Advances in mitochondrial DNA base editing technology.

Mitochondria, the energy factories of higher eukaryotes, provide energy (ATP) for life activities through aerobic respiration. They possess their own genome, mitochondrial DNA (mtDNA), which encodes 37 genes. Mutations in mtDNA cause mitochondrial diseases, and more than 100 pathogenic mutations have been identified in human mtDNA, with a total incidence rate of about 1/5000. In recent years, advances in CRISPR-based base editing technology have enabled accurate editing of nuclear genes. However, it remains a challenge to achieve precise base editing on mtDNA due to the difficulty of guide RNA in the CRISPR system passing through the mitochondrial double-membrane. In 2020, David R. Liu's group at Harvard University reported a double-stranded DNA deaminase DddA from Burkholderia cenocepacia, which was fused with the programmable transcription activator-like effector (TALE) and uracil glycosylase inhibitor (UGI) to develop DddA-derived cytosine base editors (DdCBEs). Using DdCBEs, they were able to achieve specific and efficient C?G to T?A conversion on mtDNA for the first time. In this review, we summarize the recent progress of mitochondrial base editing technology based on DddA and prospect its future application prospects. The information presented may facilitate interested researchers to grasp the principles of mitochondrial base editing, to use relevant base editors in their own studies, or to optimize mitochondrial base editors in the future.线粒体作为真核高等生物的能量工厂，通过有氧呼吸的方式为各项生命活动提供能量(ATP)。线粒体拥有一套独立于细胞核的基因组——线粒体DNA(mitochondrial DNA，mtDNA)，编码37个基因，其突变会导致线粒体疾病，目前已在人mtDNA中鉴定出了超过100种致病突变位点，总发病率约为1/5000。近年来，基于CRISPR的碱基编辑技术已经实现了对核基因组的精确编辑，然而由于CRISPR系统中的引导RNA难以通过线粒体的双层膜结构，在mtDNA上实现精确的碱基编辑仍具有较大的挑战性。2020年，美国哈佛大学David R. Liu实验室报道了一种伯克霍尔德氏菌来源的双链DNA脱氨酶DddA，将其与可编程的转录激活样效应因子(transcription activator-like effector，TALE)和尿嘧啶糖苷酶抑制剂(uracil glycosylase inhibitor，UGI)融合组装成为DdCBEs(DddA来源的胞嘧啶碱基编辑器)，首次在mtDNA上实现了特异高效的C·G到T·A的转换。本文对近几年基于DddA的线粒体碱基编辑技术的发展进行综述，并对其未来应用前景进行展望，以期为相关领域的科研人员进一步了解、使用及优化线粒体碱基编辑技术提供参考。.