A Demonstration of Mitochondrial DNA Editing with CRISPR

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.

Comments

If the damaged mitochondria quickly take over a cell when present, why has it been so hard for the SENSRF researchers to obtain cell lines that are null for a particular mitochondrial gene? I'm probably missing some detail that has been glossed over to some degree.

That being said, hopefully CRISPR will make it cheaper and easier for the SENSRF to obtain cell lines with all 13 mtDNA genes deleted.

Posted by: Jim at October 14th, 2015 1:06 AM

It looks like someone will be able to do a "Bioviva" with this therapy in the next 5-10 years.

Posted by: Abelard Lindsey at October 14th, 2015 11:45 AM

Hi Jim,

The thing is that no one AFAIK has ever identified a naturally-ocurring single-gene null mutation in the mitochondrial DNA in man or mouse. In nearly all of the human congenital mitochondriopathies originating in actual mitochondrial mutations, the underlying mutations are point mutations in the gene that cause either severe loss-of-function or toxic gain-of-function rather than a totally non-functional protein. Dr. O'Connor and SENS Research Foundation's mitochondrial mutation team struggled for many, many months trying to implement their backup-gene rescue of a mitochondrial mutation in a patient cell line that researchers thought was null for ATP8 (a protein in the electron transport chain (ETC)). It turned out that this cell line not only still had a few wild-type genomes that they had to weed out, but that the mutation that knocked out its ATP8 gene that also affects the transcription of ATP6, with which it shares an overlapping sequence within the circular mitochondrial genome.

It was only after first purifying the cell line using selection to be truly all-null, and then doing some things to compensate for the effects on ATP6, that our team was able to make rapid progress and eventually achieve a great success, which I hope will be announced along with a prominent journal publication in the coming months.

The mutant mitochondria that clonally expand and take over a small fraction of aging cells aren't single-gene null mutations either, and in their case the problem is even worse. As you'll probably recall, they are large deletions, which don't just neatly take out single genes, but tear huge swaths out of the mitochondrial genome. Moreover, such deletion mutations almost (?) always take out the genes encoding mitochondrial tRNAs, without which none of the mitochondrial genes encoding ETC proteins can be translated and turned into proteins. It is mitochondrial deletion mutations that most clearly accumulate in aging, and that are most implicated in age-related disease, debility, and systemic metabolic derangements and therefore the ones of highest biomedical priority.

Fortunately, we don't have to repair or replace the entire deleted segment of the genome: deletion mutations can be obviated by providing "backup copies" of each of the individual mitochondrial genes that encode proteins in the nucleus. (Nuclear "backup copies" will be translated using nuclear tRNAs, so we don't have to do anything to make up for the lack of tRNAs in the mitochondria).

Thus, if CRISPR really can be used to make significant modifications in mitochondrial genes, the ability to use this technology to make null mutations in mitochondrial protein-encoding genes — first in cell lines, and starting with those that encode individual mitochondrially-encoded proteins, and then proceeding to dual and multiple mutations in cells and in mice — it will contribute greatly to our ability to test and translate allotopic expression. Keeping our cellular "power plants" running cleanly with this rejuvenation biotechnology will be a key part of opening up a future free of Parkinson's disease, sarcopenia, and the less-specific frailty and metabolic derangement that plagues us as we age today.

Posted by: Michael at October 14th, 2015 12:45 PM

No comment editing here, let's try that again

Posted by: Slicer at October 14th, 2015 1:38 PM

Speaking of which, this guy (I think it's him; common name) just put down another $5k.

Posted by: Slicer at October 15th, 2015 12:01 PM

Quote:
Abelard Lindsey
"
Keeping our cellular "power plants" running cleanly with this rejuvenation biotechnology will be a key part of opening up a future free of Parkinson's disease, sarcopenia, and the less-specific frailty and metabolic derangement that plagues us as we age today.
"
Quote:
Napoleon
"
Miserable people do miserable things to extend their do
"
Result:
Interfering with multi-compartment evolution of intracellular prokaryotes is the end of the current humanity.

Posted by: ab muzzy at January 7th, 2016 12:36 AM

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