Tong Huawei, Liu Nana, Wei Yinghui, Zhou Yingsi, Li Yun, Wu Danni, Jin Ming, Cui Shuna, Li Hengbin, Li Guoling, Zhou Jingxing, Yuan Yuan, Zhang Hainan, Shi Linyu, Yao Xuan, Yang Hui
HuidaGene Therapeutics Co., Ltd., Shanghai 200131, China.
Department of Neurology and Institute of Neurology of First Affiliated Hospital, Institute of Neuroscience, and Fujian Key Laboratory of Molecular Neurology, Fujian Medical University, Fuzhou 350004, China.
Natl Sci Rev. 2023 May 16;10(8):nwad143. doi: 10.1093/nsr/nwad143. eCollection 2023 Aug.
Current DNA base editors contain nuclease and DNA deaminase that enables deamination of cytosine (C) or adenine (A), but no method for guanine (G) or thymine (T) editing is available at present. Here we developed a deaminase-free glycosylase-based guanine base editor (gGBE) with G editing ability, by fusing Cas9 nickase with engineered N-methylpurine DNA glycosylase protein (MPG). By several rounds of MPG mutagenesis via unbiased and rational screening using an intron-split EGFP reporter, we demonstrated that gGBE with engineered MPG could increase G editing efficiency by more than 1500 fold. Furthermore, this gGBE exhibited high base editing efficiency (up to 81.2%) and high G-to-T or G-to-C (i.e. G-to-Y) conversion ratio (up to 0.95) in both cultured human cells and mouse embryos. Thus, we have provided a proof-of-concept of a new base editing approach by endowing the engineered DNA glycosylase the capability to selectively excise a new type of substrate.
目前的DNA碱基编辑器包含核酸酶和DNA脱氨酶，可实现胞嘧啶（C）或腺嘌呤（A）的脱氨作用，但目前尚无对鸟嘌呤（G）或胸腺嘧啶（T）进行编辑的方法。在此，我们通过将Cas9切口酶与工程化的N-甲基嘌呤DNA糖基化酶蛋白（MPG）融合，开发了一种具有G编辑能力的无脱氨酶的基于糖基化酶的鸟嘌呤碱基编辑器（gGBE）。通过使用内含子分裂的增强绿色荧光蛋白（EGFP）报告基因，经过几轮无偏差和合理筛选的MPG诱变，我们证明，工程化MPG的gGBE可将G编辑效率提高1500倍以上。此外，这种gGBE在培养的人类细胞和小鼠胚胎中均表现出高碱基编辑效率（高达81.2%）以及高的G到T或G到C（即G到Y）转化率（高达0.95）。因此，我们通过赋予工程化DNA糖基化酶选择性切除新型底物的能力，提供了一种新的碱基编辑方法的概念验证。
