Development and optimisation of a CRISPR/CAS based method for precise non-mosaic DNA integration in Xenopus laevis

Abstract

Xenopus have 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 full genome sequencing of Xenopus genomes revealing the full extent of the genetic conservation between Xenopus and humans, it was expected that Xenopus would soon become a popular model for human 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 visualisation, quantification and tracking of proteins without the use of protein-specific antibodies, which are often unreliable or not reactive in Xenopus. This thesis describes the development and optimisation of a method for precise DNA insertion into oocytes using CRISPR/Cas9, followed by in vitro maturation and fertilisation by intracytoplasmic sperm injection, culminating in the production of embryos carrying a non-mosaic, heterozygous HA tag fused to endogenous Runx1. We also demonstrate an improvement to HDR insertion using long homology compared to short homology in embryos, carving a clear path forward to further optimising HDR insertion in oocytes. Together with other methods being developed around the globe, these techniques have great potential to strengthen the frog as a genetic model organism.

Date of Award	Jun 2021
Original language	English
Awarding Institution	University of Portsmouth
Supervisor	Matt Guille (Supervisor) & Fiona Myers (Supervisor)