CRISPR-Mediated Gene Editing: Generating Gene Knockouts or Precision Base Changes in F0 Xenopus.

CRISPR-mediated gene editing has transformed Xenopus research by enabling targeted, heritable gene disruption in both Xenopus laevis and Xenopus tropicalis. The CRISPR/Cas9 system has allowed efficient loss-of-function analysis within days of injection, overcoming the limitations of transient morpholino knockdowns. Recent advances in CRISPR-mediated base editing technology further expands this toolkit, permitting the precision generation of single-base changes in Xenopus. High editing efficiency, external development, and large clutch size make Xenopus embryos exceptionally suited for genome manipulation and phenotype screening. CRISPR-mediated gene editing in Xenopus has reproduced classic developmental phenotypes and generated robust models of human disease, including ciliopathies, congenital heart defects, skeletal dysplasias, and neurodevelopmental disorders. Disease modeling using Xenopus CRISPR mutants has provided critical insights into conserved vertebrate pathways and the pathogenic mechanisms of human gene variants. Here, we describe the complete process of producing a gene knockout or precision base changes in F0 Xenopus: target selection, design and synthesis of sgRNA, base editor selection and synthesis of base editor mRNA, microinjection into fertilized Xenopus eggs, and genotyping to assess whether gene editing has successfully occurred.