title: Single-step generation of rabbits carrying a targeted allele of the tyrosinase gene using CRISPR/Cas9.
abstract: Targeted genome editing of nonrodent mammalian species has provided the potential for highly accurate interventions into gene function in humans and the generation of useful animal models of human diseases. Here we show successful clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas)-mediated gene targeting via circular plasmid injection in rabbits. The rabbit tyrosinase gene (TYR) was effectively disrupted, and we confirmed germline transmission by pronuclear injection of a circular plasmid expressing humanized Cas9 (hCas9) and single-guide RNA. Direct injection into pronuclear stage zygotes was possible following an in vitro validation assay. Neither off-target mutagenesis nor hCas9 transgenesis was detected in any of the genetically targeted pups and embryos examined. Gene targeting with this rapid and simplified strategy will help accelerate the development of translational research using other nonrodent mammalian species.
authors.affiliation: Organization for Promotion of Tenure Track, University of Miyazaki, 5200 Kibara, Kiyotake 889-1692; RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan.
authors.forename: Arata
authors.initials: A
authors.lastname: Honda
authors.forename: Michiko
authors.initials: M
authors.lastname: Hirose
authors.forename: Tadashi
authors.initials: T
authors.lastname: Sankai
authors.forename: Lubna
authors.initials: L
authors.lastname: Yasmin
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Yuzawa
authors.forename: Kimiko
authors.initials: K
authors.lastname: Honsho
authors.forename: Haruna
authors.initials: H
authors.lastname: Izu
authors.forename: Atsushi
authors.initials: A
authors.lastname: Iguchi
authors.forename: Masahito
authors.initials: M
authors.lastname: Ikawa
authors.forename: Atsuo
authors.initials: A
authors.lastname: Ogura
mesh_terms: D000483:Alleles; D000818:Animals; D064130:CRISPR-Associated Proteins / Q000235:genetics*; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004195:Disease Models, Animal; D018390:Gene Targeting / Q000379:methods*; D014442:Monophenol Monooxygenase / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011817:Rabbits / Q000235:genetics*; D015053:Zygote
chemical_list: D064130:CRISPR-Associated Proteins; D014442:Monophenol Monooxygenase