Crispr-cas component systems, methods and compositions for sequence manipulation
AssigneesThe Broad Institute, Inc., Massachusetts Institute of Technology
InventorFeng Zhang
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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.
Claims31
1-30. (canceled)
A eukaryotic cell comprising:
- (i) a Cas9,
- (ii) a first polynucleotide sequence comprising a sequence complementary to a target DNA sequence in the eukaryotic cell and further comprising a tracr-mate sequence, and
- (iii) a second polynucleotide sequence comprising a sequence complementary to the tracr-mate sequence,
- wherein the first polynucleotide sequence and the second polynucleotide sequence are hybridized to form a hybridized polynucleotide,
- wherein the Cas9 is complexed with the hybridized polynucleotide, and
- wherein the hybridized polynucleotide and the Cas9 do not occur naturally together.
An ex vivo mammalian cell comprising:
- (i) an S. pyogenes Cas9,
- (ii) a first polynucleotide sequence comprising a sequence complementary to a target DNA sequence in the human cell and further comprising a tracr-mate sequence, and
- (iii) a second polynucleotide sequence comprising a sequence complementary to the tracr-mate sequence,
- wherein the first polynucleotide sequence and the second polynucleotide sequence are hybridized to form a hybridized polynucleotide,
- wherein the Cas9 is complexed with the hybridized polynucleotide, and
- wherein the hybridized polynucleotide and the Cas9 do not occur naturally together.
An ex vivo human cell comprising:
- (i) an S. pyogenes Cas9,
- (ii) a first polynucleotide sequence comprising a sequence complementary to a target DNA sequence in the human cell and further comprising a tracr-mate sequence, and wherein the first polynucleotide comprises one or more methylated nucleotides.
- (iii) a second polynucleotide sequence comprising a sequence complementary to the tracr-mate sequence,
- wherein the first polynucleotide sequence and the second polynucleotide sequence are hybridized to form a hybridized polynucleotide,
- wherein the Cas9 is complexed with the hybridized polynucleotide, and
- wherein the hybridized polynucleotide and the Cas9 do not occur naturally together.
Dependencies are inferred from the claim text.