Fight Aging!

Here I'll point out a technology demonstration of mitochondrial gene editing via CRISPR, something that should be of general interest, though debatable relevance to work on mitochondrial repair at the present time. The development of CRISPR, an efficient low-cost method of genetic editing, has opened a lot of doors. In the course of a few short years since the first practical demonstration, use of CRISPR has made genetic engineering projects accessible and affordable to a vastly greater number of researchers than was previously the case. As an infrastructure advance it is about as transformative as the development of induced pluripotency was for the stem cell research community. Cost and difficulty are very important determinants of the pace of progress in a field, and sharp reductions in both of those for genetic engineering suggests that the next decade is going to be very interesting indeed.

The use of transcription activator-like effector nucleases (TALENs) is one of the candidate next generation genetic engineering technologies that was developed prior to CRISPR, though work continues even now. It is promising, but clearly not starting fires to the same degree that CRISPR is: again, it is all about relative degrees of cost and difficulty. Still, you may recall that it was quite exciting to see TALENs working for mitochondrial DNA back when that was first demonstrated.

Mitchondrial DNA (mtDNA) is distinct from nuclear DNA. It is a circular genome made up of a few leftover genes that is resident in each of the hundreds of mitochondria present in every cell. Mitochondria are the evolved descendants of symbiotic bacteria, and their primary - but far from only - activity is to act as power plants, generating chemical energy store molecules that are used to power cellular activities. They still behave much like bacteria: fusing, dividing, passing molecules and even large portions of their internal structures back and forth between one another. Other processes within a cell monitor the state of mitochondria, and flag damaged ones for destruction, recycling their component parts. Somewhere in all of these interacting processes of generation and destruction, there are ways in which mitochondrial DNA can be come damaged, losing the blueprints for vital protein machinery used in some modes of energy store generation. These damaged mitochondria are in some way privileged, more able to evade destruction at the hands of quality control mechanisms despite their dysfunction. They quickly overtake the entire mitochondrial population of a cell - so quickly that researchers don't have a good view of how exactly the process happens; they only see before and after snapshots. That cell then becomes harmful and dysfunctional, exporting damaged proteins and reactive molecules into the surrounding tissue. The accumulation of such cells over time is one of the contributing causes of degenerative aging.

So as you can see, the ability to edit mitochondrial DNA to fix it is of potential interest. But what can be done here? Can the existence of these dysfunctional cells be fixed for a long enough period of time via a global gene therapy of some sort that directly delivers replacement genes to mitochondria? Or will the continued presence of broken variants just quickly overwhelm any freshly delivered working variants? After all, the damaged variants already achieved that goal in the cells they have taken over, and they are still there in large numbers. There has been sufficient doubt on that front for the research groups involved in efforts to repair damaged mitochondria to adopt other, less direct approaches. These include allotopic expression, in which copies of mitochondrial genes are placed into the cell nucleus, altered in ways that ensure the proteins produced can find their way back to the mitochondria where they are needed. Development of that approach for inherited mitochondrial diseases is at a fairly advanced stage, but it has yet to be applied to aging. With a large fall in the cost and difficulty of mitochondrial gene editing, it may be worthwhile revisiting this picture, however. I'm sure some researchers will do just that in the years ahead.

Efficient Mitochondrial Genome Editing by CRISPR/Cas9

Mitochondria play roles in many important cellular functions. Mitochondria contain their own genome, which encodes 13 proteins that are subunits of respiratory chain complexes, as well as two rRNAs and 22 mitochondrial tRNAs. Due to the critical roles of genes encoded by mtDNA, maintenance of mitochondrial genome integrity is quite important for normal cellular functions. Mitochondrial DNA are, however, constantly under mutational pressure due to oxidative stress imposed by radicals generated by oxidative phosphorylation or an imbalance in the antioxidant defense system in aging or disease processes. Damage to mtDNA, such as point mutations or deletions, contributes to or predisposes individuals to a variety of human diseases.

Despite the huge potential of mitoTALEN-mediated mtDNA editing, more user-friendly and efficient alternative methods are necessary to overcome difficulties in mtDNA modification either for correction of dysfunctional mtDNA or for producing dysfunctional mtDNA in order to create mitochondria-associated disease models.

Here we report a novel approach to generate mtDNA dysfunction with the CRISPR/Cas9 system. Cas9, widely used for genome editing, showed distribution to mitochondria as well as the nucleus. Expression of FLAG-Cas9 with gRNAs designed to target mtDNA resulted in cleavage of mtDNA and alterations in mitochondrial integrity as determined by Western blots for some mitochondrial proteins. Moreover, regular FLAG-Cas9 was modified to contain mitochondrial targeting sequence instead of nuclear localization sequence (NLS) in order to localize it to mitochondria (namely, mitoCas9). MitoCas9 robustly localized to mitochondria; together with gRNA targeting of mtDNA, specific cleavage of mtDNA was observed, demonstrating its functional application for mtDNA editing.

These results together demonstrate the successful application of CRISPR/Cas9 in mitochondrial genome editing and suggest the possibility for in vitro and in vivo manipulation of mtDNA in a site-specific manner.