CRISPR-Cas Nickase Systems, Methods And Compositions For Sequence Manipulation in Eukaryotes

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;   wherein the CRISPR enzyme comprises one or more mutations in a catalytic domain thereby rendering the CRISPR enzyme to a nickase that cleaves a single DNA strand, 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,   (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 11 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 CRISPR enzyme comprises one or more mutations in a catalytic domain thereby rendering the CRISPR enzyme to a nickase that cleaves a single DNA strand, and   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 11 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 . The composition of  claim 1  or  2 , wherein the catalytic domain is selected from the group comprising RuvCI, RuvCII, RuvCIII or HNH domain. 
     
     
         13 . The composition of  claim 1  or  2 , wherein the CRISPR enzyme comprises a mutation in a residue selected from the group consisting of D10, E762, H840, N854, N863, or D986. 
     
     
         14 . The composition of  claim 1  or  2 , wherein the CRISPR enzyme comprises a mutation selected from the group comprising D10A, E762A, H840A, N854A, N863A or D986A. 
     
     
         15 . 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   II. 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 the CRISPR enzyme comprises one or more mutations in a catalytic do am thereby rendering the CRISPR enzyme to a nickase that cleaves a single DNA strand.   
     
     
         16 . 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) 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,   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 CRISPR enzyme comprises one or more mutations in a catalytic domain thereby rendering the CRISPR enzyme to a nickase that cleaves a single DNA strand, and   wherein in the multiplexed system multiple guide sequences and a single tracr sequence is used.   
     
     
         17 . The composition of  claim 15  or  16 , wherein the first regulatory element is a polymerase III promoter. 
     
     
         18 . The composition of  claim 15  or  16 , wherein the second regulatory element is a polymerase II promoter. 
     
     
         19 . The composition of  claim 15  or  16 , wherein the third regulatory element is a polymerase III promoter. 
     
     
         20 . The composition of  claim 15  or  16 , 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. 
     
     
         21 . The composition of  claim 15  or  16 , wherein the tracr sequence exhibits at least 50% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. 
     
     
         22 . The composition of  claim 15  or  16 , wherein the CRISPR enzyme is a type 11 CRISPR system enzyme. 
     
     
         23 . The composition of  claim 15  or  16 , wherein the CRISPR enzyme is a Cas9 enzyme. 
     
     
         24 . The composition of  claim 15  or  16 , wherein the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. 
     
     
         25 . The composition of  claim 15  or  16 , wherein the guide sequence is at least 15 nucleotides in length. 
     
     
         26 . The composition of  claim 15  or  16 , wherein the catalytic domain is selected from the group comprising RuvCI, RuvCII, RuvCIII or HNH domain. 
     
     
         27 . The composition of  claim 15  or  16 , wherein the CRISPR enzyme comprises a mutation in a residue selected from the group consisting of D10, E762, H840, N854, N863, or D986. 
     
     
         28 . The composition of  claim 15  or  16 , wherein the CRISPR enzyme comprises a mutation selected from the group comprising D10A, E762A, H840A, N854A, N863A or D986A. 
     
     
         29 . A eukaryotic host cell comprising the composition of any of the preceding claims. 
     
     
         30 . An organism comprising the eukaryotic host cell of  claim 29 . 
     
     
         31 . A non-human organism comprising the eukaryotic host cell of  claim 29 . 
     
     
         32 . A kit comprising the composition of any of  claims 1  to  28  and instructions for using said kit. 
     
     
         33 . 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  28 , and   determining if the expression of the genomic locus has been altered.