CLICK: one-step generation of conditional knockout mice

Yoshiki Miyasaka; Yoshihiro Uno; Kazuto Yoshimi; Yayoi Kunihiro; Takuji Yoshimura; Tomohiro Tanaka; Harumi Ishikubo; Yuichi Hiraoka; Norihiko Takemoto; Takao Tanaka; Yoshihiro Ooguchi; Paul Skehel; Tomomi Aida; Junji Takeda; Tomoji Mashimo

doi:10.1186/s12864-018-4713-y

CLICK: one-step generation of conditional knockout mice

BMC Genomics. 2018 May 2;19(1):318. doi: 10.1186/s12864-018-4713-y.

Authors

Yoshiki Miyasaka¹, Yoshihiro Uno¹, Kazuto Yoshimi^{2

3}, Yayoi Kunihiro¹, Takuji Yoshimura¹, Tomohiro Tanaka⁴, Harumi Ishikubo⁵, Yuichi Hiraoka^{5

6}, Norihiko Takemoto⁷, Takao Tanaka⁸, Yoshihiro Ooguchi⁸, Paul Skehel⁹, Tomomi Aida^{5

6

10}, Junji Takeda¹¹, Tomoji Mashimo^{12

13}

Affiliations

¹ Institute of Experimental Animal Sciences, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan.
² Genome Editing Research and Development (R&D) Center, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan.
³ Mouse Genomics Resource Laboratory, National Institute of Genetics, Shizuoka, 411-8540, Japan.
⁴ Medical Innovation Center, Graduate School of Medicine, Kyoto University, Kyoto, 606-8507, Japan.
⁵ Laboratory of Recombinant Animals, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU), Chiyoda, Tokyo, 101-0062, Japan.
⁶ Laboratory of Molecular Neuroscience, MRI, TMDU, Tokyo, 113-8510, Japan.
⁷ Department of Otorhinolaryngology-Head and Neck Surgery, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan.
⁸ KAC Co., Ltd., Kyoto, 604-8423, Japan.
⁹ Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD, UK.
¹⁰ Present address: McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
¹¹ Department of Genome Biology, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan.
¹² Institute of Experimental Animal Sciences, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan. mashimo@iexas.med.osaka-u.ac.jp.
¹³ Genome Editing Research and Development (R&D) Center, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan. mashimo@iexas.med.osaka-u.ac.jp.

Abstract

Background: CRISPR/Cas9 enables the targeting of genes in zygotes; however, efficient approaches to create loxP-flanked (floxed) alleles remain elusive.

Results: Here, we show that the electroporation of Cas9, two gRNAs, and long single-stranded DNA (lssDNA) into zygotes, termed CLICK (CRISPR with lssDNA inducing conditional knockout alleles), enables the quick generation of floxed alleles in mice and rats.

Conclusions: The high efficiency of CLICK provides homozygous knock-ins in oocytes carrying tissue-specific Cre, which allows the one-step generation of conditional knockouts in founder (F0) mice.

Keywords: CLICK; CRISPR/Cas9; Cre-loxP system; Long single-stranded DNA; Zygote electroporation.

MeSH terms

Alleles
Animals
Base Sequence
CRISPR-Cas Systems / genetics
Genetic Engineering / methods*
Injections
Mice
Mice, Knockout
Zygote / metabolism

Abstract

MeSH terms

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