CRISPR-Cas Nickase Systems, Methods And Compositions For Sequence Manipulation in Eukaryotes
First Claim
Patent Images
1. A method for cleaving, editing, or modifying at least one target sequence adjacent to a Protospacer Adjacent Motif (PAM) in a eukaryotic cell, comprising:
- delivering one or more non-naturally occurring or engineered eukaryotic expression vectors to the eukaryotic cell,wherein the expression vectors comprise;
a) at least one nucleotide sequence encoding at least one chimeric construct comprising a tracr sequence comprising 30 or more nucleotides in length, andb) at least one nucleotide sequence encoding at least one Cas9,wherein;
components a) and b) are each operably linked to a regulatory element for transcription thereof in the eukaryotic cell and there is at least one regulatory element therefor,Cas9 comprises one or more mutations in a catalytic domain thereby rendering Cas9 a nickase that cleaves a single DNA strand,the chimeric construct comprises two or more hairpins,the chimeric construct is designed to form with Cas9 a complex,the chimeric construct comprises guide(s) designed to hybridize with the target sequence(s),the chimeric construct and Cas9 do not naturally occur together, andthe complex comprising the chimeric construct and Cas9 does not naturally occur, whereby there is cleaving, editing, or modifying of the target sequence.
0 Assignments
0 Petitions
Accused Products
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.
-
Citations
34 Claims
-
1. A method for cleaving, editing, or modifying at least one target sequence adjacent to a Protospacer Adjacent Motif (PAM) in a eukaryotic cell, comprising:
-
delivering one or more non-naturally occurring or engineered eukaryotic expression vectors to the eukaryotic cell, wherein the expression vectors comprise; a) at least one nucleotide sequence encoding at least one chimeric construct comprising a tracr sequence comprising 30 or more nucleotides in length, and b) at least one nucleotide sequence encoding at least one Cas9, wherein; components a) and b) are each operably linked to a regulatory element for transcription thereof in the eukaryotic cell and there is at least one regulatory element therefor, Cas9 comprises one or more mutations in a catalytic domain thereby rendering Cas9 a nickase that cleaves a single DNA strand, the chimeric construct comprises two or more hairpins, the chimeric construct is designed to form with Cas9 a complex, the chimeric construct comprises guide(s) designed to hybridize with the target sequence(s), the chimeric construct and Cas9 do not naturally occur together, and the complex comprising the chimeric construct and Cas9 does not naturally occur, whereby there is cleaving, editing, or modifying of the target sequence. - 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. One or more non-naturally occurring or engineered eukaryotic expression vectors comprising:
-
a) at least one nucleotide sequence encoding at least one chimeric construct comprising a tracr sequence comprising 30 or more nucleotides in length, and b) at least one nucleotide sequence encoding at least one Cas9, wherein; components a) and b) are each operably linked to a regulatory element for transcription thereof in a eukaryotic cell and there is at least one regulatory element therefor, Cas9 comprises one or more mutations in a catalytic domain thereby rendering Cas9 a nickase that cleaves a single DNA strand, the chimelic construct comprises two or more hairpins, the chimeric construct is designed to form with Cas9 a complex, the chimeric construct comprise(s) guide(s) designed to hybridize with target sequence(s) in the eukaryotic cell the chimeric construct and Cas9 do not naturally occur together, and the complex comprising the chimeric construct and Cas9 does not naturally occur.
-
-
34-69. -69. (canceled)
Specification