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

Thomas Naert1,2, Kris Vleminckx3,4,5

  • 1Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.

Methods in Molecular Biology (Clifton, N.J.)
|August 29, 2018
PubMed
Summary

This chapter updates the CRISPR/Cas9 genome engineering protocol for Xenopus tropicalis, enabling rapid generation of gene-edited mutants (crispants) for efficient gene function analysis in F0 generation.

Keywords:
CRISPR/Cas9Disease modelMultiplexTissue-specificXenopus tropicalis

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Area of Science:

  • Developmental Biology
  • Genetics
  • Molecular Biology

Background:

  • The CRISPR/Cas9 system has revolutionized genome engineering.
  • The Nakayama protocol (2014) provided foundational methods for Xenopus tropicalis.
  • Advancements are needed to refine CRISPR/Cas9 applications in Xenopus.

Purpose of the Study:

  • To provide an updated, state-of-the-art protocol for CRISPR/Cas9 genome engineering in Xenopus tropicalis.
  • To detail methods for efficient generation of F0 mosaic mutants (crispants).
  • To establish a framework for multiplex genome editing and address off-target concerns.

Main Methods:

  • Updated CRISPR/Cas9 protocol based on the 2014 Nakayama protocol.
  • Detailed guidelines for gRNA design, synthesis, and microinjection.
  • Demonstration of tissue-specific and multiplex genome editing in Xenopus tropicalis embryos.

Main Results:

  • Successful generation of F0 CRISPR/Cas9 mosaic mutants (crispants) within 1-2 workweeks.
  • Achieved high CRISPR/Cas9 efficiencies for phenotypic analysis in F0 crispants.
  • Developed a framework for simultaneous multiplex gene editing (up to four genes).

Conclusions:

  • The updated protocol enables efficient CRISPR/Cas9-mediated gene function analysis in F0 Xenopus tropicalis.
  • The methodology facilitates rapid generation of complex mutant phenotypes.
  • The protocol addresses off-target effects and leverages Xenopus tropicalis advantages for robust genome engineering.