US 11,920,128 B2
Methods, cells and organisms
Allan Bradley, Cambridge (GB); Hanif Ali, Cambridge (GB); and E-Chiang Lee, Cambridge (GB)
Assigned to Kymab Limited, Cambridge (GB)
Filed by Kymab Limited, Cambridge (GB)
Filed on Jul. 19, 2019, as Appl. No. 16/516,996.
Application 16/516,996 is a continuation of application No. 15/094,624, filed on Apr. 8, 2016, abandoned.
Application 15/094,624 is a continuation of application No. 14/490,549, filed on Sep. 18, 2014, abandoned.
Claims priority of application No. 1316560 (GB), filed on Sep. 18, 2013; and application No. 1321210 (GB), filed on Dec. 2, 2013.
Prior Publication US 2019/0338274 A1, Nov. 7, 2019
Int. Cl. A61K 48/00 (2006.01); C07K 16/00 (2006.01); C12N 5/0781 (2010.01); C12N 15/10 (2006.01); C12N 15/90 (2006.01)
CPC C12N 15/102 (2013.01) [A01K 67/0278 (2013.01); C07K 16/00 (2013.01); C12N 5/0635 (2013.01); C12N 15/907 (2013.01); A01K 2207/15 (2013.01); A01K 2217/052 (2013.01); A01K 2217/072 (2013.01); A01K 2227/105 (2013.01); A01K 2267/01 (2013.01); C07K 2317/14 (2013.01); C07K 2317/20 (2013.01); C07K 2317/24 (2013.01); C07K 2317/52 (2013.01); C07K 2317/56 (2013.01); C12N 2510/04 (2013.01); C12N 2800/80 (2013.01)] 19 Claims
 
1. A method for modifying a genome at a genomic locus of interest in a non-human mammal zygote, the method comprising assembling in vitro a ribonucleoprotein (RNP) complex, comprising contacting a purified clustered regularly interspaced short palindromic repeats (CRISPR) associated 9 (Cas9) protein and at least two single guide RNAs (sgRNAs) that hybridize to a CRISPR target sequence at the same genomic locus of interest; and injecting the RNP complex into the non-human mammal zygote; and injecting an incoming nucleic acid that comprises:
(i) a 5′ homology arm that is homologous to a 5′ target sequence at the genomic locus of interest and is homologous to a region 5′ of a first protospacer adjacent motif (PAM) sequence contained within the genomic locus of interest; and
(ii) a 3′ homology arm that is homologous to a 3′ target sequence at the genomic locus of interest and is homologous to a region 3′ of a second PAM sequence contained within the genomic locus of interest; and
(iii) optionally an insert nucleic acid sequence; wherein the method uses Cas9 mediated nucleic acid cleavage to create 5′ and 3′ cut ends in the region between the 5′ target sequence and the 3′ target sequence; wherein, following the injection step:
(a) which injection step comprises injecting an incoming nucleic acid, the genome of the zygote is modified to comprise a targeted genetic modification comprising a deletion of a region of the genomic locus of interest, wherein the targeted genetic modification is a deletion of from 1 kb to at least 100 kb; and/or
(b) which injection step comprises injecting an incoming nucleic acid, the genome of the zygote is modified to comprise a targeted genetic modification comprising insertion of the insert nucleic acid sequence at the genomic locus of interest.