Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice.
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.
Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), Tokyo, 113-8510, Japan. aida.aud@mri.tmd.ac.jp.
Tomomi
T
Aida
Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), Tokyo, 113-8510, Japan. 100571ms@tmd.ac.jp.
Keiho
K
Chiyo
Laboratory of Recombinant Animals, MRI, TMDU, Tokyo, 101-0062, Japan. kumikae.lra@mri.tmd.ac.jp.
Takako
T
Usami
Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), Tokyo, 113-8510, Japan. ishikubo.aud@mri.tmd.ac.jp.
Harumi
H
Ishikubo
Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), Tokyo, 113-8510, Japan. ma130015@tmd.ac.jp.
Risa
R
Imahashi
FASMAC Co. Ltd, Kanagawa, 243-0021, Japan. ywada@fasmac.co.jp.
Yusaku
Y
Wada
Department of Neuropsychiatry, Keio University School of Medicine, Tokyo, 160-8582, Japan. kftanaka@keio.jp.
Kenji F
KF
Tanaka
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, 739-8526, Japan. tetsushi-sakuma@hiroshima-u.ac.jp.
Tetsushi
T
Sakuma
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, 739-8526, Japan. tybig@hiroshima-u.ac.jp.
Takashi
T
Yamamoto
Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), Tokyo, 113-8510, Japan. tanaka.aud@mri.tmd.ac.jp.|The Center for Brain Integration Research (CBIR), TMDU, Tokyo, 113-8510, Japan. tanaka.aud@mri.tmd.ac.jp.|JST, CREST, Saitama, 332-0012, Japan. tanaka.aud@mri.tmd.ac.jp.
Kohichi
K
Tanaka
D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D005260:Female; D055879:Gene Knock-In Techniques; D018390:Gene Targeting; D017930:Genes, Reporter; D056426:Genetic Loci; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
D012333:RNA, Messenger; D011994:Recombinant Proteins