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CLICK: one-step generation of conditional knockout mice.

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.

SUBMITTER: Miyasaka Y

PROVIDER: S-EPMC5930688 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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CLICK: one-step generation of conditional knockout mice.

Miyasaka Yoshiki Y Uno Yoshihiro Y Yoshimi Kazuto K Kunihiro Yayoi Y Yoshimura Takuji T Tanaka Tomohiro T Ishikubo Harumi H Hiraoka Yuichi Y Takemoto Norihiko N Tanaka Takao T Ooguchi Yoshihiro Y Skehel Paul P Aida Tomomi T Takeda Junji J Mashimo Tomoji T

BMC genomics 20180502 1

<h4>Background</h4>CRISPR/Cas9 enables the targeting of genes in zygotes; however, efficient approaches to create loxP-flanked (floxed) alleles remain elusive.<h4>Results</h4>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.<h4>Conclusions</h4>The high efficiency of CLICK provides homozygous knock-in ...[more]

PMID: 29720086

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