Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice

Tomomi Aida, Keiho Chiyo, Takako Usami, Harumi Ishikubo, Risa Imahashi, Yusaku Wada, Kenji F. Tanaka, Tetsushi Sakuma, Takashi Yamamoto, Kohichi Tanaka

Research output: Contribution to journalArticlepeer-review

208 Citations (Scopus)


Although the CRISPR/Cas system has enabled one-step generation of knockout mice, low success rates of cassette knock-in limit its application range. Here we show that cloning-free, direct nuclear delivery of Cas9 protein complex with chemically synthesized dual RNAs enables highly efficient target digestion, leading to generation of knock-in mice carrying a functional cassette with up to 50% efficiency, compared with just 10% by a commonly used method consisting of Cas9 mRNA and single guide RNA. Our cloning-free CRISPR/Cas system facilitates rapid one-step generation of cassette knock-in mice, accelerating functional genomic research by providing various in vivo genetic tools.

Original languageEnglish
Article number87
JournalGenome biology
Issue number1
Publication statusPublished - 2015 Apr 29

ASJC Scopus subject areas

  • Ecology, Evolution, Behavior and Systematics
  • Genetics
  • Cell Biology


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