A Simple Protocol for Loss-of-Function Analysis in Xenopus tropicalis Founders Using the CRISPR-Cas System.

Xenopus tropicalis is a versatile model organism for studying basic biology such as developmental biology and cell biology, and for biomedical research on human diseases. Current genome editing techniques enable researchers to easily perform gene targeting in various animals. Among them, gene knockout using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system has recently become an indispensable strategy for loss-of-function analysis in vivo. Because of its ease of use, time, and cost efficiencies, CRISPR-Cas has also been applied to X. tropicalis where the gene disruption is highly efficient. In this chapter, we introduce a simple CRISPR-Cas system protocol for gene disruption in X. tropicalis. Based on our protocol, researchers can generate knock-out phenotypes within the shortest of timeframes, a week, and analyze genes of interest in founder generation.