A conditional knockout rat resource of mitochondrial protein-coding genes via a DdCBE-induced premature stop codon

Hundreds of pathogenic variants of mitochondrial DNA (mtDNA) have been reported to cause mitochondrial diseases. No curative treatments are currently available due to the shortage of cellular and animal models to understand the etiology of mitochondrial diseases and develop therapeutic strategies. However, in terms of technology, cost and time, it's impossible to install these mutations one by one in cells and animals. Knocking out the 13 mitochondrial protein-coding genes is a viable alternative. Here, we repurposed DdCBE to knock out 12 out of 13 mitochondrial protein-coding genes by incorporating a premature stop codon in cells and rats. In vitro, nearly homoplasmic knockout cells were generated for most of 12 genes, resulting in nearly depleted mtDNA encoded proteins (mtProteins) levels and impaired mitochondrial oxidative phosphorylation. In vivo, to avoid the impairment of embryonic development due to the depletion of mtProteins throughout the body, the Cre/loxP system was employed to realize mtProteins ablation in a spatial-temporal manner. We generated six conditional knockout (cKO) strains and deposited all of them to the Rat Resource Center of China (www.ratresource.com). Nd1 and Atp8 cKO strains were further characterized by crossing with Cre rats. Depletion of ND1 and ATP8 in rat heart or neurons led to heart failure or abnormal brain development, indicating that these cKO rats are proper models for studying mitochondrial diseases. In summary, for the first time, we repurposed DdCBE to deplete the rat mtProteins both in vitro and in vivo, and provided solutions and resources for researchers interested in mitochondrial diseases, paving the way to study the biological function of mtProteins and develop potential treatments for mitochondrial disorders.