One-step Generation of Phenotype-expressing Triple-knockout Mice with Heritable Mutated Alleles by the CRISPR/Cas9 System

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  • FUJII Wataru
    Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
  • ONUMA Asuka
    Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
  • SUGIURA Koji
    Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
  • NAITO Kunihiko
    Department of Animal Resource Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan

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Triple-knockout mice generated by the one-step CRISPR/Cas9 system were examined for the effects of multiple gene modifications on each phenotype and individual gene function. Sixty embryos were transferred, and 9 pups were obtained; all 9 pups had mutations on 3 loci, and 7 pups showed mutations in all-alleles. F0 mice showed knockout phenotypes or no protein expression of target genes simultaneously, and these mutations were normally inherited in the next generation.

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