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

Abstract

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.

Keywords: CRISPR/Cas9; RNA import; mitoTALEN; mitochondria; mtDNA; mtZFN.

Figure 1

Overview of Putative RNA Import into Mitochondria. (A) An overview of historically proposed mechanisms and functional roles of endogenous RNAs imported into mammalian mitochondria. Nucleus-encoded RNA is suggested to enter the mitochondrial matrix in complex with polynucleotide phosphorylase (PNPase), via the mitochondrial protein translocase of outer membrane (TOM) and translocase of inner membrane (TIM), as well as by other undescribed and undefined mechanisms of transport. Endogenous RNA species with previously proposed functional roles in mammalian mitochondria are H1 RNA (RNase P), 7-2 RNA (RNase MRP), and 5S rRNA (mt-LSU). (B) A revised overview of the proposed mechanisms and functional roles of endogenous RNAs imported into mammalian mitochondria, modified to reflect findings from recent papers concerning (i) the function of PNPase as a key constituent of the mtRNA degradasome , , (ii) replacement of 5S rRNA in mt-LSU of the mitoribosome by mitochondrial tRNA , , (iii) discovery of protein-only RNase P (PRORP) in mammalian mitochondria , and (iv) reattribution of RNase MRP activity to the nucleolus, similarly to nuclear RNase P , . Many of the RNAs ‘detected’ and ascribed mitochondrial functions are predicted to be false positives as a result of contamination of mitochondrial preparations with cytosolic RNAs or with RNAs associated with the outer membrane, such as mRNAs encoding mitochondrial proteins that undergo co-translational import into mitochondria , , . A question mark indicates unconfirmed function and/or localization of PNPase. Abbreviations: mt-LSU, mitoribosome large subunit; mtRNA, mitochondrial RNA; mt-SSU, mitoribosome small subunit.