UC San Diego

Base editors are an innovative addition to the genome editing toolbox that introduced a new genome editing strategy to the field. Instead of using double-stranded DNA breaks, base editors utilize unique types of DNA damage products featuring nucleobase mismatches with a DNA backbone nick to facilitate the efficient incorporation of single nucleotide variants into the genome of living cells. Two classes of DNA base editors current exist: cytosine deamination-derived editors and adenine deamination-derived base editors. Despite their widespread use in both basic research and therapeutic settings, the biological pathways processing base editing intermediates remain unclear. Chapter 1 provides a general introduction to base editors. Chapter 2 presents the elucidation of the genetic mechanisms governing cytosine base editing outcomes through CRISPRi screens. This work demonstrates that competition and collaboration among various DNA repair proteins from different pathways shape cytosine base editing outcomes. It provides a deeper understanding of the mechanisms of cytosine base editing and sets a foundation for understanding the genetic mechanisms of other base editors. All current base editors rely on deamination chemistry to introduce DNA damage products that serve as their mutagenic intermediates. In chapter 3, the development of a G•C to T•A base editor utilizing methylation chemistry to introduce N1-methylguanine as the mutagenic intermediate was described. This project sets a precedent for harnessing non-deamination chemistry to perform genome editing and demonstrates a novel strategy for the development of new base editors.