Can Mitochondrial DNA be CRISPRized: Pro and Contra

Abstract

Mitochondria represent a chimera of macromolecules encoded either in the organellar genome, mtDNA, or in the nuclear one. If the pathway of protein targeting to different sub-compartments of mitochondria was relatively well studied, import of small noncoding RNAs into mammalian mitochondria still awaits mechanistic explanations and its functional issues are often not understood thus raising polemics. At the same time, RNA mitochondrial import pathway has an obvious attractiveness as it appears as a unique natural mechanism permitting to address nucleic acids into the organelles. Deciphering the function(s) of imported RNAs inside the mitochondria is extremely complicated due to their relatively low abundance, which suggests their regulatory role. We previously demonstrated that mitochondrial targeting of small noncoding RNAs able to specifically anneal with the mutant mitochondrial DNA led to a decrease of the mtDNA heteroplasmy level by inhibiting mutant mtDNA replication. We then demonstrated that increasing level of expression of such antireplicative recombinant RNAs increases significantly the antireplicative effect. In this report, we present a new data investigating the possibility to establish a CRISPR-Cas9 system targeting mtDNA exploiting of the pathway of RNA import into mitochondria. Mitochondrially addressed Cas9 versions and a set of mitochondrially targeted guide RNAs were tested in vitro and in vivo and their effect on mtDNA copy number was demonstrated. So far, the system appeared as more complicated for use than previously found for nuclear DNA, because only application of a pair of guide RNAs produced the effect of mtDNA depletion. We discuss, in a critical way, these results and put them in a broader context of polemics concerning the possibilities of manipulation of mtDNA in mammalians. The findings described here prove the potential of the RNA import pathway as a tool for studying mtDNA and for future therapy of mitochondrial disorders. © The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 70(12):1233-1239, 2018.

Adapting CRISPR/Cas9 System for Targeting Mitochondrial Genome

“…Our results show an enhanced delivery of gRNA with RP-loop. A recent study has shown that guide RNA with short hairpin structures can promote mitochondrial import and specific cleavage of mtDNA albeit at low level due to limited import into mitochondrial matrix (41,42). This study further strengthens our hypothesis that specific hairpin structures that are involved in the delivery of nuclear encoded RNA can serve as potential adapter for delivery and PNPase may serve as conduit for channeling this recombinant RNA into mitochondrial matrix.…”

DNA‐editing enzymes as potential treatments for heteroplasmic mtDNA diseases

“…Because there is no natural mechanism for the import of RNA into mitochondria, the mechanism involved was puzzling. Extensive discussions on the possibility of having a CRISPR‐like system working in mammalian mitochondria have been discussed [49,50]. Again, the main limitation is the ability to import RNA into mitochondria, which in itself is a controversial topic [50].…”

“…Extensive discussions on the possibility of having a CRISPR‐like system working in mammalian mitochondria have been discussed [49,50]. Again, the main limitation is the ability to import RNA into mitochondria, which in itself is a controversial topic [50]. The use of a CRISPR‐like architecture in mitochondria would be very powerful, particularly because of the recent advances in DNA base editing tools [51].…”

Risk of mitochondrial deletions is affected by the global secondary structure of the human mitochondrial genome

“…The limitation on ubiquitous use of these systems is their two-component nature: RGENs include both protein and RNA parts. Successful delivery of RNA moieties into mitochondria (as a part of RGEN) is very complicated, but there is an experimental proof of its possibility (Jo et al 2015;Loutre et al 2018;Bian et al 2019) . Thus, in the future, there are all the prerequisites that mtDNA editing technology can be implemented, and even after some time, introduced into the clinic as ART, as it has happened with mitochondrial donation technology (Figure 2).…”