CRISPR-Cas9-Based Functional Analysis in Amphibians: Xenopus laevis, Xenopus tropicalis, and Pleurodeles waltl.

Abstract

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.