Base editors (BEs) introduce base substitutions without double-strand DNA cleavage. Besides precise substitutions, BEs generate low-frequency ‘stochastic’ byproducts through unclear mechanisms. Here, we performed in-depth outcome profiling and genetic dissection, revealing that C-to-G BEs (CGBEs) generate substantial amounts of intermediate double-strand breaks (DSBs), which are at the centre of several byproducts. Imperfect DSB end-joining leads to small deletions via end-resection, templated insertions or aberrant transversions during end fill-in. Chromosomal translocations were detected between the editing target and off-targets of Cas9/deaminase origin. Genetic screenings of DNA repair factors disclosed a central role of abasic site processing in DSB formation. 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 HMCES-aided CGBEs hold promise as safer tools.

碱基编辑器 (BE) 引入碱基替换，无需双链 DNA 切割。除了精确的替代之外，BE 还通过不清楚的机制产生低频“随机”副产品。在这里，我们进行了深入的结果分析和基因解剖，揭示了 C 到 G BE (CGBE) 产生大量的中间双链断裂 (DSB)，这是几种副产物的核心。不完美的 DSB 末端连接会通过末端切除、模板化插入或末端填充过程中的异常颠换而导致小缺失。在 Cas9/脱氨酶起源的编辑靶标和脱靶之间检测到染色体易位。DNA 修复因子的遗传筛选揭示了脱碱基位点加工在 DSB 形成中的核心作用。自杀酶 HMCES 对脱碱基位点的屏蔽减少了 CGBE 引发的 DSB，从而提供了一种有效的方法来最大限度地减少 DSB 触发事件而不影响替换。这项工作表明，CGBE 可以引发有害的中间 DSB，因此需要仔细考虑治疗应用，并且 HMCES 辅助的 CGBE 有希望成为更安全的工具。