Systematic Engineering of Loss-of-Function Alleles in the Zebrafish Mitochondrial Proteome
- 1Department of Pediatrics, Dell Pediatric Research Institute, Dell Medical School, The University of Texas at Austin, Texas, USA
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.
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BibTeX
@misc{sabharwal2026systematic,
title = {{Systematic Engineering of Loss-of-Function Alleles in the Zebrafish Mitochondrial Proteome}},
author = {A. Sabharwal and M. R. Ali and K. S. Mitra and J. Morisue and J. Klein and R. Sarkar and K. Savage and L. R. Thulung and A. Zumbrock and C. Petree and S. R. Castillo and M. Mota and G. K. Varshney and K. J. Clark and E. Nakamuru-Ogiso and S. C. Ekker},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.14.751483},
url = {https://www.biorxiv.org/content/10.64898/2026.09.14.751483v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Systematic Engineering of Loss-of-Function Alleles in the Zebrafish Mitochondrial Proteome
AU - A. Sabharwal
AU - M. R. Ali
AU - K. S. Mitra
AU - J. Morisue
AU - J. Klein
AU - R. Sarkar
AU - K. Savage
AU - L. R. Thulung
AU - A. Zumbrock
AU - C. Petree
AU - S. R. Castillo
AU - M. Mota
AU - G. K. Varshney
AU - K. J. Clark
AU - E. Nakamuru-Ogiso
AU - S. C. Ekker
PY - 2026
DA - 2026/09/14
PB - bioRxiv
DO - 10.64898/2026.09.14.751483
UR - https://www.biorxiv.org/content/10.64898/2026.09.14.751483v1
AB - 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.
ER -