CRISPR-Cas9-Based Functional Analysis in Amphibians: Xenopus laevis, Xenopus tropicalis, and Pleurodeles waltl
CRISPR-Cas9-Based Functional Analysis in Amphibians: Xenopus laevis, Xenopus tropicalis, and Pleurodeles waltl
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基于 CRISPR-Cas9 的两栖动物功能分析：非洲爪蟾、热带爪蟾和侧耳藻
DOI：
10.1007/978-1-0716-3016-7_26
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Suzuki Ken-ichi T.
中科院分区：
文献类型：
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作者：
Suzuki Miyuki;Iida Midori;Hayashi Toshinori;Suzuki Ken-ichi T.
从基础生物学到人类疾病的生物医学研究，两栖动物对我们的知识做出了许多根本性的贡献。目前基于CRISPR-Cas系统的基因组编辑工具使我们能够在体内甚至在非模式生物中进行基因功能分析。我们在此介绍了一种高效且简单的基因敲除方法，该方法可以无缝地用于三种不同的两栖动物：非洲爪蟾、热带爪蟾和华氏侧耳。由于它使用Cas9核糖核蛋白复合物（RNP）进行注射，这种无克隆的方法使研究人员能够在一周内获得具有几乎完全敲除表型的创始胚胎。为了评估体细胞突变率及其与Cas9 rnp注射胚胎（criscrisp）表型的相关性，我们还提出了使用集合扩增子测序和用户友好的基于web的工具的准确和经济高效的基因分型方法。
Amphibians have made many fundamental contributions to our knowledge, from basic biology to biomedical research on human diseases. Current genome editing tools based on the CRISPR-Cas system enable us to perform gene functional analysis in vivo, even in non-model organisms. We introduce here a highly efficient and easy protocol for gene knockout, which can be used in three different amphibians seamlessly:Xenopus laevis,Xenopus tropicalis, andPleurodeles waltl. As it utilizes Cas9 ribonucleoprotein complex (RNP) for injection, this cloning-free method enables researchers to obtain founder embryos with a nearly complete knockout phenotype within a week. To evaluate somatic mutation rate and its correlation to the phenotype of a Cas9 RNP-injected embryo (crispant), we also present accurate and cost-effective genotyping methods using pooled amplicon-sequencing and a user-friendly web-based tool.