) a tracr sequence, and   II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences (NLSs) in the proximity of a terminus of the CRISPR enzyme,   wherein (a), (b) and (c) are arranged in a 5′ to 3′ orientation,   wherein components I and II are located on the same or different vectors of the system,   wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence,   wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,   wherein the chiRNA polynucleotide sequence comprises two or more hairpins, and   wherein in the multiplexed system multiple chiRNA polynucleotide sequences are used.   
     
     
         3 . The composition of  claim 1  or  2 , wherein the first regulatory element is a polymerase III promoter. 
     
     
         4 . The composition of  claim 1  or  2 , wherein the second regulatory element is a polymerase II promoter. 
     
     
         5 . The composition of  claim 1  or  2 , wherein the CRISPR enzyme comprises one or more NLSs of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. 
     
     
         6 . The composition of  claim 1  or  2 , 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  or  2 , wherein the CRISPR enzyme is a type II CRISPR system enzyme. 
     
     
         8 . The composition of  claim 1  or  2 , wherein the CRISPR enzyme is a Cas9 enzyme. 
     
     
         9 . The composition of  claim 1  or  2 , wherein the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. 
     
     
         10 . The composition of  claim 1  or  2 , wherein the guide sequence is at least 15 nucleotides in length. 
     
     
         11 . The composition of  claim 1  or  2 , wherein the chimeric RNA polynucleotide sequence 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) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and   (b) a tracr mate sequence,   II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences (NLSs) in the proximity of a terminus of the CRISPR enzyme, and   III. a third regulatory element operably linked to a tracr sequence,   wherein components I, II and III are located on the same or different vectors of the system,   wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and   wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence.   
     
     
         13 . A multiplexed CRISPR enzyme system, wherein the system comprises a vector system comprising one or more vectors comprising
 I. a first regulatory element operably linked to   (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and   (b) tracr mate sequence,   II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences (NLSs) in the proximity of a terminus of the CRISPR enzyme, and   III. a third regulatory element operably linked to a tracr sequence,   wherein components I, II and III are located on the same or different vectors of the system,   wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence,   wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, and   wherein in the multiplexed system multiple guide sequences and a single tracr sequence is used.   
     
     
         14 . The composition of  claim 12  or  13 , wherein the first regulatory element is a polymerase III promoter. 
     
     
         15 . The composition of  claim 12  or  13 , wherein the second regulatory element is a polymerase II promoter. 
     
     
         16 . The composition of  claim 12  or  13 , wherein the third regulatory element is a polymerase III promoter. 
     
     
         17 . The composition of  claim 12  or  13 , wherein the CRISPR enzyme comprises one or more NLSs of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. 
     
     
         18 . The composition of  claim 12  or  13 , 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  or  13 , wherein the CRISPR enzyme is a type II CRISPR system enzyme. 
     
     
         20 . The composition of  claim 12  or  13 , wherein the CRISPR enzyme is a Cas9 enzyme. 
     
     
         21 . The composition of  claim 12  or  13 , wherein the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. 
     
     
         22 . The composition of  claim 12  or  13 , wherein the guide sequence is at least 15 nucleotides in length. 
     
     
         23 . A eukaryotic host cell comprising the composition of any of the preceding claims. 
     
     
         24 . An organism comprising the eukaryotic host cell of  claim 23 . 
     
     
         25 . A non-human organism comprising the eukaryotic host cell of  claim 23 . 
     
     
         26 . A kit comprising the composition of any of  claims 1  to  22  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 any of  claims 1  to  22 , 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. 
     
     
         30 . The method of selecting one or more prokaryotic cell(s) by introducing one or more mutations in a gene in the 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;
 wherein the editing template comprises the one or more mutations that abolish CRISPR enzyme cleavage; 
   allowing homologous recombination of the editing template with the target polynucleotide in the cell(s) to be selected;   allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide within said gene, wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence within the 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 induces cell death, 
   thereby allowing one or more prokaryotic cell(s) in which one or more mutations have been introduced to be selected.   
     
     
         31 . The method of  claim 30 , wherein the CRISPR enzyme is a type II CRISPR system enzyme 
     
     
         32 . The method of  claim 31 , wherein the CRISPR enzyme is Cas9. 
     
     
         33 . A vector system comprising one or more vectors, wherein the system comprises
 a. a first regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting a guide sequence upstream of the tracr mate sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence; and   b. a second regulatory element operably linked to an enzyme-coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence;   wherein components (a) and (b) are located on the same or different vectors of the system.   
     
     
         34 . The vector system of  claim 33 , wherein component (a) further comprises the tracr sequence downstream of the tracr mate sequence under the control of the first regulatory element. 
     
     
         35 . The vector system of  claim 33 , wherein component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a CRISPR complex to a different target sequence in a eukaryotic cell. 
     
     
         36 . The vector system of  claim 35 , wherein the system comprises the tracr sequence under the control of a third regulatory element. 
     
     
         37 . The vector system of  claim 33 , wherein the tracr sequence exhibits at least 50% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. 
     
     
         38 . The vector system of  claim 33 , wherein the CRISPR enzyme comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. 
     
     
         39 . The vector system of  claim 33 , wherein the CRISPR enzyme is a type II CRISPR system enzyme. 
     
     
         40 . The vector system of  claim 33 , wherein the CRISPR enzyme is a Cas9 enzyme. 
     
     
         41 . The vector system of  claim 33 , wherein the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. 
     
     
         42 . The vector system of  claim 33 , wherein the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. 
     
     
         43 . The vector system of  claim 33 , wherein the CRISPR enzyme lacks DNA strand cleavage activity. 
     
     
         44 . The vector system of  claim 33 , wherein the first regulatory element is a polymerase III promoter. 
     
     
         45 . The vector system of  claim 33 , wherein the second regulatory element is a polymerase II promoter. 
     
     
         46 . The vector system of  claim 36 , wherein the third regulatory element is a polymerase III promoter. 
     
     
         47 . The vector system of  claim 33 , wherein the guide sequence is at least 15 nucleotides in length. 
     
     
         48 . The vector system of  claim 33 , wherein fewer than 50% of the nucleotides of the guide sequence participate in self-complementary base-pairing when optimally folded. 
     
     
         49 . A vector comprising a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising one or more nuclear localization sequences, wherein said regulatory element drives transcription of the CRISPR enzyme in a eukaryotic cell such that said CRISPR enzyme accumulates in a detectable amount in the nucleus of the eukaryotic cell. 
     
     
         50 . The vector of  claim 49 , wherein said regulatory element is a polymerase II promoter. 
     
     
         51 . The vector of  claim 49 , wherein said CRISPR enzyme is a type II CRISPR system enzyme. 
     
     
         52 . The vector of  claim 49 , wherein said CRISPR enzyme is a Cas9 enzyme. 
     
     
         53 . A method of modifying a target polynucleotide in a eukaryotic cell, the method comprising allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence. 
     
     
         54 . The method of  claim 53 , wherein said cleavage comprises cleaving two strands at the location of the target sequence by said CRISPR enzyme. 
     
     
         55 . The method of  claim 53 , wherein said cleavage results in decreased transcription of a target gene. 
     
     
         56 . The method of  claim 53 , further comprising repairing said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein said repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide. 
     
     
         57 . The method of  claim 56 , wherein said mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence. 
     
     
         58 . The method of  claim 53 , further comprising delivering one or more vectors to said eukaryotic cell, wherein the one or more vectors drive expression of one or more of: the CRISPR enzyme, the guide sequence linked to the tracr mate sequence, and the tracr sequence. 
     
     
         59 . The method of  claim 58 , wherein said vectors are delivered to the eukaryotic cell in a subject. 
     
     
         60 . The method of  claim 53 , wherein said modifying takes place in said eukaryotic cell in a cell culture. 
     
     
         61 . The method of  claim 53 , further comprising isolating said eukaryotic cell from a subject prior to said modifying. 
     
     
         62 . The method of  claim 61 , further comprising returning said eukaryotic cell and/or cells derived therefrom to said subject.