C-to-G editing generates double-strand breaks causing deletion, transversion and translocation.

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. © 2024. The Author(s), under exclusive licence to Springer Nature Limited.

Citation

Min Emma Huang, Yining Qin, Yafang Shang, Qian Hao, Chuanzong Zhan, Chaoyang Lian, Simin Luo, Liu Daisy Liu, Senxin Zhang, Yu Zhang, Yang Wo, Niu Li, Shuheng Wu, Tuantuan Gui, Binbin Wang, Yifeng Luo, Yanni Cai, Xiaojing Liu, Ziye Xu, Pengfei Dai, Simiao Li, Liang Zhang, Junchao Dong, Jian Wang, Xiaoqi Zheng, Yingjie Xu, Yihua Sun, Wei Wu, Leng-Siew Yeap, Fei-Long Meng. C-to-G editing generates double-strand breaks causing deletion, transversion and translocation. Nature cell biology. 2024 Feb;26(2):294-304

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PMID: 38263276

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