Elucidating the genetic mechanisms governing cytosine base editing outcomes through CRISPRi screens

Cytosine base editors (CBEs) enable programmable and efficient substitutions of cytosine bases without relying on double-stranded breaks. CBEs introduce a unique type of DNA damage intermediate into the genome, featuring a U:G mismatch with a DNA nick 5 upstream from the mismatch on the G-containing strand. Cellular processing of this DNA damage intermediate leads to two major mutational outcomes (C:G to T:A and C:G to G:C point mutations), and two minor outcomes (C:G to A:T point mutations and C:G to indels). While it is known that the uracil intermediate is excised by the DNA repair protein uracil N-glycosylase (UNG), other factors involved in processing this unique intermediate are not well-characterized. In this work, we couple fluorescent reporters for C:G to T:A and C:G to G:C base editing activity with CRISPRi screens to measure the effects of knockdown of 2,015 individual DNA processing genes on these two editing outcomes. We observe and validate for the first time that the mismatch repair (MMR) recognition complex MutSa (the MSH2/MSH6 heterodimer) facilitates C:G to T:A outcomes. We additionally find that ligase 3 (LIG3), a single-strand break ligase, inhibits the C:G to T:A outcome, potentially via ligation of the Cas9n-induced nick. In addition, we find that RFWD3, an E3 ubiquitin ligase, mediates a translesion synthesis pathway that specifically leads to the C:G to G:C outcome. Finally, we find that XPF (encoded by ERCC4), a 3-flap endonuclease, is involved in repairing the intermediate back to the original C:G base pair. Our results demonstrate that competition and collaboration among various DNA repair proteins from different pathways shape cytosine base editing outcomes. This work provides a deeper understanding of the mechanisms cytosine base editing and sets a foundation for understanding the genetic mechanisms of other base editors.