State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.
College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China.
Nat Biotechnol. 2024 Jun;42(6):936-945. doi: 10.1038/s41587-023-01910-9. Epub 2023 Aug 28.
Transcription-activator-like effector (TALE)-based tools for base editing of nuclear and organellar DNA rely on double-stranded DNA deaminases, which edit substrate bases on both strands of DNA, reducing editing precision. Here, we present CyDENT base editing, a CRISPR-free, strand-selective, modular base editor. CyDENT comprises a pair of TALEs fused with a FokI nickase, a single-strand-specific cytidine deaminase and an exonuclease to generate a single-stranded DNA substrate for deamination. We demonstrate effective base editing in nuclear, mitochondrial and chloroplast genomes. At certain mitochondrial sites, we show editing efficiencies of 14% and strand specificity of 95%. Furthermore, by exchanging the CyDENT deaminase with one that prefers editing GC motifs, we demonstrate up to 20% mitochondrial base editing at sites that are otherwise inaccessible to editing by other methods. The modular nature of CyDENT enables a suite of bespoke base editors for various applications.
基于转录激活因子样效应物（TALE）的核和细胞器 DNA 碱基编辑工具依赖于双链 DNA 脱氨酶，该酶在 DNA 的两条链上编辑底物碱基，从而降低编辑精度。在这里，我们提出了 CyDENT 碱基编辑，这是一种无 CRISPR、链选择性、模块化的碱基编辑器。CyDENT 由一对 TALEs 与 FokI 切口酶、单链特异性胞嘧啶脱氨酶和核酸外切酶融合而成，可生成用于脱氨的单链 DNA 底物。我们在核、线粒体和叶绿体基因组中证明了有效的碱基编辑。在某些线粒体位点，我们展示了 14%的编辑效率和 95%的链特异性。此外，通过用偏好编辑 GC 基序的 CyDENT 脱氨酶替换，我们在其他方法无法编辑的位点上展示了高达 20%的线粒体碱基编辑。CyDENT 的模块化性质使其能够为各种应用提供一系列定制的碱基编辑器。
