CRISPR-Cas systems and methods for altering expression of gene products
First Claim
1. A method of altering expression of at least one gene product comprising introducing into a eukaryotic cell containing, and expressing a DNA molecule having a target sequence and encoding the gene product an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system comprising one or more vectors comprising:
- a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the target sequence; and
b) a second regulatory element operable in a eukaryotic cell 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,the CRISPR-Cas system comprises one or more nuclear localization signals (NLSs),the guide RNA comprises a tracr sequence which is 30 or more nucleotides in length, andthe Cas9 protein comprises one or more mutations in a catalytic domain,whereby the guide RNA targets and hybridizes to the target sequence, whereby expression of the at least one gene product is altered; and
, wherein the Cas9 protein and the guide RNA do not naturally occur together.
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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.
285 Citations
28 Claims
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1. A method of altering expression of at least one gene product comprising introducing into a eukaryotic cell containing, and expressing a DNA molecule having a target sequence and encoding the gene product an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system comprising one or more vectors comprising:
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a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with the target sequence; and b) a second regulatory element operable in a eukaryotic cell 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, the CRISPR-Cas system comprises one or more nuclear localization signals (NLSs), the guide RNA comprises a tracr sequence which is 30 or more nucleotides in length, and the Cas9 protein comprises one or more mutations in a catalytic domain, whereby the guide RNA targets and hybridizes to the target sequence, whereby expression of the at least one gene product is altered; and
, wherein the Cas9 protein and the guide RNA do not naturally occur together. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. An engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system comprising one or more vectors comprising:
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a) a first regulatory element operable in a eukaryotic cell operably linked to at least one nucleotide sequence encoding a CRISPR-Cas system guide RNA that hybridizes with a target sequence, and b) a second regulatory element operable in a eukaryotic cell 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, the CRISPR-Cas system comprises one or more nuclear localization signals (NLSs), the guide RNA comprises a tracr sequence which is 30 or more nucleotides in length, and the Cas9 protein comprises one or more mutations in a catalytic domain, whereby the guide RNA targets and hybridizes to the target sequence, whereby expression of at least one gene product is altered; and
, wherein the Cas9 protein and the guide RNA do not naturally occur together. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. An engineered, programmable, non-naturally occurring Type II CRISP R-Cas system comprising a Cas9 protein and at least one guide RNA that targets and hybridizes to a target sequence of a DNA molecule in a eukaryotic cell, wherein the DNA molecule encodes and the eukaryotic cell expresses at least one gene product, wherein the Cas9 protein comprises one or more mutations in a catalytic domain, wherein the CRISPR-Cas system comprises one or more NLSs and the guide RNA comprises a tracr sequence which is 30 or more nucleotides in length, whereby expression of the at least one gene product is altered;
- and, wherein the Cas9protein and the guide RNA do not naturally occur together.
- View Dependent Claims (22, 23, 24, 25, 26, 27, 28)
Specification