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

CLICK: one-step generation of conditional knockout mice.

Author information

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

PMID:
29720086
PMCID:
PMC5930688
DOI:
10.1186/s12864-018-4713-y
[Indexed for MEDLINE]
Free PMC Article

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