Single-step generation of rabbits carrying a targeted allele of the tyrosinase gene using CRISPR/Cas9

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Author(s)

    • HONDA Arata
    • Organization for Promotion of Tenure Track, University of Miyazaki, 5200, Kibara, Kiyotake, Miyazaki 889-1692, Japan| RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan
    • OGURA Atsuo
    • RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan
    • SANKAI Tadashi
    • Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Tsukuba, Ibaraki 305-0843, Japan
    • YASMIN Lubna
    • Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Tsukuba, Ibaraki 305-0843, Japan
    • YUZAWA Kazuaki
    • Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Tsukuba, Ibaraki 305-0843, Japan
    • HONSHO Kimiko
    • Organization for Promotion of Tenure Track, University of Miyazaki, 5200, Kibara, Kiyotake, Miyazaki 889-1692, Japan
    • IZU Haruna
    • Organization for Promotion of Tenure Track, University of Miyazaki, 5200, Kibara, Kiyotake, Miyazaki 889-1692, Japan
    • IGUCHI Atsushi
    • Interdisciplinary Research Organization, University of Miyazaki, Miyazaki 889-1692, Japan
    • IKAWA Masahito
    • Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 556-0871, Japan

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 (<i>TYR</i>) 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 <i>in vitro</i> 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.

Journal

  • Experimental Animals

    Experimental Animals 64(1), 31-37, 2015

    Japanese Association for Laboratory Animal Science

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