title: Feasibility for a large scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes.
abstract: The recombinant clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas system has opened a new era for mammalian genome editing. Here, we constructed pX330 plasmids expressing humanized Cas9 (hCas9) and single guide RNAs (sgRNAs) against mouse genes and validated them both in vitro and in vivo. When we randomly chose 291 target sequences within protein coding regions of 73 genes, an average number of off-target candidates (exact match 13 nucleotides from 3' target and NGG) found by Bowtie software was 9.2 ± 21.0 (~1.8 times more than the estimated value, 5.2). We next validated their activity by observing green fluorescence reconstituted by homology dependent repair (HDR) of an EGFP expression cassette in HEK293T cells. Of the pX330 plasmids tested, 81.8% (238/291) were found to be functional in vitro. We finally injected the validated pX330 plasmids into mouse zygotes in its circular form against 32 genes (including two genes previously tested) and obtained mutant mice at a 52.9 ± 22.3% (100/196) mutation frequency. Among the pups carrying mutations on the autosomes, 43.6% (47/96) carried the mutations in both alleles. When off-target candidate sites were examined in 63 mutant mice, 0.8% (3/382) were mutated. We conclude that our method provides a simple, efficient, and cost-effective way for mammalian gene editing that is applicable for large scale mutagenesis in mammals.
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871; Graduate School of Medicine, Osaka University, Suita, Osaka, 565-0871, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Mashiko
authors.forename: Samantha A M
authors.initials: SA
authors.lastname: Young
authors.forename: Masanaga
authors.initials: M
authors.lastname: Muto
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Kato
authors.forename: Kaori
authors.initials: K
authors.lastname: Nozawa
authors.forename: Masaki
authors.initials: M
authors.lastname: Ogawa
authors.forename: Taichi
authors.initials: T
authors.lastname: Noda
authors.forename: Yeon-Joo
authors.initials: YJ
authors.lastname: Kim
authors.forename: Yuhkoh
authors.initials: Y
authors.lastname: Satouh
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Fujihara
authors.forename: Masahito
authors.initials: M
authors.lastname: Ikawa
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D005240:Feasibility Studies; D018390:Gene Targeting / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008817:Mice, Mutant Strains; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D015053:Zygote / Q000378:metabolism*
keywords: Eucomm; International Knockout Mouse Consortium; International Mouse Phenotyping Consortium; Knockout mouse project, KOMP; knockout