美国斯隆凯特琳学院Agnel Sfeir研究团队揭示了通过重建人类线粒体末端连接来工程mtDNA缺失。该项研究成果发表在2025年3月10日出版的《细胞》上。
课题组研究人员通过共表达末端连接（end-joining， EJ）机制和靶向内切酶，在人类细胞中设计了mtDNA缺失。利用线粒体EJ （mito-EJ）和mito-ScaI，该课题组研究人员在全谱异质性中产生了一组含有~3.5kb mtDNA缺失的克隆细胞系。对这些细胞的研究揭示了一个约75%基因组缺失的临界阈值，超过这个阈值，在含半乳糖的培养基中会观察到氧化磷酸化（OXPHOS）蛋白耗损、代谢破坏和生长受损。单细胞多组学分析确定了两种不同的核基因解除管制反应：一种在缺失阈值触发，另一种逐渐响应异质性。最终，该课题组证明了他们的方法能够建立跨细胞类型的疾病相关mtDNA缺失的模型，并可以为靶向治疗的开发提供信息。
研究人员表示，最近在线粒体DNA （mtDNA）基因操作方面的突破使得精确的碱基替换和有效地消除携带致病突变的基因组成为可能。然而，重建与线粒体肌病相关的mtDNA缺失仍然具有挑战性。
附：英文原文
Title: Engineering mtDNA deletions by reconstituting end joining in human mitochondria
Author: Yi Fu, Max Land, Tamar Kavlashvili, Ruobing Cui, Minsoo Kim, Emily DeBitetto, Toby Lieber, Keun Woo Ryu, Elim Choi, Ignas Masilionis, Rahul Saha, Meril Takizawa, Daphne Baker, Marco Tigano, Caleb A. Lareau, Ed Reznik, Roshan Sharma, Ronan Chaligne, Craig B. Thompson, Dana Pe’er, Agnel Sfeir
Issue&Volume: 2025-03-10
Abstract: Recent breakthroughs in the genetic manipulation of mitochondrial DNA (mtDNA) have enabled precise base substitutions and the efficient elimination of genomes carrying pathogenic mutations. However, reconstituting mtDNA deletions linked to mitochondrial myopathies remains challenging. Here, we engineered mtDNA deletions in human cells by co-expressing end-joining (EJ) machinery and targeted endonucleases. Using mitochondrial EJ (mito-EJ) and mito-ScaI, we generated a panel of clonal cell lines harboring a ～3.5 kb mtDNA deletion across the full spectrum of heteroplasmy. Investigating these cells revealed a critical threshold of ～75% deleted genomes, beyond which oxidative phosphorylation (OXPHOS) protein depletion, metabolic disruption, and impaired growth in galactose-containing media were observed. Single-cell multiomic profiling identified two distinct nuclear gene deregulation responses: one triggered at the deletion threshold and another progressively responding to heteroplasmy. Ultimately, we show that our method enables the modeling of disease-associated mtDNA deletions across cell types and could inform the development of targeted therapies.
DOI: 10.1016/j.cell.2025.02.009
Source: https://www.cell.com/cell/abstract/S0092-8674(25)00194-1