US 11,697,827 B2
Systems and methods for gene modification
Richard Stoner, San Jose, CA (US); Travis Maures, Pacifica, CA (US); and David Conant, San Francisco, CA (US)
Assigned to Synthego Corporation, Redwood City, CA (US)
Filed by Synthego Corporation, Redwood City, CA (US)
Filed on Dec. 9, 2020, as Appl. No. 17/116,791.
Application 17/116,791 is a continuation of application No. 16/418,893, filed on May 21, 2019, granted, now 11,345,932.
Application 16/418,893 is a continuation of application No. PCT/US2019/032735, filed on May 16, 2019.
Claims priority of provisional application 62/672,437, filed on May 16, 2018.
Prior Publication US 2021/0180055 A1, Jun. 17, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/00 (2006.01); C12N 15/90 (2006.01); C12N 9/22 (2006.01); C12N 15/10 (2006.01); C12N 15/113 (2010.01); C12N 15/11 (2006.01)
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.