US 12,331,328 B2
CRISPR/Cas9-mediated exon-skipping approach for USH2A-associated usher syndrome
Eric A. Pierce, Boston, MA (US); Carrie Marie Margulies, Newton, MA (US); Nachiket D. Pendse, Quincy, MA (US); Sebastian W. Gloskowski, Malden, MA (US); Qin Liu, Boston, MA (US); and Morgan L. Maeder, Cambridge, MA (US)
Assigned to Massachusetts Eye and Ear Infirmary, Boston, MA (US); and Editas Medicine, Inc., Cambridge, MA (US)
Appl. No. 17/040,629
Filed by Massachusetts Eye and Ear Infirmary, Boston, MA (US); and Editas Medicine, Inc., Cambridge, MA (US)
PCT Filed Mar. 25, 2019, PCT No. PCT/US2019/023934
§ 371(c)(1), (2) Date Sep. 23, 2020,
PCT Pub. No. WO2019/183641, PCT Pub. Date Sep. 26, 2019.
Claims priority of provisional application 62/794,402, filed on Jan. 18, 2019.
Claims priority of provisional application 62/647,578, filed on Mar. 23, 2018.
Prior Publication US 2021/0115419 A1, Apr. 22, 2021
Int. Cl. C12N 9/22 (2006.01); A61K 48/00 (2006.01); C12N 15/113 (2010.01); C12N 15/86 (2006.01)
CPC C12N 9/22 (2013.01) [A61K 48/0016 (2013.01); C12N 15/113 (2013.01); C12N 15/86 (2013.01); C12N 2310/20 (2017.05); C12N 2320/33 (2013.01); C12N 2750/14143 (2013.01)] 17 Claims
OG exemplary drawing
 
11. A method of altering the genome of a cell, the method comprising contacting the cell with a CRISPR-Cas9 nuclease and gRNAs to form a first double strand break within intron 12 of the human USH2A gene and a second double strand break within intron 13 of the human USH2A gene, wherein the first double strand break is formed between nucleotides 216,248,383 and 216,248,639 of human chromosome 1 and the second double strand break is formed between nucleotides 216,245,292 and 216,246,542 of human chromosome 1, wherein the first and second double strand breaks are then repaired by the cell, thereby removing exon 13 of the USH2A gene on human chromosome 1, and wherein the step of forming the first strand break comprises contacting the cell with a first gRNA and the step of forming the second strand break comprises contacting the cell with a second gRNA, and wherein the first gRNA and the second gRNA comprise SEQ ID NO: 54 and SEQ ID NO: 125; SEQ ID NO: 54 and SEQ ID NO: 129; SEQ ID NO: 54 and SEQ ID NO: 130; SEQ ID NO: 54 and SEQ ID NO: 134; SEQ ID NO: 54 and SEQ ID NO: 135; SEQ ID NO: 68 and SEQ ID NO: 125; SEQ ID NO: 68 and SEQ ID NO: 129; SEQ ID NO: 68 and SEQ ID NO: 130; SEQ ID NO: 68 and SEQ ID NO: 134; or SEQ ID NO: 68 and SEQ ID NO: 135, respectively.