CLICK: one-step generation of conditional knockout mice

• DOI: 10.1186/s12864-018-4713-y
• Publication Date: 2018-05-02T03:51:34Z
• Authors: 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
• Keywords: Mice, Knockout; 0301 basic medicine; Cre-loxP system; /dk/atira/pure/subjectarea/asjc/1300/1311; Base Sequence; Zygote; QH426-470; Long single-stranded DNA; Injections; Mice; 03 medical and health sciences; Genetics; Animals; CRISPR-Cas Systems; Genetic Engineering; CRISPR/Cas9; Zygote electroporation; CLICK; TP248.13-248.65; Alleles; /dk/atira/pure/subjectarea/asjc/1300/1305; Biotechnology; Research Article

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