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

Published on Dec 1, 2015in Genome Biology14.03
· DOI :10.1186/s13059-015-0653-x
Tomomi Aida11
Estimated H-index: 11
(Tokyo Medical and Dental University),
Keiho Chiyo1
Estimated H-index: 1
(Tokyo Medical and Dental University)
+ 7 AuthorsKohichi Tanaka51
Estimated H-index: 51
(Tokyo Medical and Dental University)
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.
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  • Citations (122)
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