CPC C12N 15/902 (2013.01) [C12N 9/22 (2013.01); C12N 15/102 (2013.01); C12N 15/11 (2013.01); C12N 15/113 (2013.01); C12N 2310/20 (2017.05); C12N 2310/315 (2013.01); C12N 2310/321 (2013.01); C12N 2800/80 (2013.01)] | 14 Claims |
1. A system for modifying a gene within a cell, comprising:
(a) a first complex comprising a first gRNA and a first nuclease capable of generating double-stranded breaks;
(b) a second complex comprising a second gRNA and a second nuclease capable of generating double-stranded breaks; and
(c) a third complex comprising a third gRNA and a third nuclease capable of generating double-stranded breaks; wherein
(i) the first gRNA is configured to hybridize to a first genomic region of interest within the gene in the cell,
(ii) the second gRNA is configured to hybridize to a second genomic region of interest within the gene in the cell, and
(iii) the third guide RNA is configured to hybridize to a third genomic region of interest within the gene in the cell,
wherein the first gRNA, the second gRNA, and the third gRNA are each a different sequence, and wherein the first gRNA is configured to hybridize to a first binding site that is 30-80 base pairs apart from a second binding site hybridizable to the second gRNA and the second gRNA is configured to hybridize to the second binding site that is 30-80 base pairs apart from a third binding site hybridizable to the third gRNA to allow for modifying the gene with three double-stranded breaks;
wherein the first nuclease, the second nuclease, and the third nuclease each comprises a Cas9 nuclease, and
wherein the first complex, the second complex, and the third complex are provided in a single composition.
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