DddA-derived cytosine base editors (DdCBEs), composed of the split interbacterial toxin DddAtox, transcription activator-like effector (TALE), and uracil glycosylase inhibitor (UGI), enable targeted C-to-T base conversions in mitochondrial DNA (mtDNA). Here, we demonstrate highly efficient mtDNA editing in mouse embryos using custom-designed DdCBEs. We target the mitochondrial gene, MT-ND5(ND5), which encodes a subunit of NADH dehydrogenase that catalyzes NADH dehydration and electron transfer to ubiquinone, to obtain several mtDNA mutations, including m.G12918A associated with human mitochondrial diseases and m.C12336T that incorporates a premature stop codon, creating mitochondrial disease models in mice and demonstrating a potential for the treatment of mitochondrial disorders.
Mitochondrial DNA plays a critical role in cellular respiration via the mitochondrial oxidative phosphorylation (OXPHOS) system. Because the OXPHOS system is essential for survival, mutations in mtDNA cause severe malfunctions in multiple organs and muscles, especially in high-energy demand tissues.
Typically, in humans with a mitochondrial disease, wild-type (WT) and mutant mtDNA with single-base mutations coexist in a cell, resulting in a heteroplasmic state of the mtDNA population.
The balance between mutant and WT mtDNA determines the development of mitochondrial diseases with clinical phenotypes. Programmable nucleases have been used to cleave a mutant mtDNA, but not the WT mtDNA to reduce the mutant mtDNA population in vitro and in vivo,But these nucleases cannot induce or revert a specific mutation in mtDNA, possibly because DNA double-strand breaks are not efficiently repaired in mitochondria by nonhomologous end joining or homologous recombination, unlike those in the nucleus.
Mok et al. recently developed a base editing approach using the bacterial cytidine deaminase toxin, DddAtox, to demonstrate efficient C-to-T base conversions in vitro. In this approach, split DddAtox nontoxic halves fused to transcription activator-like effector (TALE) proteins, which can be custom-designed to recognize predetermined target DNA sequences,form a functional cytosine deaminase within the editing window to induce C-to-T base editing at the target site in mtDNA.
Fig.: Schematic illustration for assembling DdCBE and its mitochondrial DNA editing.
In this study, we investigate whether DdCBEs can achieve mtDNA base editing in vivo to create animal models with mitochondrial mutations and to show germline transmission of the resulting mitochondrial mutations in mice.
Lee, H., Lee, S., Baek, G. et al. Mitochondrial DNA editing in mice with DddA-TALE fusion deaminases. Nat Commun 12, 1190 (2021). https://doi.org/10.1038/s41467-021-21464-1