CRISPR–Cas9 Mutagenesis in Xenopus tropicalis for Phenotypic Analyses in the F0 Generation and Beyond

Abstract

CRISPR–Cas9 mutagenesis is being widely used to create targeted loss-of-function mutations in the diploid frog Xenopus tropicalis. Here we describe a simple mutagenesis protocol using microinjection of Cas9 protein or mRNA, together with synthetic guide RNAs (sgRNAs) targeting specific DNA sequences, into the early embryo. Cas9-catalyzed double-strand breaks undergo error-prone repair, resulting in production of short insertions and/or deletions. Thus, careful selection of target sites can lead to mutations that impair normal function of the protein product. CRISPR–Cas9 can be used to create either mosaic loss-of-function Xenopus embryos that display F0 generation phenotypes or mutant lines for downstream analysis. In addition to describing how to mutagenize genes using CRISPR–Cas9, we also discuss a simple method to determine the mutagenesis efficiency, some potential problems that can arise, and possible solutions to overcome them. The protocol described here should be applicable to other amphibians and, in principle, many other organisms.