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 method for cleaving, editing, or modifying at least one target sequence adjacent to a Protospacer Adjacent Motif (PAM) in a eukaryotic cell, comprising:
 delivering one or more non-naturally occurring or engineered eukaryotic expression vectors to the eukaryotic cell,   wherein the expression vectors comprise:   a) at least one nucleotide sequence encoding at least one chimeric construct comprising a tracr sequence comprising 30 or more nucleotides in length, and   b) at least one nucleotide sequence encoding at least one Cas9,   
       wherein:
 components a) and b) are each operably linked to a regulatory element for transcription thereof in the eukaryotic cell and there is at least one regulatory element therefor, 
 Cas9 comprises one or more mutations in a catalytic domain thereby rendering Cas9 a nickase that cleaves a single DNA strand, 
 the chimeric construct comprises two or more hairpins, 
 the chimeric construct is designed to form with Cas9 a complex, 
 the chimeric construct comprises guide(s) designed to hybridize with the target sequence(s), 
 the chimeric construct and Cas9 do not naturally occur together, and 
 the complex comprising the chimeric construct and Cas9 does not naturally occur, whereby there is cleaving, editing, or modifying of the target sequence. 
 
     
     
         2 . The method of  claim 1 , wherein components a) and b) are on a single vector and the delivering comprises delivering the single vector. 
     
     
         3 . The method of  claim 1 , wherein the method comprises a multiplexed method for cleaving, editing, or modifying more than one target sequence. 
     
     
         4 . The method of  claim 1 , wherein at least one gene target comprises the at least one target sequence and the method is for modulating transcription or altering expression of the gene target. 
     
     
         5 . The method of  claim 1 , wherein the regulatory element comprises a polymerase III promoter. 
     
     
         6 . The method of  claim 1 , wherein the regulatory element comprises a polymerase II promoter. 
     
     
         7 . The method of  claim 1 , wherein the chimeric construct includes a tracr mate sequence, the tracr sequence comprises 30-50 nucleotides or more than 50 nucleotides, and exhibits at least 50% sequence complementarity along the length of the tracr mate. 
     
     
         8 . The method of  claim 1 , wherein component b) is codon-optimized for expression in the eukaryotic cell. 
     
     
         9 . The method of  claim 1 , wherein the guide includes a guide sequence comprising at least 15 nucleotides. 
     
     
         10 . The method of  claim 1 , wherein the chimeric construct comprises two, three, four or five hairpins. 
     
     
         11 . The method of  claim 1 , wherein the catalytic domain comprises RuvCI, RuvCII, RuvCIII or HNH domain. 
     
     
         12 . The method of  claim 1 , wherein the mutation(s) comprise a mutation in a residue selected from the group consisting of D10, E762, H840, N854, N863, and D986, with reference to  S. pyogenes  Cas9. 
     
     
         13 . The method of  claim 1 , wherein the mutation(s) comprise D1OA, E762A, H840A, N854A, N863A or D986A, with reference to  S. pyogenes  Cas9. 
     
     
         14 . The method of  claim 1 , wherein Cas9 includes at least one nuclear localization signal (NLS). 
     
     
         15 . The method of  claim 14 , wherein Cas9 includes at least one NLS at or in the proximity of the C-terminus of Cas9, or at least one NLS at or in the proximity of the N-terminus of Cas9, or at least one NLS at or in the proximity of the C-terminus of Cas9 and at least one NLS at or in the proximity of the N-terminus of Cas9. 
     
     
         16 . The method of  claim 1 , wherein the method is for cleaving the target sequence. 
     
     
         17 . The method of  claim 1 , wherein the method is for editing the target sequence. 
     
     
         18 . The method of  claim 1 , wherein the method is for modifying the target sequence. 
     
     
         19 . The method of  claim 4 , wherein the method is for modulating transcription of the gene target. 
     
     
         20 . The method of  claim 4 , wherein the method is for altering expression of the gene target. 
     
     
         21 . The method of  claim 1 , wherein the expression vector(s) comprise(s) a lentivirus, an adenovirus or an adeno-associated virus. 
     
     
         22 . The method of  claim 1 , wherein the eukaryotic cell comprises a plant cell. 
     
     
         23 . The method of  claim 1 , wherein the eukaryotic cell comprises a mammalian cell. 
     
     
         24 . The method of  claim 1 , wherein Cas9 includes one or more effector domains comprising 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. 
     
     
         25 . The method of  claim 1 , wherein there is at least one first regulatory element for transcription of component a) and at least one second regulatory element for transcription of component b). 
     
     
         26 . The method of  claim 3 , wherein the multiplexed method comprises the chimeric construct designed to hybridize with more than one target sequence comprising component a) comprising coding for more than one chimeric construct. 
     
     
         27 . The method of  claim 26 , wherein there is more than one regulatory element for transcription of the more than one chimeric construct of component a). 
     
     
         28 . The method of  claim 24 , wherein the one or more effector domains comprises a nuclease domain. 
     
     
         29 . The method of  claim 23 , wherein the method includes isolating the mammalian cell from a subject prior to the delivering. 
     
     
         30 . The method of  claim 1 , including delivering a donor polynucleotide. 
     
     
         31 . The method of  claim 23 , wherein the mammalian cell is a human cell. 
     
     
         32 . The method of  claim 1 , wherein the chimeric construct comprises one or more modified nucleotides, nucleotide analogs or non-nucleotide components. 
     
     
         33 . One or more non-naturally occurring or engineered eukaryotic expression vectors comprising:
 a) at least one nucleotide sequence encoding at least one chimeric construct comprising a tracr sequence comprising 30 or more nucleotides in length, and   b) at least one nucleotide sequence encoding at least one Cas9,   
       wherein:
 components a) and b) are each operably linked to a regulatory element for transcription thereof in a eukaryotic cell and there is at least one regulatory element therefor, 
 Cas9 comprises one or more mutations in a catalytic domain thereby rendering Cas9 a nickase that cleaves a single DNA strand, 
 the chimelic construct comprises two or more hairpins, 
 the chimeric construct is designed to form with Cas9 a complex, 
 the chimeric construct comprise(s) guide(s) designed to hybridize with target sequence(s) in the eukaryotic cell 
 the chimeric construct and Cas9 do not naturally occur together, and 
 the complex comprising the chimeric construct and Cas9 does not naturally occur. 
 
     
     
         34 .- 69 . (canceled)