Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.
Department of Bioscience and Bioinformatics, School of Computer Science and Systems Engineering, Kyushu Institute of Technology, Kitakyushu, Fukuoka, Japan.
Methods Mol Biol. 2023;2637:341-357. doi: 10.1007/978-1-0716-3016-7_26.
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, and Pleurodeles 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.
两栖动物在基础生物学和人类疾病的生物医学研究等方面做出了许多重要贡献。目前基于 CRISPR-Cas 系统的基因组编辑工具使我们能够在体内进行基因功能分析，甚至在非模式生物中也能进行。我们在这里介绍一种高效且简单的基因敲除方法，该方法可在三种不同的两栖动物中无缝使用：非洲爪蟾（Xenopus laevis）、热带爪蟾（Xenopus tropicalis）和欧洲林蛙（Pleurodeles waltl）。由于该方法利用 Cas9 核糖核蛋白复合物（RNP）进行注射，因此这种无克隆方法可使研究人员在一周内获得具有近乎完全敲除表型的奠基者胚胎。为了评估 Cas9 RNP 注射胚胎（crispant）的体细胞突变率及其与表型的相关性，我们还提供了使用合并扩增子测序和用户友好的基于网络的工具进行准确且具有成本效益的基因分型方法。
