The accumulation of mutant mitochondrial DNA (mtDNA) often leads to disease. Here, Bacman et al. engineered TALENs (transcription activator-like effector nucleases) so that they localize to mitochondria and cleave known pathogenic mtDNA mutations. The efficacy of mitochondrion-targeted TALENs (mitoTALENs) was tested by designing them to cleave mutant mtDNA carrying a large common deletion that is found in ageing tissues and in 30% of patients with mtDNA deletions. As TALENs function as dimers to bind and cleave specific DNA target sequences, each mitoTALEN monomer was designed to bind to a wild-type sequence flanking this deletion. As such, the monomers were only close enough to dimerize on and cleave mtDNA carrying this deletion. mitoTALEN expression in patient-derived cells was shown to permanently reduce the levels of pathogenic mtDNAs. Although the safety of mitoTALENs remains to be tested, this technique could potentially be used to treat mitochondrial diseases.