Patent ID: 11920128
Assignee: KYMAB LIMITED
Field: Biotechnology (Chemistry)
Classification: CPC C  A | IPC A  C

Claim 0:
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.