ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH FUNCTIONAL DOMAINS
First Claim
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1. A non-naturally occurring or engineered composition for altering expression of at least one gene product in a eukaryotic cell comprising:
- a delivery system operably configured to deliver CRISPR-Cas complex components or polynucleotide sequences encoding said components into a eukaryotic cell, wherein said CRISPR-Cas complex is operable in a eukaryotic cell containing a DNA molecule having a target sequence adjacent to a Protospacer Adjacent Motif (PAM);
said composition comprising;
I. one or more CRISPR-Cas complex polynucleotide sequences comprising or encoding for expression in the eukaryotic cell;
(a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell,(b) a tracr mate sequence, and(c) a tracr sequence, andII. a CRISPR enzyme or a polynucleotide encoding a CRISPR enzyme for expression in the eukaryotic cell;
wherein the tracr mate sequence hybridizes to the tracr sequence,the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence,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 CRISPR enzyme comprises one or more mutations such that the enzyme has altered nuclease activity compared to the wild-type enzyme, andthe CRISPR enzyme further comprises one or more heterologous functional domain(s), whereby expression of the at least one gene product is altered through the CRISPR-Cas system acting as to the DNA molecule comprising the guide RNA directing sequence-specific binding of the CRISPR-Cas system and PAM recognition.
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Abstract
The invention provides for engineering and optimization of 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 with additional functional domains. Also provided are methods of directing CRISPR complex formation in prokaryotic and eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity.
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34 Claims
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1. A non-naturally occurring or engineered composition for altering expression of at least one gene product in a eukaryotic cell comprising:
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a delivery system operably configured to deliver CRISPR-Cas complex components or polynucleotide sequences encoding said components into a eukaryotic cell, wherein said CRISPR-Cas complex is operable in a eukaryotic cell containing a DNA molecule having a target sequence adjacent to a Protospacer Adjacent Motif (PAM);
said composition comprising;I. one or more CRISPR-Cas complex polynucleotide sequences comprising or encoding for expression in the eukaryotic cell; (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, (b) a tracr mate sequence, and (c) a tracr sequence, and II. a CRISPR enzyme or a polynucleotide encoding a CRISPR enzyme for expression in the eukaryotic cell; wherein the tracr mate sequence hybridizes to the tracr sequence, the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, 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 CRISPR enzyme comprises one or more mutations such that the enzyme has altered nuclease activity compared to the wild-type enzyme, and the CRISPR enzyme further comprises one or more heterologous functional domain(s), whereby expression of the at least one gene product is altered through the CRISPR-Cas system acting as to the DNA molecule comprising the guide RNA directing sequence-specific binding of the CRISPR-Cas system and PAM recognition. - 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, 31, 32, 33, 34)
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Specification