CRISPR/Cas9 gene disruption studies in F₀ Xenopus tadpoles: understanding development and disease in the frog

CRISPR/Cas9 has become the favorite method for gene knockouts in a range of vertebrate model organisms due to its ease of use and versatility. Gene-specific guide RNAs can be designed to a unique genomic sequence and used to target the Cas9 endonuclease, which causes a double-stranded break at the desired locus. Repair of the breaks through non-homologous end joining often results in the deletion or insertion of several nucleotides, which frequently result in nonsense mutations. Xenopus frogs have long been an excellent model organism in which to study gene function, and they have proven to be useful in gene-editing experiments, especially the diploid species, X. tropicalis. In this chapter, we present our protocols for gene disruption in Xenopus, which we regularly use to investigate developmental processes and model human genetic disease.