UC San Diego

Progress in next-generation sequencing (NGS) technologies has streamlined the detection of human genetic variations, and the identification of clinically actionable genes and pathogenic mutations has transformed precision medicine. However, only a small fraction of identified human genetic variants have been assigned a clinical classification or functionally characterized. This highlights the importance of investigating genetic variants and obtaining mechanistic insight into disease etiology and progression. Base editing is a new precision genome editing methodology that utilizes native DNA repair pathways within living cells to either fix or install genetic variants. In Chapter 2, we detail our findings which aim to provide an optimized protocol in utilizing base editing technology. Then in Chapter 3, we detail how we harness base editing to overcome many of the limitations of traditional genome editing to generate both homozygous and heterozygous isogenic cell lines of four MUTYH variants. To date, these were the first reports of successful generation of isogenic cell lines containing MUTYH variants and also the first to functionally characterize them within living cells. Finally, in Chapter 4, non-thesis-related university service garnered throughout the primary author and researcher’s tenure is briefly discussed as this work has also led to both personal and professional advances.