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

Ira L. Blitz; Takuya Nakayama

doi:10.1101/pdb.prot106971

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

Ira L. Blitz1,3 and
Takuya Nakayama2,3

¹Department of Developmental and Cell Biology, University of California, Irvine, California 92697, USA;
²Department of Biology, University of Virginia, Charlottesville, Virginia 22904, USA

↵3Correspondence: ilblitz{at}uci.edu; tn8t{at}virginia.edu

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.