CLICK: one-step generation of conditional knockout mice

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 l...

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Autores principales: Miyasaka, Yoshiki, Uno, Yoshihiro, Yoshimi, Kazuto, Kunihiro, Yayoi, Yoshimura, Takuji, Tanaka, Tomohiro, Ishikubo, Harumi, Hiraoka, Yuichi, Takemoto, Norihiko, Tanaka, Takao, Ooguchi, Yoshihiro, Skehel, Paul, Aida, Tomomi, Takeda, Junji, Mashimo, Tomoji
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2018
Materias:
Research Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930688/
https://www.ncbi.nlm.nih.gov/pubmed/29720086
http://dx.doi.org/10.1186/s12864-018-4713-y
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author Miyasaka, Yoshiki
Uno, Yoshihiro
Yoshimi, Kazuto
Kunihiro, Yayoi
Yoshimura, Takuji
Tanaka, Tomohiro
Ishikubo, Harumi
Hiraoka, Yuichi
Takemoto, Norihiko
Tanaka, Takao
Ooguchi, Yoshihiro
Skehel, Paul
Aida, Tomomi
Takeda, Junji
Mashimo, Tomoji
author_facet Miyasaka, Yoshiki
Uno, Yoshihiro
Yoshimi, Kazuto
Kunihiro, Yayoi
Yoshimura, Takuji
Tanaka, Tomohiro
Ishikubo, Harumi
Hiraoka, Yuichi
Takemoto, Norihiko
Tanaka, Takao
Ooguchi, Yoshihiro
Skehel, Paul
Aida, Tomomi
Takeda, Junji
Mashimo, Tomoji
author_sort Miyasaka, Yoshiki
collection PubMed
description 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12864-018-4713-y) contains supplementary material, which is available to authorized users.
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spelling pubmed-59306882018-05-09 CLICK: one-step generation of conditional knockout mice Miyasaka, Yoshiki Uno, Yoshihiro Yoshimi, Kazuto Kunihiro, Yayoi Yoshimura, Takuji Tanaka, Tomohiro Ishikubo, Harumi Hiraoka, Yuichi Takemoto, Norihiko Tanaka, Takao Ooguchi, Yoshihiro Skehel, Paul Aida, Tomomi Takeda, Junji Mashimo, Tomoji BMC Genomics Research Article 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12864-018-4713-y) contains supplementary material, which is available to authorized users. BioMed Central 2018-05-02 /pmc/articles/PMC5930688/ /pubmed/29720086 http://dx.doi.org/10.1186/s12864-018-4713-y Text en © The Author(s). 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Miyasaka, Yoshiki
Uno, Yoshihiro
Yoshimi, Kazuto
Kunihiro, Yayoi
Yoshimura, Takuji
Tanaka, Tomohiro
Ishikubo, Harumi
Hiraoka, Yuichi
Takemoto, Norihiko
Tanaka, Takao
Ooguchi, Yoshihiro
Skehel, Paul
Aida, Tomomi
Takeda, Junji
Mashimo, Tomoji
CLICK: one-step generation of conditional knockout mice
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 Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930688/
https://www.ncbi.nlm.nih.gov/pubmed/29720086
http://dx.doi.org/10.1186/s12864-018-4713-y
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