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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)
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.
  • References (48)
  • Citations (122)
References48
Newest
#1Tomomi Aida (Tokyo Medical and Dental University)H-Index: 11
#2Junichi Yoshida (Tokyo Medical and Dental University)H-Index: 1
Last.Hidenori Aizawa (Tokyo Medical and Dental University)H-Index: 20
view all 19 authors...
#1Shengdar Q. Tsai (Harvard University)H-Index: 24
#2Zongli Zheng (KI: Karolinska Institutet)H-Index: 24
Last.Long P. Le (Harvard University)H-Index: 19
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#1Priti Singh (Cornell University)H-Index: 2
#2John C. Schimenti (Cornell University)H-Index: 45
Last.Ewelina Bolcun-Filas (Cornell University)H-Index: 11
view all 3 authors...
#1Le Cong (MIT: Massachusetts Institute of Technology)H-Index: 15
#2Feng Zhang (MIT: Massachusetts Institute of Technology)H-Index: 104
#1Jennifer A. Doudna (University of California, Berkeley)H-Index: 92
#2Emmanuelle Charpentier (MHH: Hannover Medical School)H-Index: 28
Cited By122
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#1Wakako Kumita (Central Institute for Experimental Animals)H-Index: 2
#2Kenya Sato (Central Institute for Experimental Animals)H-Index: 3
Last.Yasubumi Sakakibara (Keio: Keio University)H-Index: 29
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#1Mashito Sakai (UCSD: University of California, San Diego)H-Index: 7
#2Mashito Sakai (UCSD: University of California, San Diego)H-Index: 1
Last.Zihou Deng (MSK: Memorial Sloan Kettering Cancer Center)H-Index: 1
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#1Mieradili Mulati (Tokyo Medical and Dental University)H-Index: 2
#2Yutaka Kobayashi (Tokyo Medical and Dental University)
Last.Masato Yuasa (Tokyo Medical and Dental University)H-Index: 10
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#1Hideki Ukai (UTokyo: University of Tokyo)H-Index: 8
#2Hideki Ukai (UTokyo: University of Tokyo)
Last.Hiroki R. Ueda (UTokyo: University of Tokyo)H-Index: 43
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#1Channabasavaiah B. Gurumurthy (UNMC: University of Nebraska Medical Center)H-Index: 15
#2Masahiro Sato (Kadai: Kagoshima University)H-Index: 30
Last.Shinichi Nakagawa (Hokkaido University)
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#1Nanyang Xiao (Fujian Normal University)
#2Jingjing Wei (Fujian Normal University)
Last.Qi Chen (Fujian Normal University)
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