Cell synchronization experiments are consistent with adenine and cytosine base editors reliance on base excision repair

Base editors (BEs) are genome editing agents that install point mutations with high efficiency and specificity. Because of their reliance on uracil and inosine DNA damage intermediates (rather than double-strand DNA breaks, or DSBs), it has been hypothesized that BEs are more ubiquitous than DSB-reliant genome editing methods, which require DNA repair processes that are only active during certain phases of the cell cycle. We report here the first systematic study of the cell cycle-dependence of base editing via cell synchronization experiments. We find that nickase derived BEs (which introduce DNA backbone nicks opposite the uracil or inosine base) function independently of the cell cycle, while non-nicking BEs are highly dependent on S phase. We observe that endogenous expression levels of DNA damage repair pathways are sufficient to process base editing intermediates into desired editing outcomes. Finally, we analyze the cell cycle-dependence of DNA damage repair genes using a single cell RNAseq dataset to produce the first quantitative assessment of changes in DNA repair pathway expression levels with respect to the cell cycle and relate these data to our cell synchronization experiments. Overall, our study suggests that the most widely used nickase derived BEs rely on the single-stranded break base excision repair pathway to install point mutations, and trends in CG to non-TA editing by cytosine base editors mirror expression level trends in certain translesion synthesis polymerases.