Cas9-based genome editing in Xenopus tropicalis

Methods Enzymol. 2014;546:355-75. doi: 10.1016/B978-0-12-801185-0.00017-9.

Abstract

Xenopus tropicalis has been developed as a model organism for developmental biology, providing a system offering both modern genetics and classical embryology. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR/Cas) system for genome modification has provided an additional tool for Xenopus researchers to achieve simple and efficient targeted mutagenesis. Here, we provide insights into experimental design and procedures permitting successful application of this technique to Xenopus researchers, and offer a general strategy for performing loss-of-function assays in F0 and subsequently F1 embryos.

Keywords: Amphibian mutagenesis; CRISPR; Disease model; Genome engineering; Loss-of-function; Off-target effects; Targeted mutagenesis; sgRNA design.

Publication types

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

MeSH terms

  • Animals
  • Base Sequence
  • CRISPR-Cas Systems
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Embryo, Nonmammalian / metabolism
  • Gene Targeting / methods*
  • Genetic Engineering / methods
  • Genome
  • Molecular Sequence Data
  • Mutagenesis*
  • RNA, Guide / genetics
  • Xenopus / embryology*
  • Xenopus / genetics*

Substances

  • RNA, Guide