Development and Applications of CRISPR/Cas9 Genome Editing Technology

Abstract

The ability to edit the genome of organism can positively impact biomedical research. Using genome editing we can dissect the function of gene(s) and their regulatory elements and, more importantly, facilitate modelling of human genetic diseases to understand their pathology and develop treatments. The recently developed CRISPR/Cas9 genome editing platform has become rapidly and widely used in biomedical research due to its ease of use, highly efficiency and low cost. The research conducted during my PhD attempted to further develop this technology and apply it to a range of biological questions that are of interest to the Thomas laboratory. I showed that this technology can be applied to generate a gene-swap mouse model to study functional redundancy between closely related transcription factors in SOXB1 family, SOX2 and SOX3. By swapping Sox3 with Sox2 in vivo we showed that the presence of Sox2 in the absence of Sox3 rescues Sox3-null phenotypes. This finding provides strong and direct evidence that they are functionally redundant. I also develop CRISPR/Cas9-based strategies to allow targeted elimination of an entire chromosome. These strategies termed centromere removal and chromosome shredding could facilitate efficient chromosome deletion as shown by successful elimination of the Y chromosome in mouse ES cells and zygotes. We also contribute to the development of CRISPR/Cas9 toolbox by generating plasmids that can express dual gRNAs and other required components such as the Cas9 or Cas9-nickase as well as selection markers within a single plasmid. Interestingly, our vector design allows facile generation of two unique guides in a simple one-step reaction, rendering these plasmids user-friendly for researchers requiring simultaneous expression of two gRNAs. Targeting two sites can be achieved with very high efficiency using these plasmids, which will be made freely available to all researchers via the Addgene plasmid repository. Lastly, my research show that large deletions are frequently generated as the repair outcome of CRISPR/Cas9-mediated cleavage. The large deletions contain mostly microhomology sequences at the break junctions and are generated via DNA resection, indicating an alternative end joining mechanism underlies their generation. This study reveals an underestimated yet common repair outcome that researchers should be aware of to avoid genotyping misinterpretation. Collectively, the studies in this thesis have contributed to the development of CRISPR/Cas9 genome editing technology by providing valuable tools and new knowledge about DNA repair. In addition, I demonstrate that CRISPR/Cas9 technology can be applied to diverse biological questions including functional redundancy of developmental transcription factors and targeted chromosomal ablation