Homo sapiens Raw sequence reads. Homo sapiens

Base editors introduce base substitutions without double-strand DNA cleavage. Besides the precise substitutions, BEs generate low-frequency "stochastic" byproducts through unclear mechanisms. Here, we applied in-depth outcome profiling and genetic screening to show that C-to-G BEs (CGBEs) generate substantial amounts of intermediate double-strand breaks (DSBs), which are at the center of several byproducts. Imperfect end-joining leads to small deletions via end-resection, templated insertions or aberrant transversions during end fill-in. Chromosomal translocations between the CGBE editing target and off-targets of Cas9/deaminase-origin were also detected. Outcome-screening of DNA repair factors revealed a central role of abasic site processing in DSB formation, and the shielding of abasic sites by the suicide-enzyme HMCES reduced CGBE-initiated DSBs, providing an effective way to minimize DSB-triggered events without affecting substitutions. This work demonstrates that CGBEs can initiate deleterious intermediate DSBs and therefore require careful consideration for therapeutic applications, and that the HMCES-aided CGBEs are potentially safer tools.