Mitochondrial Genome Engineering: The Revolution May Not Be CRISPR-Ized

In recent years mitochondrial DNA (mtDNA) has transitioned to greater prominence across diverse areas of biology and medicine. The recognition of mitochondria as a major biochemical hub, contributions of mitochondrial dysfunction to various diseases, and several high-profile attempts to prevent hereditary mtDNA disease through mitochondrial replacement therapy have roused interest in the organellar genome. Subsequently, attempts to manipulate mtDNA have been galvanized, although with few robust advances and much controversy. Re-engineered protein-only nucleases such as mtZFN and mitoTALEN function effectively in mammalian mitochondria, although efficient delivery of nucleic acids into the organelle remains elusive. Such an achievement, in concert with a mitochondria-adapted CRISPR/Cas9 platform, could prompt a revolution in mitochondrial genome engineering and biological understanding. However, the existence of an endogenous mechanism for nucleic acid import into mammalian mitochondria, a prerequisite for mitochondrial CRISPR/Cas9 gene editing, remains controversial. Engineering of mammalian mtDNA has been hampered by an inability to import nucleic acids into mitochondria. A limited toolkit exists for manipulation of mammalian mtDNA, relying on protein-only nucleolysis and heteroplasmy-shifting approaches. Although present in lower metazoans, the weight of evidence against an efficient endogenous RNA import mechanism in mammalian mitochondria is considerable. Controversially, the application of CRISPR/Cas9 for manipulation of mammalian mtDNA in human cells has been reported.