Glycosylase-based base editors for efficient T-to-G and C-to-G editing in mammalian cells
Glycosylase-based base editors for efficient T-to-G and C-to-G editing in mammalian cells
复制标题
DOI：
10.1038/s41587-023-02050-w
复制
发表时间：
2024-01
影响因子：
46.9
通讯作者：
Lijun Ye;Dongdong Zhao;Ju Li;Yiran Wang;Bo Li;Yuanzhao Yang;Xueting Hou;Huibin Wang;
中科院分区：
文献类型：
--
作者：
Lijun Ye;Dongdong Zhao;Ju Li;Yiran Wang;Bo Li;Yuanzhao Yang;Xueting Hou;Huibin Wang;
碱基编辑器显示出治疗人类遗传疾病的希望，但目前大多数系统使用脱氨酶，这会导致脱靶效应，并且在编辑类型上受到限制。在这项研究中，我们构建了胞嘧啶（DAF-CBE）和胸腺嘧啶（DAF-TBE）的脱氨酶无碱基编辑器，它们分别只包含胞嘧啶- dna或胸腺嘧啶- dna糖基酶（CDG/TDG）变体，与Cas9酶连接。通过定向进化的多轮诱变，大肠杆菌共聚集了两种具有增强碱基转化活性的变体cdg - ncas9和tdg - ncas9，其C-to-A的效率高达58.7%，T-to-A的效率高达54.3%。DAF-BEs在哺乳动物细胞中实现C-to-G/T-to-G编辑，具有最小的cas9依赖性和非cas9依赖性脱靶效应以及最小的RNA脱靶效应。额外的工程产生了DAF-CBE2/DAF-TBE2，它们从原间隔器的5 '端到中间的编辑窗口发生了变化，C-to-G/T-to-G编辑效率分别提高了3.5倍和1.2倍。与基本编辑器或cgbe相比，DAF-BEs扩展了碱基编辑器的转换类型，具有相似的效率、更小的大小和更低的脱靶效果。
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 inEscherichia coligenerated 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.