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...
| Main Authors: | , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2018-05-01
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| Series: | BMC Genomics |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12864-018-4713-y |
| _version_ | 1818301709873577984 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Yoshiki Miyasaka |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-13T05:27:20Z |
| format | Article |
| id | doaj.art-226795467b9f47eebef2789b9696428e |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-13T05:27:20Z |
| publishDate | 2018-05-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-226795467b9f47eebef2789b9696428e2022-12-21T23:58:09ZengBMCBMC Genomics1471-21642018-05-011911810.1186/s12864-018-4713-yCLICK: one-step generation of conditional knockout miceYoshiki Miyasaka0Yoshihiro Uno1Kazuto Yoshimi2Yayoi Kunihiro3Takuji Yoshimura4Tomohiro Tanaka5Harumi Ishikubo6Yuichi Hiraoka7Norihiko Takemoto8Takao Tanaka9Yoshihiro Ooguchi10Paul Skehel11Tomomi Aida12Junji Takeda13Tomoji Mashimo14Institute of Experimental Animal Sciences, Graduate School of Medicine, Osaka UniversityInstitute of Experimental Animal Sciences, Graduate School of Medicine, Osaka UniversityGenome Editing Research and Development (R&D) Center, Graduate School of Medicine, Osaka UniversityInstitute of Experimental Animal Sciences, Graduate School of Medicine, Osaka UniversityInstitute of Experimental Animal Sciences, Graduate School of Medicine, Osaka UniversityMedical Innovation Center, Graduate School of Medicine, Kyoto UniversityLaboratory of Recombinant Animals, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU)Laboratory of Recombinant Animals, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU)Department of Otorhinolaryngology-Head and Neck Surgery, Graduate School of Medicine, Osaka UniversityKAC Co., Ltd.KAC Co., Ltd.Centre for Integrative Physiology, University of EdinburghLaboratory of Recombinant Animals, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU)Department of Genome Biology, Graduate School of Medicine, Osaka UniversityInstitute of Experimental Animal Sciences, Graduate School of Medicine, Osaka UniversityAbstract 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.http://link.springer.com/article/10.1186/s12864-018-4713-yCRISPR/Cas9CLICKZygote electroporationLong single-stranded DNACre-loxP system |
| spellingShingle | 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 CLICK: one-step generation of conditional knockout mice BMC Genomics CRISPR/Cas9 CLICK Zygote electroporation Long single-stranded DNA Cre-loxP system |
| title | CLICK: one-step generation of conditional knockout mice |
| title_full | CLICK: one-step generation of conditional knockout mice |
| title_fullStr | CLICK: one-step generation of conditional knockout mice |
| title_full_unstemmed | CLICK: one-step generation of conditional knockout mice |
| title_short | CLICK: one-step generation of conditional knockout mice |
| title_sort | click one step generation of conditional knockout mice |
| topic | CRISPR/Cas9 CLICK Zygote electroporation Long single-stranded DNA Cre-loxP system |
| url | http://link.springer.com/article/10.1186/s12864-018-4713-y |
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