A CRISPR/Cas-Based Method for Precise DNA integration in Xenopus laevis oocytes followed by intracytoplasmic sperm injection (ICSI) fertilization

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)peer-review


Xenopus has long had a reputation for being a powerful model organism for use in developmental cell and biochemistry research. With the advent of gene-editing technologies, and the full genome sequencing of Xenopus genomes revealing the extent of the genetic conservation between Xenopus and humans, Xenopus has the potential to become an ideal model for human genetic disease. However, the inability to produce non-mosaic, precise DNA insertions through homology directed repair has limited the strength of Xenopus this field. Furthermore, it has prevented researchers from taking full advantage of fusion tagging, a method for directly tagging genes with either epitope or fluorescent tags, allowing the visualization, quantification, and tracking of proteins without the use of protein-specific antibodies. Here, we describe a method for precise DNA insertion into oocytes using CRISPR/Cas9, followed by in vitro maturation and fertilization by intracytoplasmic sperm injection (ICSI), culminating in the production of embryos carrying a non-mosaic, heterozygous insertion.
Original languageEnglish
Title of host publicationDNA Manipulation and Analysis
EditorsGarry Scarlett
Place of PublicationNew York
PublisherHumana Press
Number of pages13
ISBN (Electronic)9781071630044
ISBN (Print)9781071630037
Publication statusPublished - 1 Mar 2023

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • Xenopus
  • precise insertion
  • oocytes
  • ICSI
  • sperm nuclei
  • disease model
  • epitope tag

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