A CRISPR/Cas-Based Method for Precise DNA Integration in Xenopus laevis Oocytes Followed by Intracytoplasmic Sperm Injection (ICSI) Fertilization

Abstract

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.