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Genome Biol. 2015 Apr 29;16:87. doi: 10.1186/s13059-015-0653-x.

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

Author information

1
Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), Tokyo, 113-8510, Japan. aida.aud@mri.tmd.ac.jp.
2
Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), Tokyo, 113-8510, Japan. 100571ms@tmd.ac.jp.
3
Laboratory of Recombinant Animals, MRI, TMDU, Tokyo, 101-0062, Japan. kumikae.lra@mri.tmd.ac.jp.
4
Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), Tokyo, 113-8510, Japan. ishikubo.aud@mri.tmd.ac.jp.
5
Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), Tokyo, 113-8510, Japan. ma130015@tmd.ac.jp.
6
FASMAC Co. Ltd, Kanagawa, 243-0021, Japan. ywada@fasmac.co.jp.
7
Department of Neuropsychiatry, Keio University School of Medicine, Tokyo, 160-8582, Japan. kftanaka@keio.jp.
8
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, 739-8526, Japan. tetsushi-sakuma@hiroshima-u.ac.jp.
9
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, 739-8526, Japan. tybig@hiroshima-u.ac.jp.
10
Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), Tokyo, 113-8510, Japan. tanaka.aud@mri.tmd.ac.jp.
11
The Center for Brain Integration Research (CBIR), TMDU, Tokyo, 113-8510, Japan. tanaka.aud@mri.tmd.ac.jp.
12
JST, CREST, Saitama, 332-0012, Japan. tanaka.aud@mri.tmd.ac.jp.

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.

PMID:
25924609
PMCID:
PMC4414275
DOI:
10.1186/s13059-015-0653-x
[Indexed for MEDLINE]
Free PMC Article
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