近日，中国科学院天津工业生物技术研究所毕昌昊等研究人员合作发现，基于糖基化酶的碱基编辑器可在哺乳动物细胞中进行高效的T-G和C-G编辑。该研究于2024年1月2日在线发表于国际一流学术期刊《自然—生物技术》。

研究人员为胞嘧啶（DAF-CBE）和胸腺嘧啶（DAF-TBE）构建了不含脱氨基酶的碱基编辑器，它们分别只包含一个胞嘧啶-DNA 或一个胸腺嘧啶-DNA 糖基化酶（CDG/TDG）变体，并与Cas9缺口酶相连。通过在大肠杆菌中进行多轮定向演化诱变，产生了两种具有增强碱基转换活性的变体（CDG-nCas9 和 TDG-nCas9），C-to-A转换效率高达58.7%，T-to-A转换效率高达54.3%。DAF-BE在哺乳动物细胞中实现了C-to-G/T-to-G编辑，Cas9依赖性和Cas9非依赖性脱靶效应以及RNA脱靶效应极小。

DAF-CBE2/DAF-TBE2经过进一步工程化后，从原间隔序列的5′端到中间的编辑窗口发生了改变，C-to-G/T-to-G编辑效率分别提高了3.5倍和1.2倍。与先导编辑或CGBE相比，DAF-BE以相似的效率、更小的尺寸和更低的脱靶效应扩展了碱基编辑器的转换类型。

研究人员表示，碱基编辑器有望治疗人类遗传疾病，但目前大多数系统都使用脱氨酶，这会导致脱靶效应，而且编辑类型有限。

附：英文原文

Title: Glycosylase-based base editors for efficient T-to-G and C-to-G editing in mammalian cells

Author: Ye, Lijun, Zhao, Dongdong, Li, Ju, Wang, Yiran, Li, Bo, Yang, Yuanzhao, Hou, Xueting, Wang, Huibin, Wei, Zhandong, Liu, Xiaoqi, Li, Yaqiu, Li, Siwei, Liu, Yajing, Zhang, Xueli, Bi, Changhao

Issue&Volume: 2024-01-02

Abstract: Base editors show promise for treating human genetic diseases, but most current systems use deaminases, which cause off-target effects and are limited in editing type. In this study, we constructed deaminase-free base editors for cytosine (DAF-CBE) and thymine (DAF-TBE), which contain only a cytosine-DNA or a thymine-DNA glycosylase (CDG/TDG) variant, respectively, tethered to a Cas9 nickase. Multiple rounds of mutagenesis by directed evolution in Escherichia coli generated two variants with enhanced base-converting activity—CDG-nCas9 and TDG-nCas9—with efficiencies of up to 58.7% for C-to-A and 54.3% for T-to-A. DAF-BEs achieve C-to-G/T-to-G editing in mammalian cells with minimal Cas9-dependent and Cas9-independent off-target effects as well as minimal RNA off-target effects. Additional engineering resulted in DAF-CBE2/DAF-TBE2, which exhibit altered editing windows from the 5′ end to the middle of the protospacer and increased C-to-G/T-to-G editing efficiency of 3.5-fold and 1.2-fold, respectively. Compared to prime editing or CGBEs, DAF-BEs expand conversion types of base editors with similar efficiencies, smaller sizes and lower off-target effects.

DOI: 10.1038/s41587-023-02050-w

Source: https://www.nature.com/articles/s41587-023-02050-w