Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis

Genesis. 2013 Dec;51(12):835-43. doi: 10.1002/dvg.22720.

Abstract

We have assessed the efficacy of the recently developed CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system for genome modification in the amphibian Xenopus tropicalis. As a model experiment, targeted mutations of the tyrosinase gene were verified, showing the expected albinism phenotype in injected embryos. We further tested this technology by interrupting the six3 gene, which is required for proper eye and brain formation. Expected eye and brain phenotypes were observed when inducing mutations in the six3 coding regions, as well as when deleting the gene promoter by dual targeting. We describe here a standardized protocol for genome editing using this system. This simple and fast method to edit the genome provides a powerful new reverse genetics tool for Xenopus researchers.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brain / metabolism
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Embryo, Nonmammalian / metabolism
  • Eye / metabolism
  • Eye Proteins / genetics*
  • Eye Proteins / metabolism
  • Genetic Loci
  • Genome
  • Germ Cells / metabolism
  • Homeodomain Proteins / genetics*
  • Homeodomain Proteins / metabolism
  • Mutagenesis, Site-Directed*
  • Nerve Tissue Proteins / genetics*
  • Nerve Tissue Proteins / metabolism
  • Phenotype
  • Xenopus / embryology*
  • Xenopus / genetics*
  • Xenopus Proteins / genetics
  • Xenopus Proteins / metabolism

Substances

  • Eye Proteins
  • Homeodomain Proteins
  • Nerve Tissue Proteins
  • Sine oculis homeobox homolog 3 protein
  • Xenopus Proteins