Center for Genome Engineering, Institute for Basic Science, Daejeon 34126, Republic of Korea; Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea.
Center for Genome Engineering, Institute for Basic Science, Daejeon 34126, Republic of Korea.
Cell. 2022 May 12;185(10):1764-1776.e12. doi: 10.1016/j.cell.2022.03.039. Epub 2022 Apr 25.
Mitochondrial DNA (mtDNA) editing paves the way for disease modeling of mitochondrial genetic disorders in cell lines and animals and also for the treatment of these diseases in the future. Bacterial cytidine deaminase DddA-derived cytosine base editors (DdCBEs) enabling mtDNA editing, however, are largely limited to C-to-T conversions in the 5'-TC context (e.g., TC-to-TT conversions), suitable for generating merely 1/8 of all possible transition (purine-to-purine and pyrimidine-to-pyrimidine) mutations. Here, we present transcription-activator-like effector (TALE)-linked deaminases (TALEDs), composed of custom-designed TALE DNA-binding arrays, a catalytically impaired, full-length DddA variant or split DddA originated from Burkholderia cenocepacia, and an engineered deoxyadenosine deaminase derived from the E. coli TadA protein, which induce targeted A-to-G editing in human mitochondria. Custom-designed TALEDs were highly efficient in human cells, catalyzing A-to-G conversions at a total of 17 target sites in various mitochondrial genes with editing frequencies of up to 49%.
线粒体 DNA（mtDNA）编辑为在细胞系和动物中对线粒体遗传疾病进行疾病建模以及将来治疗这些疾病铺平了道路。然而，源自细菌胞嘧啶脱氨酶 DddA 的胞嘧啶碱基编辑器（DdCBE）主要局限于 5'-TC 环境中的 C-to-T 转换（例如，TC-to-TT 转换），仅适合产生所有可能的转换（嘌呤到嘌呤和嘧啶到嘧啶）突变的 1/8。在这里，我们提出了转录激活因子样效应物（TALE）连接的脱氨酶（TALED），它由定制设计的 TALE DNA 结合阵列、催化失活的全长 DddA 变体或源自伯克霍尔德菌的全长 DddA 组成源自 Burkholderia cenocepacia 的分裂 DddA，以及源自大肠杆菌 TadA 蛋白的工程化脱氧腺苷脱氨酶，可诱导人线粒体中的靶向 A-to-G 编辑。定制设计的 TALED 在人类细胞中非常有效，可在各种线粒体基因的总共 17 个靶标位点催化 A-to-G 转换，编辑频率高达 49%。
