A conditional knockout rat resource of mitochondrial protein-coding genes via a DdCBE-induced premature stop codon
Lei Tan, Xiaolong Qi, Weining Kong, Jiachuan Jin, Dan Lü, Xu Zhang, Yue Wang, Siting Wang, Wei Dong, Xudong Shi, Wei Chen, Jianying Wang, Keru Li, Yuan Xie, Lijuan Gao, Feifei Guan, Kai Gao, Chaojun Li, Cheng Wang, Zhibin Hu, Lianfeng Zhang, Xuejiang Guo, Bin Shen, Yuanwu Ma
Abstract
Hundreds of pathogenic variants of mitochondrial DNA (mtDNA) have been reported to cause mitochondrial diseases, which still lack effective treatments. It is a huge challenge to install these mutations one by one. We repurposed the DddA-derived cytosine base editor to incorporate a premature stop codon in the mtProtein-coding genes to ablate mitochondrial proteins encoded in the mtDNA (mtProteins) instead of installing pathogenic variants and generated a library of both cell and rat resources with mtProtein depletion. In vitro, we depleted 12 of 13 mtProtein-coding genes with high efficiency and specificity, resulting in decreased mtProtein levels and impaired oxidative phosphorylation. Moreover, we generated six conditional knockout rat strains to ablate mtProteins using Cre/loxP system . Mitochondrially encoded ATP synthase membrane subunit 8 and NADH:ubiquinone oxidoreductase core subunit 1 were specifically depleted in heart cells or neurons, resulting in heart failure or abnormal brain development. Our work provides cell and rat resources for studying the function of mtProtein-coding genes and therapeutic strategies.