title: Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein.
abstract: The CRISPR/Cas9 system has rapidly advanced targeted genome editing technologies. However, its efficiency in targeting with constructs in mouse zygotes via homology directed repair (HDR) remains low. Here, we systematically explored optimal parameters for targeting constructs in mouse zygotes via HDR using mouse embryonic stem cells as a model system. We characterized several parameters, including single guide RNA cleavage activity and the length and symmetry of homology arms in the construct, and we compared the targeting efficiency between Cas9, Cas9nickase, and dCas9-FokI. We then applied the optimized conditions to zygotes, delivering Cas9 as either mRNA or protein. We found that Cas9 nucleo-protein complex promotes highly efficient, multiplexed targeting of circular constructs containing reporter genes and floxed exons. This approach allows for a one-step zygote injection procedure targeting multiple genes to generate conditional alleles via homologous recombination, and simultaneous knockout of corresponding genes in non-targeted alleles via non-homologous end joining.
authors.affiliation: University of California, San Francisco Benioff Children's Hospital Oakland Research Institute, Oakland, CA, 94609, USA.
authors.forename: Chris J
authors.initials: CJ
authors.lastname: Jung
authors.affiliation: Gladstone Institutes, San Francisco, CA, 94158, USA.
authors.forename: Junli
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Wellcome Trust Sanger Institute, Cambridge, CB10 1SA, UK.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Trenchard
authors.affiliation: Mouse Biology Program, University of California, Davis, CA, 95618, USA.
authors.forename: Kent C
authors.initials: KC
authors.lastname: Lloyd
authors.affiliation: University of California, San Francisco Benioff Children's Hospital Oakland Research Institute, Oakland, CA, 94609, USA.
authors.forename: David B
authors.initials: DB
authors.lastname: West
authors.affiliation: Wellcome Trust Sanger Institute, Cambridge, CB10 1SA, UK.
authors.forename: Barry
authors.initials: B
authors.lastname: Rosen
authors.affiliation: University of California, San Francisco Benioff Children's Hospital Oakland Research Institute, Oakland, CA, 94609, USA. pdejong@chori.org.
authors.forename: Pieter J
authors.identifier: 0000-0002-7972-5260
authors.initials: PJ
authors.lastname: de Jong
mesh_terms: D000483:Alleles; D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D059766:DNA End-Joining Repair / Q000235:genetics; D005091:Exons; D018390:Gene Targeting / Q000379:methods*; D059765:Homologous Recombination / Q000235:genetics*; D051379:Mice; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D015053:Zygote / Q000254:growth & development
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger
keywords: CRISPR; Gene targeting; Transgenic mouse model