Systematic Engineering of Loss-of-Function Alleles in the Zebrafish Mitochondrial Proteome

Authors

Sabharwal, A., Ali, M. R., Mitra, K. S., Morisue, J., Klein, J., Sarkar, R., Savage, K., Thulung, L. R., Zumbrock, A., Petree, C., Castillo, S. R., Mota, M., Varshney, G. K., Clark, K. J., Nakamuru-Ogiso, E., Ekker, S. C.

Abstract

Pathogenic variants in the 13 protein-coding genes of the mitochondrial genome underlie clinically and biochemically heterogeneous disorders. Most mtDNA-encoded genes lack defined loss-of-function (LOF) models in vivo. To address this gap, we have generated Z-Terminator, a systematic in vivo atlas of loss-of-function alleles covering all the mtDNA-encoded OXPHOS subunits in zebrafish (Danio rerio). We used mitochondrial TALE base editors to introduce premature termination codon (PTC) alleles via C-to-T transitions across Complexes I, III, IV, and V. Larvae harboring PTC alleles displayed bioenergetic defects and elevated lactate. While mtDNA mutations are associated with sensorineural hearing loss, the cellular basis has remained unclear. We show that engineered mtDNA LOF alleles directly impair hair cell function in proportion to heteroplasmy in a living vertebrate. We investigated the germline transmission and tissue-specific heteroplasmy of these LOF alleles and observed that a subset of variants was transmitted to the F1 generation and displayed distinct mutant loads across organs. These findings establish Z-Terminator as a vertebrate platform for interrogating the role of mtDNA protein-coding genes in cellular dysfunction and for elucidating the pathophysiology of mitochondrial disorders.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 18 Sep 2026.

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