title: Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA.
abstract: CRISPR/Cas mediated genome editing has been successfully demonstrated in mammalian cells and further applications for generating mutant mice were reported by injecting humanized Cas9 (hCas) mRNA and single guide RNA into fertilized eggs. Here we inject the circular plasmids expressing hCas9 and sgRNA into mouse zygotes and obtained mutant mice within a month. When we targeted the Cetn1 locus, 58.8% (10/17) of the pups carried the mutations and six of them were homozygously mutated. Co-injection of the plasmids targeting different loci resulted in the successful removal of the flanked region in two out of three mutant pups. The efficient mutagenesis was also observed at the Prm1 locus. Among the 46 offspring carrying CRISPR/Cas plasmid mediated mutations, only two of them carried the hCas9 transgene. The pronuclear injection of circular plasmid expressing hCas9/sgRNA complex is a rapid, simple, and reproducible method for targeted mutagenesis.
authors.affiliation: 1] Research Institute for Microbial Diseases [2] Graduate School of Medicine [3].
authors.forename: Daisuke
authors.initials: D
authors.lastname: Mashiko
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Fujihara
authors.forename: Yuhkoh
authors.initials: Y
authors.lastname: Satouh
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Miyata
authors.forename: Ayako
authors.initials: A
authors.lastname: Isotani
authors.forename: Masahito
authors.initials: M
authors.lastname: Ikawa
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002460:Cell Line; D000067470:Cellular Reprogramming Techniques / Q000379:methods*; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics; D008845:Microinjections; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011479:Protamines / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D002135:Calcium-Binding Proteins; D002868:Chromosomal Proteins, Non-Histone; C513176:Prm1 protein, mouse; D011479:Protamines; D000094704:RNA, Guide, CRISPR-Cas Systems; C056608:caltractin; D004720:Endonucleases