ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED GUIDE COMPOSITIONS FOR SEQUENCE MANIPULATION
First Claim
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1. 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:
- 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, the guide RNA comprising a guide sequence, a tracr sequence, and a tracr mate sequence,b) a second regulatory element operably linked to a nucleotide sequence encoding a Type II Cas9 protein, said protein comprising a nuclear localization signal (NLS);
wherein components (a) and (b) are located on same or different vectors of the system,wherein the tracr sequence is 30 or more nucleotides in length, andwhereby 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.
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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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12 Claims
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1. 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, the guide RNA comprising a guide sequence, a tracr sequence, and a tracr mate sequence, b) a second regulatory element operably linked to a nucleotide sequence encoding a Type II Cas9 protein, said protein comprising a nuclear localization signal (NLS); wherein components (a) and (b) are located on same or different vectors of the system, wherein the tracr sequence is 30 or more nucleotides in length, and 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 (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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Specification