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 . An engineered composition comprising a vector system comprising one or more eukaryotic expression vectors comprising
 I. a nucleotide sequence encoding a CRISPR-Cas complex chimeric RNA (chiRNA) having two or more hairpins and comprising   (a) a guide sequence capable of hybridizing to a target eukaryotic genome DNA sequence adjacent to a Protospacer Adjacent Motif (PAM) in a eukaryotic cell   (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 one or more nuclear localization signals (NLSs) in the proximity of a terminus of the Cas9 and one or more mutations in a catalytic domain thereby rendering it a nickase that cleaves a single DNA strand,   wherein:
 components I and II are located on the same or different vectors of the system, and 
 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. 
   
     
     
         2 . A CRISPR composition for multiplexing, wherein the composition comprises a vector system comprising one or more eukaryotic expression vectors comprising
 I. a nucleotide sequence encoding more than one CRISPR-Cas complex   each having two or more hairpins and comprising
 (a) a guide sequence capable of hybridizing to a different target eukaryotic genome DNA sequence adjacent to a PAM in a eukaryotic, 
 (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 one or more NLSs in the proximity of a terminus of the Cas9 and one or more mutations in a catalytic domain thereby rendering it a nickase that cleaves a single DNA strand,
 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. 
 
   
     
     
         3 . The composition of  claim 1 , wherein the regulatory element operably linked to component I comprises a polymerase III promoter. 
     
     
         4 . The composition of  claim 1 , wherein the regulatory element operably linked to component II 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 50% 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 chiRNA 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 . An engineered composition comprising a vector system comprising one or more vectors comprising
 I. a nucleotide sequence encoding (a) a guide sequence capable of hybridizing to a eukaryotic genome DNA target sequence adjacent to a PAM in a eukaryotic cell, and (b) a tracr mate sequence,   II. a nucleotide sequence encoding a Cas9 comprising one or more NLSs in the proximity of a terminus of the Cas9 and one or more mutations in a catalytic domain thereby rendering it a nickase that cleaves a single DNA strand, and   III. a nucleotide sequence encoding 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, and 
 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. 
   
     
     
         14 . A CRISPR composition for multiplexing, wherein the composition comprises a vector system comprising one or more vectors comprising
 I. (a) nucleotide sequences encoding more than one guide sequences each capable of hybridizing to a different eukaryotic genome DNA target sequence adjacent to a PAM in a eukaryotic cell, and (b) a tracr mate sequence,   II. a nucleotide sequence encoding a Cas9 comprising one or more NLSs in the proximity of a terminus of the Cas9 and one or more mutations in a catalytic domain thereby rendering it a nickase that cleaves a single DNA strand, and   III. a nucleotide sequence encoding 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, and 
 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. 
   
     
     
         15 . The composition of  claim 13 , wherein the regulatory element operably linked to component I comprises a polymerase III promoter. 
     
     
         16 . The composition of  claim 13 , wherein the regulatory element operably linked to component II comprises a polymerase II promoter. 
     
     
         17 . The composition of  claim 13 , wherein the regulatory element operably linked to component III comprises a polymerase III promoter. 
     
     
         18 . The composition of  claim 13 , 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. 
     
     
         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 a 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 , 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 one or more heterologous 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 eukaryotic genome DNA sequence adjacent to a PAM 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 Cas9 comprising one or more NLSs, wherein said regulatory element is configured to drive transcription of the Cas9 in a eukaryotic cell in an amount effective to accumulate the Cas9 in the nucleus of the eukaryotic cell; wherein the Cas9 comprises one or more mutations in a catalytic domain thereby rendering the Cas9 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 Cas9; wherein the Cas9 comprises one or more mutations in a catalytic domain thereby rendering the Cas9 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 component I is operably linked to a first regulatory element and component II is operably linked to a second regulatory element. 
     
     
         51 . The composition of  claim 13  wherein component I is operably linked to a first regulatory element, component II is operably linked to a second regulatory element, and component III is operably linked to a third regulatory element. 
     
     
         52 . The composition of  claim 1  wherein the target eukaryotic genome DNA sequence adjacent to a PAM in a eukaryotic cell is a target mammalian genome DNA sequence adjacent to a PAM in a mammalian cell. 
     
     
         53 . A method of modifying the target mammalian genome DNA sequence adjacent to a PAM in the mammalian cell, the method comprising contacting the mammalian cell with the composition of  claim 52  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. 
     
     
         54 . The method of  claim 53  wherein the modifying obtains increased expression as to a product of the mammalian cell, in comparison with a cell that has not been subjected to the method. 
     
     
         55 . 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. 
     
     
         56 . An isolated cell having increased expression as to a product of the cell, from the method of  claim 54 , 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. 
     
     
         57 . An isolated cell having increased expression as to a product of the cell, from the method of  claim 55 , 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. 
     
     
         58 . An isolated cell line of the cell of  claim 56 . 
     
     
         59 . An isolated cell line of the cell of  claim 57 .