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Protocols for transgenesis at a safe harbor site in the Xenopus laevis genome using CRISPR-Cas9.

ABSTRACT: We have established a new transgenesis protocol based on CRISPR-Cas9, "New and Easy XenopusTransgenesis (NEXTrans)," and identified a novel safe harbor site in African clawed frogs, Xenopus laevis. We describe steps in detail for the construction of NEXTrans plasmid and guide RNA, CRISPR-Cas9-mediated NEXTrans plasmid integration into the locus, and its validation by genomic PCR. This improved strategy allows us to simply generate transgenic animals that stably express the transgene. For complete details on the use and execution of this protocol, please refer to Shibata et al. (2022).¹.

SUBMITTER: Shibata Y

PROVIDER: S-EPMC10511863 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Protocols for transgenesis at a safe harbor site in the Xenopus laevis genome using CRISPR-Cas9.

Shibata Yuki Y Okumura Akinori A Mochii Makoto M Suzuki Ken-Ichi T KT

STAR protocols 20230628 3

We have established a new transgenesis protocol based on CRISPR-Cas9, "New and Easy XenopusTransgenesis (NEXTrans)," and identified a novel safe harbor site in African clawed frogs, Xenopus laevis. We describe steps in detail for the construction of NEXTrans plasmid and guide RNA, CRISPR-Cas9-mediated NEXTrans plasmid integration into the locus, and its validation by genomic PCR. This improved strategy allows us to simply generate transgenic animals that stably express the transgene. For complet ...[more]

PMID: 37389994

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