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

• Published in: Nature cell biology Vol. 26; no. 2; pp. 294 - 304
• Main Authors: Huang, Min Emma, Qin, Yining, Shang, Yafang, Hao, Qian, Zhan, Chuanzong, Lian, Chaoyang, Luo, Simin, Liu, Liu Daisy, Zhang, Senxin, Zhang, Yu, Wo, Yang, Li, Niu, Wu, Shuheng, Gui, Tuantuan, Wang, Binbin, Luo, Yifeng, Cai, Yanni, Liu, Xiaojing, Xu, Ziye, Dai, Pengfei, Li, Simiao, Zhang, Liang, Dong, Junchao, Wang, Jian, Zheng, Xiaoqi, Xu, Yingjie, Sun, Yihua, Wu, Wei, Yeap, Leng-Siew, Meng, Fei-Long
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-02-2024; Nature Publishing Group
• Subjects: 13; 13/109; 13/31; 14/19; 38/1; 38/22; 38/23; 38/77; 38/90; 45/29; 631/1647/1511; 631/208/4041/3196; 631/208/737/211; 82; 82/51; 82/83; Alkanesulfonic Acids; Biomedical and Life Sciences; Byproducts; Cancer Research; Cell Biology; Chromosome translocations; CRISPR-Cas Systems; Deoxyribonucleic acid; Developmental Biology; DNA; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA repair; DNA Repair - genetics; Editing; Gene deletion; Genetic screening; Humans; Life Sciences; Stem Cells; Stochasticity; Suicide; Target detection; technical-report; Therapeutic applications; Translocation; Translocation, Genetic; Transversion; Yeast
• ISSN: 1465-7392; 1476-4679
• DOI: 10.1038/s41556-023-01342-2

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.
Huang, Qin, Shang et al. profile double-strand breaks (DSBs) generated by C-to-G base editors (CGBEs) and find that HMCES protects abasic sites and reduces CGBE-triggered DSBs.