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

  • Genome Biol. 2015 Apr 29;16(1):87. doi: 10.1186/s13059-015-0653-x.
Tomomi Aida  1 ,  Keiho Chiyo  2 ,  Takako Usami  3 ,  Harumi Ishikubo  4 ,  Risa Imahashi  5 ,  Yusaku Wada  6 ,  Kenji F Tanaka  7 ,  Tetsushi Sakuma  8 ,  Takashi Yamamoto  9 ,  Kohichi Tanaka  10  11  12
Affiliations
  • 1. Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), Tokyo, 113-8510, Japan. [email protected].
  • 2. Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), Tokyo, 113-8510, Japan. [email protected].
  • 3. Laboratory of Recombinant Animals, MRI, TMDU, Tokyo, 101-0062, Japan. [email protected].
  • 4. Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), Tokyo, 113-8510, Japan. [email protected].
  • 5. Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), Tokyo, 113-8510, Japan. [email protected].
  • 6. FASMAC Co. Ltd, Kanagawa, 243-0021, Japan. [email protected].
  • 7. Department of Neuropsychiatry, Keio University School of Medicine, Tokyo, 160-8582, Japan. [email protected].
  • 8. Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, 739-8526, Japan. [email protected].
  • 9. Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, 739-8526, Japan. [email protected].
  • 10. Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), Tokyo, 113-8510, Japan. [email protected].
  • 11. The Center for Brain Integration Research (CBIR), TMDU, Tokyo, 113-8510, Japan. [email protected].
  • 12. JST, CREST, Saitama, 332-0012, Japan. [email protected].
PMID: 25924609 DOI: 10.1186/s13059-015-0653-x
Abstract

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.