Functional analyses of mitochondria have been hampered by few effective approaches to manipulate mtDNA and a lack of existing animal models. Recently TALE-derived base editors are shown to induce T-to-C  and G-to-A sequence changes in mtDNA. We report here the mitochondrial FusX TALE Base Editor (FusXTBE) to facilitate broad-based access to TALE mitochondrial base editing technology. TALE Writer is a de novo in silico design tool to map potential mtDNA base editing sites. Zebrafish embryos were used as a pioneering in vivo test system, with FusXTBE inducing 90+% editing efficiency in mtDNA loci for both protein-coding and tRNA genes as examples of near-complete induction of mtDNA heteroplasmy in vivo. Gene editing specificity as precise as a single nucleotide was observed for a protein-coding gene. Non-destructive genotyping enables single animal mtDNA analyses for downstream biological functional genomics applications. For example, introduction of a premature stop codon in the mt-co3 gene significantly downregulated the activity of complex IV of the mitochondrial respiratory chain in addition to elevation of lactate indicting towards mitochondrial dysfunction in the edited larvae. We have expanded this system to human work in vitro including primary cells, showing near-complete mtDNA editing using this advanced MitoFusXTBE system. Together, this work supports the potential development of the MitoFusXTBE for exploring important questions in mitochondrial biology and genetics, demonstrates the zebrafish as a new animal model system for mtDNA disorders, and opens the door for therapeutic development.