Systems and methods for gene modification

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.