As a platform capable of mtDNA heteroplasmy manipulation, mitochondrially targeted zinc finger-nuclease (mtZFN) technology holds significant potential for the future of mitochondrial genome engineering, in both laboratory and clinic. Recent work highlights the importance of finely-controlled mtZFN levels in mitochondria, permitting far greater mtDNA heteroplasmy modification efficiencies than observed in early applications. An initial approach, differential fluorescence activated cell sorting (dFACS), allowing selection of transfected cells expressing various levels of mtZFN, demonstrated improved heteroplasmy modification. A further, key optimisation has been the use of an engineered hammerhead ribozyme as means for dynamic regulation of mtZFN expression. Protocols detailing these transformative optimisations are described in this chapter.