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.

1 . A non-naturally occurring or engineered composition comprising a vector system comprising one or more eukaryotic expression vectors comprising
 I. a CRISPR-Cas complex chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises   (a) a guide sequence capable of hybridizing to a target eukaryotic genome DNA sequence in a eukaryotic cell, wherein the target sequence is adjacent to a DNA Protospacer Adjacent Motif (PAM),   (b) a tracr mate sequence, and   (c) a tracr sequence comprising 30 or more nucleotides in length, and   II. a nucleotide sequence encoding a Cas9 comprising at least one or more nuclear localization signals (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, 
 components I and II each are operably linked to a regulatory element for transcription thereof in the eukaryotic cell and there can be one or more regulatory elements for transcription of components I and II, 
 when the one or more vectors is introduced into the eukaryotic cell, components I and II are transcribed in the eukaryotic cell, 
 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, there is PAM recognition, eukaryotic genome DNA cleavage and altered gene expression in the eukaryotic cell, 
 the CRISPR complex comprises the Cas9 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, 
 the Cas9 comprises one or more mutations in a catalytic domain thereby rendering the Cas9 a nickase that cleaves a single DNA strand, and 
   wherein the chimeric RNA polynucleotide sequence comprises two or more hairpins.   
     
     
         2 . A multiplexed CRISPR system composition, wherein the system comprises a vector system comprising one or more eukaryotic expression vectors comprising
 I. more than one CRISPR-Cas complex chimeric RNA (chiRNA) polynucleotide sequence,
 wherein each polynucleotide sequence comprises 
 (a) a guide sequence capable of hybridizing to a target eukaryotic genome DNA sequence in a eukaryotic cell adjacent to a DNA Protospacer Adjacent Motif (PAM),
 wherein each guide sequence of each of the more than one polynucleotide sequences is capable of hybridizing to a different target eukaryotic genome DNA sequence in the eukaryotic cell: each target sequence being adjacent to a DNA Protospacer Adjacent Motif (PAM), 
 
 (b) a tracr mate sequence, and 
 (c) a tracr sequence, and 
   II. a nucleotide sequence encoding a Cas9 comprising at least one or more nuclear localization signals (NLSs) in the proximity of a terminus of the Cas9,
 wherein,
 each of components I and II are operably linked to a regulatory element for transcription thereof in the eukaryotic cell and there can be one or more regulatory elements for transcription of components I and II, 
 when the one or more vectors is introduced into the eukaryotic cell, components I and II are transcribed in the eukaryotic cell, 
 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, there is PAM recognition, eukaryotic genome DNA cleavage and altered gene expression in the eukaryotic cell, 
 the CRISPR complex comprises the Cas9 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 
 each chimeric RNA polynucleotide sequence comprises two or more hairpins, 
 each guide RNA comprises a tracr sequence comprising 30 or more nucleotides in length, and 
 
 the Cas9 comprises one or more mutations in a catalytic domain thereby rendering the Cas9 a nickase that cleaves a single DNA strand. 
   
     
     
         3 . The composition of  claim 1 , wherein the regulatory element as to the polynucleotide sequence comprises a polymerase III promoter. 
     
     
         4 . The composition of  claim 1 , wherein the regulatory element as to the nucleotide sequence encoding the Cas9 comprises a polymerase II promoter. 
     
     
         5 . The composition of  claim 1 , wherein the guide sequence comprises about or more than about 10-75 nucleotides in length, and the tracr sequence comprises about or more than about 30-50 nucleotides in length, and the tracr sequence exhibits at least 500/a sequence complementarity along the length of the tracr mate. 
     
     
         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. 
     
     
         7 . The composition of  claim 1 , wherein the nucleotide sequence encoding the Cas9 is codon-optimized for expression in a eukaryotic cell. 
     
     
         8 . The composition of  claim 1 , wherein the guide sequence is at least 15 nucleotides in length. 
     
     
         9 . The composition of  claim 1 , wherein the chimeric RNA polynucleotide sequence comprises two, three, four or five hairpins. 
     
     
         10 . The composition of  claim 1 , wherein the catalytic domain comprises RuvCI, RuvCII, RuvCIII or HNH domain. 
     
     
         11 . The composition of  claim 1 , wherein the Cas9 comprises a mutation in a residue selected from the group consisting of D10, E762, H840, N854, N863, or D986, with reference to  S. pyogenes  Cas9. 
     
     
         12 . The composition of  claim 1 , wherein the Cas9 comprises a mutation comprising D10A, E762A, H840A, N854A, N863A or D986A, with reference to  S. pyogenes  Cas9. 
     
     
         13 . A non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising
 I. (a) a guide sequence capable of hybridizing to a eukaryotic genome DNA target sequence in a eukaryotic cell, wherein the target sequence is adjacent to a DNA Protospacer Adjacent Motif (PAM), and   (b) a tracr mate sequence,   II. a nucleotide sequence encoding a Cas9 comprising at least one or more nuclear localization signals (NLSs) in the proximity of a terminus of the Cas9, and   III. a tracr sequence comprising 30 or more nucleotides in length,   wherein:
 components I, II and III are located on the same or different vectors of the system, 
 components I, II and III are each operably linked to a regulatory element and there can be one or more regulatory elements for transcription of components I, II and III, 
 when the one or more vectors is introduced into the eukaryotic cell, components I, II and III are transcribed in the eukaryotic cell, 
 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 there is PAM recognition, eukaryotic genome DNA cleavage and altered gene expression in the eukaryotic cell, 
 the CRISPR complex comprises the Cas9 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 
 the Cas9 comprises one or more mutations in a catalytic domain thereby rendering the Cas9 a nickase that cleaves a single DNA strand. 
   
     
     
         14 . A multiplexed CRISPR enzyme system composition, wherein the system comprises a vector system comprising one or more vectors comprising
 I. (a) more than one guide sequence, wherein a guide sequence is capable of hybridizing to a eukaryotic genome DNA target sequence in a eukaryotic cell adjacent to a DNA Protospacer Adjacent Motif (PAM), and each guide sequence of the more than one guide sequences is capable of hybridizing to a different target eukaryotic genome DNA sequence in the eukaryotic cell; each target sequence being adjacent to a DNA Protospacer Adjacent Motif (PAM), and   (b) a tracr mate sequence,   II. a nucleotide sequence encoding a Cas9 comprising at least one or more nuclear localization signals (NLSs) in the proximity of a terminus of the Cas9, and   III. a tracr sequence comprising 30 or more nucleotides in length,   wherein:
 components I, II and III are located on the same or different vectors of the system, 
 each of components I, II and III are operably linked to a regulatory element for transcription thereof in the eukaryotic cell and there can be one or more regulatory elements therefor, 
 when the one or more vectors is introduced into the eukaryotic cell, components I, II and III are transcribed in the eukaryotic cell, 
 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 there is PAM recognition, DNA cleavage and altered gene expression in the eukaryotic cell, 
 the CRISPR complex comprises the Cas9 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 
 the Cas9 comprises one or more mutations in a catalytic domain thereby rendering the Cas9 a nickase that cleaves a single DNA strand. 
   
     
     
         15 . The composition of  claim 13 , wherein the regulatory element associated with component I comprises a polymerase III promoter. 
     
     
         16 . The composition of  claim 13 , wherein the regulatory element associated with the nucleotide sequence encoding the Cas9 comprises a polymerase II promoter. 
     
     
         17 . The composition of  claim 13 , wherein the regulatory element associated with the component III comprises a polymerase III promoter. 
     
     
         18 . The composition of  claim 13 , the guide sequence comprises about or more than about 10-75 nucleotides in length, and the tracr sequence comprises about or more than about 30-50 nucleotides in length, and the tracr sequence exhibits at least 50% sequence complementarity along the length of the tracr mate. 
     
     
         19 . The composition of  claim 13 , wherein the tracr sequence exhibits at least 50% of sequence complementarity along the length of the tracr mate sequence. 
     
     
         20 . The composition of  claim 13 , wherein the nucleotide sequence encoding the Cas9 is codon-optimized for expression in a eukaryotic cell. 
     
     
         21 . The composition of  claim 13 , wherein the guide sequence is at least 15 nucleotides in length. 
     
     
         22 . The composition of  claim 13 , wherein the catalytic domain comprises RuvCI, RuvCII, RuvCIII or HNH domain. 
     
     
         23 . The composition of  claim 13 , wherein the Cas9 comprises a mutation in a residue selected from the group consisting of D10, E762, H840, N854, N863, or D986, with reference to  S. pyogenes  Cas9. 
     
     
         24 . The composition of  claim 13 , wherein the Cas9 comprises a mutation comprising D10A, E762A, H840A, N854A, N863A or D986A, with reference to  S. pyogenes  Cas9. 
     
     
         25 . An isolated eukaryotic host cell comprising the composition of  claim 1 . 
     
     
         26 . A non-human animal comprising the eukaryotic host cell of  claim 25 . 
     
     
         27 . A non-human organism or plant comprising the eukaryotic host cell of  claim 25 . 
     
     
         28 . A kit comprising the composition of  claim 1 , and instructions for using said kit. 
     
     
         29 . A method of altering the expression of a genomic locus of interest in a eukaryotic cell comprising
 contacting the eukaryotic cell with the composition of  claim 1  and thereby delivering the one or more vectors and allowing the CRISPR-Cas complex to form and bind to target and   determining if the expression of the genomic locus has been altered.   
     
     
         30 . The composition of  claim 1  or  2 , wherein components I and II are on the same vector. 
     
     
         31 . An isolated eukaryotic cell or a eukaryotic cell of a non-human organism comprising the composition of  claim 30 . 
     
     
         32 . The composition of  claim 13 , wherein components I, II and III are on the same vector. 
     
     
         33 . An isolated eukaryotic cell or a eukaryotic cell of a non-human organism comprising the composition of  claim 32 . 
     
     
         34 . The composition of  claim 1 , wherein the Cas9 comprises a fusion of a Cas9 protein and one or more effector domains. 
     
     
         35 . The composition according to  claim 34 , wherein the one or more effector domains comprises a transposase domain, integrase domain, recombinase domain, resolvase domain, invertase domain, protease domain, DNA methyltransferase domain, DNA demethylase domain, histone acetylase domain, histone deacetylases domain, nuclease domain, repressor domain, activator domain, transcription-protein recruiting domain, cellular uptake activity associated domain, nucleic acid binding domain or antibody presentation domain. 
     
     
         36 . A method of modifying the target genome DNA sequence in the eukaryotic cell, the method comprising contacting the eukaryotic cell with the composition of  claim 1  and thereby delivering the one or more vectors to the eukaryotic cell and allowing a CRISPR complex to form and bind to the target to effect DNA cleavage of said target thereby modifying the target. 
     
     
         37 . The method of  claim 36 , wherein said one or more vectors are delivered to the eukaryotic cell in a subject. 
     
     
         38 . The method of  claim 36 , wherein said modifying takes place in said eukaryotic cell in a cell culture. 
     
     
         39 . The method of  claim 36 , further comprising isolating said eukaryotic cell from a subject prior to said modifying. 
     
     
         40 . The method of  claim 39 , further comprising returning said eukaryotic cell and/or progeny cell(s) therefrom to said subject after said modifying. 
     
     
         41 . An isolated eukaryotic cell or a eukaryotic cell of a non-human organism from the method of  claim 36 . 
     
     
         42 . A vector comprising a regulatory element operably linked to a coding sequence encoding a Cas 9 comprising one or more nuclear localization signals (NLSs), wherein said regulatory element drives transcription of the Cas 9 in a eukaryotic cell such that said Cas 9 accumulates in a detectable amount in the nucleus of the eukaryotic cell; wherein the Cas 9 comprises one or more mutations in a catalytic domain thereby rendering the Cas 9 a nickase that cleaves a single DNA strand. 
     
     
         43 . The vector of  claim 42 , wherein said regulatory element is a polymerase II promoter. 
     
     
         44 . An isolated eukaryotic cell or a eukaryotic cell of a non-human organism comprising the vector of  claim 42 . 
     
     
         45 . An isolated eukaryotic cell or a eukaryotic cell of a non-human organism comprising a Cas 9; wherein the Cas 9 comprises one or more mutations in a catalytic domain thereby rendering the Cas 9 to a nickase that cleaves a single DNA strand. 
     
     
         46 . An isolated eukaryotic cell culture from the cell of  claim 31 . 
     
     
         47 . An isolated eukaryotic cell culture from the cell of  claim 33 . 
     
     
         48 . An isolated eukaryotic cell culture from the cell of  claim 41 . 
     
     
         49 . An isolated eukaryotic cell culture from the cell of  claim 44 . 
     
     
         50 . The composition of  claim 1  wherein as to component I there is a first regulatory element and as to component II there is a second regulatory element. 
     
     
         51 . The composition of  claim 13  wherein as to component I there is a first regulatory element, as to component II there is a second regulatory element, and as to component III there is a third regulatory element. 
     
     
         52 . A non-naturally occurring or engineered composition comprising a single eukaryotic expression vector comprising
 I. a CRISPR-Cas complex chimeric RNA (chiRNA) polynucleotide sequence,
 wherein the polynucleotide sequence comprises 
 (a) a guide sequence capable of hybridizing to a target mammalian genome DNA sequence in a mammalian cell, wherein the target sequence is adjacent to a DNA Protospacer Adjacent Motif (PAM), 
 (b) a tracr mate sequence, and 
 (c) a tracr sequence, and 
   II. a nucleotide sequence encoding a Cas9 comprising at least one or more nuclear localization signals (NLSs) in the proximity of a terminus of the Cas9,
 wherein:
 components I and II are each operably linked to a regulatory element for transcription thereof in the mammalian cell and the vector includes at least one regulatory element therefor, 
 when the vector is introduced into the mammalian cell, components I and II are transcribed in the mammalian cell, 
 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, there is PAM recognition, mammalian genome DNA cleavage and altered gene expression in the mammalian cell, 
 the CRISPR complex comprises the Cas9 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, 
 the chimeric RNA polynucleotide sequence comprises two or more hairpins, 
 the guide RNA comprises a tracr sequence comprising 30 or more nucleotides in length, and 
 the Cas9 comprises one or more mutations in a catalytic domain thereby rendering the Cas9 a nickase that cleaves a single DNA strand. 
 
   
     
     
         53 . A multiplexed CRISPR system comprising
 a eukaryotic expression vector comprising:   I. more than one CRISPR-Cas complex chimeric RNA (chiRNA) polynucleotide sequence,
 wherein each polynucleotide sequence comprises 
 (a) a guide sequence capable of hybridizing to a target mammalian genome DNA sequence in a mammalian cell adjacent to a DNA Protospacer Adjacent Motif (PAM),
 wherein each guide sequence of each of the more than one polynucleotide sequences is capable of hybridizing to a different target mammalian genome DNA sequence in the mammalian cell; each target sequence being adjacent to a DNA Protospacer Adjacent Motif (PAM), 
 
 (b) a tracr mate sequence, and 
 (c) a tracr sequence, and 
   II. a nucleotide sequence encoding a Cas9 comprising at least one or more nuclear localization signals (NLSs) in the proximity of a terminus of the Cas9,
 wherein:
 each of components I and II are operably linked to a regulatory element for transcription thereof in the mammalian cell and the vector includes at least one regulatory element therefor, 
 when the vector is introduced into the mammalian cell, components I and II are transcribed in the mammalian cell, 
 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, there is PAM recognition, multiplexed editing within a single genome including mammalian genome DNA cleavage at more than one loci and altered gene expression in the mammalian cell, 
 the CRISPR complex comprises the Cas9 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 
 each chimeric RNA polynucleotide sequence comprises two or more hairpins and 
 each guide RNA comprises a tracr sequence comprising 30 or more nucleotides in length, and 
 the Cas9 comprises one or more mutations in a catalytic domain thereby rendering the Cas9 a nickase that cleaves a single DNA strand. 
 
   
     
     
         54 . A non-naturally occurring or engineered composition comprising a single eukaryotic expression vector comprising:
 I. (a) a guide sequence capable of hybridizing to a mammalian genome DNA target sequence in a mammalian cell, wherein the target sequence is adjacent to a DNA Protospacer Adjacent Motif (PAM), and   (b) a tracr mate sequence,   II. a nucleotide sequence encoding a Cas9 comprising at least one or more nuclear localization signals (NLSs) in the proximity of a terminus of the Cas9, and   III. a tracr sequence, wherein the tracr sequence comprises 30 or more nucleotides in length,   wherein:
 components I, II and III are each operably linked to a regulatory element and the vector includes one or more regulatory elements for transcription of components I, II and III 
 when the vector is introduced into the mammalian cell, components I, II and III are transcribed in the mammalian cell, 
 when transcribed, the tract mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, there is PAM recognition, mammalian genome DNA cleavage and altered gene expression in the mammalian cell, 
 the CRISPR complex comprises the Cas9 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 
 the chimeric RNA polynucleotide sequence comprises two or more hairpins and 
 the guide RNA comprises a tracr sequence comprising 30 or more nucleotides in length, and 
 the Cas9 comprises one or more mutations in a catalytic domain thereby rendering the Cas9 a nickase that cleaves a single DNA strand. 
   
     
     
         55 . A multiplexed CRISPR enzyme system composition, wherein the system comprises a single eukaryotic expression vector comprising:
 I. (a) more than one guide sequence, wherein a guide sequence is capable of hybridizing to a mammalian genome DNA target sequence in a mammalian cell adjacent to a DNA Protospacer Adjacent Motif (PAM), and each guide sequence of the more than one guide sequences is capable of hybridizing to a different target mammalian genome DNA sequence in the mammalian cell; each target sequence being adjacent to a DNA Protospacer Adjacent Motif (PAM),   (b) a tracr mate sequence,   II. a nucleotide sequence encoding a Cas9 comprising at least one or more nuclear localization signals (NLSs) in the proximity of a terminus of the Cas9, and   III. a tracr sequence, wherein the tracr sequence comprises 30 or more nucleotides in length,   wherein:
 components I, II and III are each of components I, II and III are operably linked to a regulatory element for transcription thereof in the mammalian cell and the vector includes at least one regulatory element therefor, 
 when the vector is introduced into the mammalian cell, components I, II and III are transcribed in the mammalian cell, 
 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, there is PAM recognition, multiplexed editing within a single genome including mammalian genome DNA cleavage at more than one loci and altered gene expression in the mammalian cell, 
 the CRISPR complex comprises the Cas9 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 
 each chimeric RNA polynucleotide sequence comprises two or more hairpins and 
 each guide RNA comprises a tracr sequence comprising 30 or more nucleotides in length, and 
 the Cas9 comprises one or more mutations in a catalytic domain thereby rendering the Cas9 a nickase that cleaves a single DNA strand. 
   
     
     
         56 . The composition of  claim 52 , wherein the regulatory element as to the polynucleotide sequence comprises a polymerase III promoter. 
     
     
         57 . The composition of  claim 52 , wherein the regulatory element as to the nucleotide sequence encoding the Cas9 comprises a polymerase II promoter. 
     
     
         58 . The composition of  claim 54 , wherein the regulatory element associated with component I comprises a polymerase III promoter. 
     
     
         59 . The composition of  claim 54 , wherein the regulatory element associated with the Cas9 comprises a polymerase II promoter. 
     
     
         60 . The composition of  claim 54 , wherein the regulatory element associated with the component III comprises a polymerase III promoter. 
     
     
         61 . The composition of  claim 52  wherein as to component I there is a first regulatory element and as to component II there is a second regulatory element. 
     
     
         62 . The composition of  claim 54  wherein as to component I there is a first regulatory element, as to component II there is a second regulatory element, and as to component III there is a third regulatory element. 
     
     
         63 . The composition of  claim 52 , wherein the guide sequence comprises about or more than about 10-75 nucleotides in length, and the tracr sequence comprises about or more than about 30-50 nucleotides in length, and the tracr sequence exhibits at least 50% sequence complementarity along the length of the tracr mate. 
     
     
         64 . The composition of  claim 52 , wherein the tracr sequence exhibits at least 50% of sequence complementarity along the length of the tracr mate sequence. 
     
     
         65 . The composition of  claim 52 , wherein the nucleotide sequence encoding the Cas9 is codon-optimized for expression in a eukaryotic cell. 
     
     
         66 . The composition of  claim 52 , wherein the guide sequence is at least 15 nucleotides in length. 
     
     
         67 . The composition of  claim 52 , wherein the chimeric RNA polynucleotide sequence comprises two, three, four or five hairpins. 
     
     
         68 . The composition of  claim 52 , wherein the catalytic domain comprises RuvCI, RuvCII, RuvCIII or HNH domain. 
     
     
         69 . The composition of  claim 52 , wherein the Cas9 comprises a mutation in a residue selected from the group consisting of D10, E762, H840, N854, N863, or D986, with reference to  S. pyogenes  Cas9. 
     
     
         70 . The composition of  claim 52 , wherein the Cas9 comprises a mutation comprising D10A, E762A, H840A, N854A, N863A or D986A, with reference to  S. pyogenes  Cas9. 
     
     
         71 . An isolated mammalian host cell comprising the composition of  claim 52 . 
     
     
         72 . A non-human animal comprising the mammalian host cell of  claim 71 . 
     
     
         73 . A kit comprising the composition of  claim 52 , and instructions for using said kit. 
     
     
         74 . A method of altering the expression of a genomic locus of interest in a mammalian cell comprising
 contacting the cell with the composition of  claim 52  and thereby delivering the vector and allowing the CRISPR-Cas complex to form and bind to target, and   determining if the expression of the genomic locus has been altered.   
     
     
         75 . The composition according to  claim 52  wherein the Cas9 comprises a fusion of a Cas9 protein and one or more effector domains. 
     
     
         76 . The composition according to  claim 75 , wherein the one or more effector domains comprises a transposase domain, integrase domain, recombinase domain, resolvase domain, invertase domain, protease domain, DNA methyltransferase domain, DNA demethylase domain, histone acetylase domain, histone deacetylases domain, nuclease domain, repressor domain, activator domain, transcription-protein recruiting domain, cellular uptake activity associated domain, nucleic acid binding domain or antibody presentation domain. 
     
     
         77 . The composition or system according to  claim 76 , wherein the one or more effector domains comprises a nuclease domain. 
     
     
         78 . The method of  claim 74 , including DNA cleavage resulting in decreased transcription of a gene. 
     
     
         79 . The method of  claim 74  wherein altering expression comprises genome editing. 
     
     
         80 . The composition of  claim 1 , wherein altered expression comprises genome editing. 
     
     
         81 . The composition of  claim 2  wherein the DNA cleavage and altered gene expression comprises multiplexed editing within a single genome including mammalian genome DNA cleavage at more than one loci. 
     
     
         82 . A method of modifying the target genome DNA sequence in the mammalian cell, the method comprising contacting the mammalian cell with the composition of  claim 52  with the cell and thereby delivering the vector to the mammalian cell and allowing a CRISPR complex to form and bind to the target to effect DNA cleavage of said target thereby modifying the target. 
     
     
         83 . The method of  claim 82  wherein the modifying obtains increased expression as to a product of the cell, in comparison with a cell that has not been subjected to the method. 
     
     
         84 . The method of  claim 36  wherein the modifying obtains increased expression as to a product of the cell, in comparison with a cell that has not been subjected to the method. 
     
     
         85 . An isolated cell having increased expression as to a product of the cell, from the method of  claim 83 , wherein the increased expression is in comparison with a cell that has not been subjected to method modifying the target genome DNA sequence in the cell. 
     
     
         86 . An isolated cell having increased expression as to a product of the cell, from the method of  claim 84 , wherein the increased expression is in comparison with a cell that has not been subjected to method modifying the target genome DNA sequence in the cell. 
     
     
         87 . An isolated cell line of the cell of  claim 85 . 
     
     
         88 . An isolated cell line of the cell of  claim 86 .