CRISPR-CAS COMPONENT SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION
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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.
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Citations
60 Claims
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1-34. -34. (canceled)
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35. An engineered, non-naturally occurring Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) vector system comprising one or more vectors comprising:
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a) a first regulatory element operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas system guide RNAs that hybridize with target sequences in polynucleotide loci in a eukaryotic cell, b) a second regulatory element operably linked to a nucleotide sequence encoding a Type II Cas9 protein, wherein components (a) and (b) are located on same or different vectors of the system, wherein the guide RNA comprises a tracr sequence which is 30 or more nucleotides in length, whereby the one or more guide RNAs target the polynucleotide loci in a eukaryotic cell and the Cas9 protein cleaves the polynucleotide loci, whereby sequence of the polynucleotide loci is modified; and
, wherein the Cas9 protein and the one or more guide RNAs do not naturally occur together. - View Dependent Claims (36, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48)
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49. A CRISPR-Cas system-mediated method for:
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a) site specific gene knockout; b) site-specific genome editing; c) DNA sequence-specific interference;
ord) multiplexed genome engineering; comprising introducing into a eukaryotic cell containing a target sequence in a polynucleotide locus an engineered, non-naturally occurring CRISPR-Cas system comprising one or more vectors comprising; a) a first regulatory element operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas system guide RNAs that hybridize with target sequences in polynucleotide loci in a eukaryotic cell, b) a second regulatory element operably linked to a nucleotide sequence encoding a Type II Cas9 protein, wherein components (a) and (b) are located on same or different vectors of the system, wherein the guide RNA comprises a tracr sequence which is 30 or more nucleotides in length, whereby the one or more guide RNAs target the polynucleotide loci in a eukaryotic cell and the Cas9 protein cleaves the polynucleotide loci, whereby a) site specific gene knockout;
b) site-specific genome editing;
c) DNA sequence-specific interference;
or d) multiplexed genome engineering takes place; and
, wherein the Cas9 protein and the one or more guide RNAs do not naturally occur together.- View Dependent Claims (50, 51, 52, 53, 54, 55, 56, 57, 58, 59)
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60. A CRISPR-Cas system-mediated method for the production of a non-human transgenic animal or transgenic plant, the method comprising introducing into a non-human animal or plant cell containing a target sequence in a polynucleotide locus an engineered, non-naturally occurring CRISPR-Cas system comprising one or more vectors comprising:
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a) a first regulatory element operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas system guide RNAs that hybridize with target sequences in polynucleotide loci in a non-human animal or plant cell, b) a second regulatory element operably linked to a nucleotide sequence encoding a Type II Cas9 protein, wherein components (a) and (b) are located on same or different vectors of the system, wherein the guide RNA comprises a tracr sequence which is 30 or more nucleotides in length, whereby the one or more guide RNAs target the polynucleotide loci in a non-human animal or plant cell and the Cas9 protein cleaves the polynucleotide loci, whereby sequence of the polynucleotide loci is modified; and
, wherein the Cas9 protein and the one or more guide RNAs do not naturally occur together.
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Specification