Methods for CRISPR/Cas9 Xenopus tropicalis Tissue-Specific Multiplex Genome Engineering

Methods Mol Biol. 2018;1865:33-54. doi: 10.1007/978-1-4939-8784-9_3.

Abstract

In this chapter, we convey a state-of-the art update to the 2014 Nakayama protocol for CRISPR/Cas9 genome engineering in Xenopus tropicalis (X. tropicalis). We discuss in depth, gRNA design software and rules, gRNA synthesis, and procedures for tissue- and tissue-specific CRISPR/Cas9 genome editing by targeted microinjection in X. tropicalis embryos. We demonstrate the methodology by which any standard equipped Xenopus researcher with microinjection experience can generate F0 CRISPR/Cas9 mediated mosaic mutants (crispants) within one to two work-week(s). The described methodology allows CRISPR/Cas9 efficiencies to be high enough to read out phenotypic consequences, and thus perform gene function analysis, in the F0 crispant. Additionally, we provide the framework for performing multiplex tissue-specific CRISPR/Cas9 experiments generating crispants mosaic mutant in up to four genes simultaneously, which can be of importance for Laevis researchers aiming to target by CRISPR/Cas9 both the S and L homeolog of a gene simultaneously. Finally, we discuss off-target concerns, how to minimize these and ways to rapidly bypass reviewer off-target critique by exploiting the advantages of X. tropicalis.

Keywords: CRISPR/Cas9; Disease model; Multiplex; Tissue-specific; Xenopus tropicalis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems / genetics*
  • Gene Editing
  • Genetic Engineering / methods*
  • Genome*
  • Microinjections
  • Monophenol Monooxygenase / metabolism
  • Organ Specificity / genetics*
  • RNA, Guide / genetics
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Ribonucleoproteins / metabolism
  • Xenopus / embryology
  • Xenopus / genetics*

Substances

  • RNA, Guide
  • RNA, Messenger
  • Ribonucleoproteins
  • Monophenol Monooxygenase
  • CRISPR-Associated Protein 9