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.

1 . 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, 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. 
 
   
     
     
         2 . A multiplexed CRISPR system comprising a single 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, 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 
 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. 
 
   
     
     
         3 . The composition of  claim 1 , wherein the regulatory element comprises a polymerase III promoter. 
     
     
         4 . The composition of  claim 1 , wherein the regulatory element 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 chimeric RNA polynucleotide sequence comprises two, three, four or five hairpins 
     
     
         10 . 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 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, 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. 
   
     
     
         11 . A multiplexed CRISPR system, 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,
 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, 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 
 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. 
   
     
     
         12 . The composition of  claim 10 , wherein the regulatory element comprises a polymerase III promoter. 
     
     
         13 . The composition of  claim 10 , wherein the regulatory element comprises a polymerase II promoter. 
     
     
         14 . The composition of  claim 1 , wherein the vector comprises more than one regulatory element for transcription of components I and II or components I, II and III. 
     
     
         15 . The composition of  claim 10 , 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. 
     
     
         16 . The composition of  claim 10 , wherein the tracr sequence exhibits at least 50% of sequence complementarity along the length of the tracr mate sequence. 
     
     
         17 . The composition of  claim 10 , wherein the nucleotide sequence encoding the Cas9 is codon-optimized for expression in a eukaryotic cell. 
     
     
         18 . The composition of  claim 10 , wherein the guide sequence is at least 15 nucleotides in length. 
     
     
         19 . An isolated mammalian cell comprising the composition of  claim 1 . 
     
     
         20 . A non-human animal comprising the mammalian cell of  claim 19 . 
     
     
         21 . A non-human organism comprising the mammalian cell of  claim 19 . 
     
     
         22 . A kit comprising the composition of  claim 1  and instructions for using said kit. 
     
     
         23 . 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  claim 1 , and   determining if the expression of the genomic locus has been altered.   
     
     
         24 . 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 trace 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 CRISP 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. 
   
     
     
         25 . The vector of  claim 24 , wherein the tracr sequence is downstream of the tracr mate sequence and both are under the control of the same regulatory element. 
     
     
         26 . The vector of  claim 24 , wherein component (a) further comprises two or more guide sequences, 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, there is PAM recognition, mammalian genome DNA cleavage and altered gene expression in the mammalian cell.   
     
     
         27 . The vector of  claim 26 , wherein the tracr sequence is under the control of a regulatory element separate from that controlling guide sequence transcription. 
     
     
         28 . The vector of  claim 24 , wherein the tracr sequence exhibits at least 50% of sequence complementarity along the length of the tracr mate sequence. 
     
     
         29 . The vector of  claim 24 , wherein the nucleotide sequence encoding the Cas9 is codon-optimized for expression in a eukaryotic cell. 
     
     
         30 . The vector of  claim 24 , wherein the Cas9 directs cleavage of one or two strands at the location of the target sequence. 
     
     
         31 . The vector of  claim 24 , wherein the regulatory element comprises a polymerase III promoter. 
     
     
         32 . The vector of  claim 24 , wherein the regulatory element comprises a polymerase II promoter. 
     
     
         33 . The vector of  claim 27 , wherein comprising more than one regulatory element for transcription of components I and II. 
     
     
         34 . The vector of  claim 24 , 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. 
     
     
         35 . The vector of  claim 24 , wherein fewer than 50% of the nucleotides of the guide sequence participate in self-complementary base-pairing. 
     
     
         36 . The composition of  claim 1  wherein the vector comprises an adenovirus, a lentivirus or an adeno-associated virus. 
     
     
         37 . The composition of  claim 1 , wherein said cleavage comprises cleaving two strands at the location of the target sequence. 
     
     
         38 . The composition of  claim 1 , wherein said cleavage results in decreased transcription of a target gene. 
     
     
         39 . The method of  claim 23 , further comprising repairing cleaved DNA 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. 
     
     
         40 . The method of  claim 39 , wherein said mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence. 
     
     
         41 . The method of  claim 23 , comprising delivering said vector to the mammalian cell in a subject. 
     
     
         42 . The method of  claim 23 , wherein comprising delivering said vector to a mammalian cell in a cell culture. 
     
     
         43 . The method of  claim 42 , comprising isolating said mammalian cell from a subject prior to said delivering. 
     
     
         44 . The method of  claim 43 , further comprising returning said mammalian cell and/or progeny cell(s) therefrom to said subject. 
     
     
         45 . The composition according to  claim 1  wherein the Cas9 comprises a fusion of a Cas9 protein and one or more effector domains. 
     
     
         46 . The composition or system or vector according to  claim 45 , 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. 
     
     
         47 . A mammalian cell comprising composition, system or vector of  claim 36 . 
     
     
         48 . The method of  claim 23  including cleaving two DNA strands at the location of the target sequence. 
     
     
         49 . The method of claim including DNA cleavage resulting in decreased transcription of a gene. 
     
     
         50 . The method of  claim 23  wherein altering expression comprises genome editing. 
     
     
         51 . The composition of  claim 1  wherein altered expression comprises genome editing. 
     
     
         52 . The system 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. 
     
     
         53 . The composition or system or vector according to  claim 46 , wherein the one or more effector domains comprises a nuclease domain. 
     
     
         54 . The composition  claim 1  as to component I there is a first regulatory element and as to component II there is a second regulatory element. 
     
     
         55 . The composition of  claim 10 , 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.