CRISPR-CAS SYSTEMS AND METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTS
First Claim
1. A non-naturally occurring or engineered composition comprising a delivery system operably configured to deliver an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) complex to a eukaryotic cell containing a DNA molecule having a target sequence adjacent to a Protospacer Adjacent Motif (PAM), the system comprising:
- I. one or more regulatory elements operably linked to one or more CRISPR-Cas complex polynucleotide sequences comprising(a) a guide sequence capable of hybridizing to the target sequence in the eukaryotic cell,(b) a tracr mate sequence, and(c) a tracr sequence, andII. a second regulatory element operably linked to an enzyme-coding sequence encoding a Type II CRISPR enzyme,whereinthe CRISPR complex comprises the CRISPR enzyme 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,the guide sequence comprises more than about 10 nucleotides in length, andthe tracr sequence comprises more than about 30 nucleotides in length, andwhen transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence and PAM recognition, andthe tracr sequence exhibits at least 50% sequence complementarity along the length of the tracr mate, andthe CRISPR enzyme and the guide RNA do not naturally occur together.
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Accused Products
Abstract
The invention provides for systems, methods, and compositions for altering expression of target gene sequences and related gene products. 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 utilizing the CRISPR-Cas system.
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Citations
30 Claims
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1. A non-naturally occurring or engineered composition comprising a delivery system operably configured to deliver an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) complex to a eukaryotic cell containing a DNA molecule having a target sequence adjacent to a Protospacer Adjacent Motif (PAM), the system comprising:
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I. one or more regulatory elements operably linked to one or more CRISPR-Cas complex polynucleotide sequences comprising (a) a guide sequence capable of hybridizing to the target sequence in the eukaryotic cell, (b) a tracr mate sequence, and (c) a tracr sequence, and II. a second regulatory element operably linked to an enzyme-coding sequence encoding a Type II CRISPR enzyme, wherein the CRISPR complex comprises the CRISPR enzyme 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, the guide sequence comprises more than about 10 nucleotides in length, and the tracr sequence comprises more than about 30 nucleotides in length, and when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence and PAM recognition, and the tracr sequence exhibits at least 50% sequence complementarity along the length of the tracr mate, and the CRISPR enzyme and the guide RNA do not naturally occur together. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
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Specification