This is an audio long read from nature.
In this episode, faulty mitochondria cause deadly diseases.
Fixing them is about to get a lot easier.
Written by Gemma Conroy and read by me, Benjamin Thompson.
CRISPR gene editing has made its way into every corner of modern biology, but not into every corner of the cell.
Although researchers have used these systems to develop treatments for sickle cell anemia and blood cancers, to unlock the secrets of multicellularity and to discover the role of thousands of overlooked proteins, there's one place CRISPR can't easily reach – mitochondria.
The rings of DNA inside mitochondria are inaccessible to these techniques, which means that precise edits to mitochondrial DNA – mtDNA remain frustratingly out of reach.
Mitochondria missed the CRISPR-Cas9 revolution that exploded 12 years ago, says Mikhail Minshuk, a geneticist at the University of Cambridge UK.
But researchers are eager to access this DNA, says Minshuk.
Mitochondria are bean-shaped organelles that power cells and have myriad other cellular tasks.
Exploring their DNA is essential for understanding the energy production and exchange that underlies metabolic health.
And more than 300 mutations in this DNA cause mitochondrial diseases.
Incurable genetic disorders with a wide range of symptoms that can rob people of their sight and hearing, trigger muscle problems and spark seizures.
These disorders affect roughly 1 in 5,000 people.
Because CRISPR can't help with these problems, researchers have been looking for other ways to precisely edit the mitochondrial genome.
And the past few years have brought some success.
The tools are already proving to be a boon for creating accurate animal models of mitochondrial diseases.
The progress has been remarkable, says Jinsu Kim, a chemical biologist who develops mtDNA editing tools at the Korea Advanced Institute of Science and Technology in Daejeon, South Korea.
If researchers can make mtDNA editing safe and accurate enough, it could eventually be used to treat and even cure these genetic conditions.
It would be a medical breakthrough, says Kim.
The exact origins of mitochondria are murky, but the leading theory holds that the organelle story started around 15 billion years ago, when a single-celled microorganism called an archaeon gobbled up a roaming bacterium that survived inside its host.
This event marks the beginning of the eukaryotes, the large group of organisms including plants, animals and fungi, in which cells contain organelles that are enclosed inside membranes.
The swallowed bacterium retained its characteristic circular DNA as it settled into its new home, but over time it sacrificed most of its genes to the nuclear genome of its host.
In the evolutionary lineage that gave rise to humans and other animals.
This genetic transfer whittled the resident bacterium's genome down to just 37 genes that code for 13 proteins involved in energy production, turning it into a specialised organelle.
The small amount of mitochondrial DNA that's stuck around in animals differs in key ways from nuclear DNA, which in humans encodes around 20000 genes.
For a start, mtDNA is typically inherited solely from the mother.
There can be several copies of mtDNA in each mitochondrion, and the organelle has its own built-in machinery for making RNA and proteins from that DNA.
Mitochondrial DNA is also much more error-prone, with a mutation rate estimated to be 10 to 20 times greater than that of nuclear DNA.
This is in part because it has to contend with a barrage of damaging reactive oxygen species, unstable molecules that are generated in mitochondria during normal energy production.
But it's also because it doesn't have histones, the proteins that protect and package nuclear DNA.
Compared with its counterpart in the nucleus, mtDNA's toolkit for repairing itself is rudimentary.
The nucleus is quick to fix a snapped DNA strand using an arsenal of repair mechanisms, but mitochondria can only mend some defects.
They often simply throw away their broken DNA.
This difference limits the options for gene editing tools, because nearly all such tools for nuclear DNA use its inherent repair pathways.
It has been notoriously challenging to develop approaches for modifying mitochondrial DNA, says Stephen Ecker, a molecular biologist at the University of Texas at Austin.
Its bacterial origins are revealed when you start trying to edit it, he says.
The most crucial hurdle for scientists trying to tinker with the mitochondrial genome is that it is locked behind a wall of membranes that doesn't allow external nucleic acids to pass into the organelle.
Although there have been hints that CRISPR-based gene editing tools, which rely on RNA to guide them to the correct sequence, might be able to overcome these barriers, many researchers remain unconvinced.
Still, there are other ways in.
More than a decade before CRISPR became a research tool, mitochondria researchers began experimenting with other editing tools that could cross mitochondrial membranes and coax the organelles into ditching their problematic DNA.
Every cell contains a vast number of mitochondrial genomes, because cells contain thousands of mitochondria and each one can carry several copies of mtDNA.
Healthy and mutated mtDNA often coexist, a state known as heteroplasmy.
It's when the proportion of mutated mtDNA reaches 60 to 80 in a particular tissue or cell type that mitochondrial diseases manifest.
If researchers could reduce the faulty copies of mtDNA in cells, they could eliminate the resulting disease.
So they turned to enzymes called zinc finger nucleases.
ZFNs, and transcription activator-like effexor nucleases, or TALONs, to snip the double-stranded empty DNA.
Whereas targeted snipping of nuclear DNA cajoles the cut DNA strands to glue themselves back together without the harmful mutation.
The cut DNA in mitochondria is simply cast out.
This elimination triggers the remaining intact copies to replicate themselves so that the correct level of mtDNA is maintained.
In most cases, the mutated copies will be reduced to an acceptable level as the normal copies are replicated.
That's going to make up for what you're destroying, says Carlos Moraes, a geneticist at the University of Miami in Florida.
Although there has been some progress for this approach, it hasn't made its way out of the laboratory.
And even if it did reach the clinic, the technique would be powerless against diseases caused by mutations that are often present in all copies of a person's mtDNA, such as Leber's hereditary optic neuropathy or LHON, a rare condition that causes rapid vision loss.
What researchers need are tools that do more than cut DNA, but that don't rely on guide RNA.
When CRISPR-Cas9 emerged as a tool in 2012, it became the go-to gene editor for all kinds of application.
A guide.
RNA directs the Cas9 enzyme to a specific DNA sequence.
Where the enzyme does the cutting, genetic changes are introduced as the DNA repairs itself.
The approach became even more useful in 2016 when David Liu, a chemical biologist at the Broad Institute of MIT and Harvard in Cambridge Massachusetts, and his colleagues introduced a more precise technique called base editing.
In this case, researchers modify the Cas9 enzyme and rely on another enzyme called a deaminase to convert one DNA base letter to another, such as cytosine or C to thymine T or adenine A to guanine G.
Although base editing and other CRISPR techniques took off for nuclear DNA, Liu and other research teams couldn't get it working on mtDNA.
Because CRISPR's guide RNA doesn't readily pass through a mitochondrion's double membrane.
Using precise tools on mtDNA remained a pipe dream.
We did not have much success, says Liu.
A solution materialised in 2018, when Joseph Mougeot, a microbiologist then at the University of Washington in Seattle, and his colleagues stumbled across a toxin made by the bacterium Burkholderia sinusopasia.
This enzyme, a deadly weapon against other bacteria, wreaks havoc by ultimately converting base C to T across the bacterial genome.
Mougeot, now based at Yale University in New Haven Connecticut, emailed Liu asking whether the enzyme called DDDA would be of any use to him.
I knew exactly what it might be used for.
Base editing, mtDNA, says Lou.
But switching every C to a T would be lethal to cells.
Lou and his colleagues set out to, quote, tame the beast, end quote.
They split DDDA into two inactive pieces, so that the enzyme would do its handiwork on mtDNA only when the pieces were brought together in a particular orientation.
And instead of using guide RNA Liu and his colleagues, modified proteins found in talons to direct the DDDA segments to their target sequences.
Using their mtDNA base, editor Liu and his team introduced five mitochondrial mutations into human cells in the lab.
The adrenaline rush that came with accomplishing such a technical feat is still vivid for Beverly Mock, a chemical biologist, who was a graduate student on the team and is now at the Agency for Science, Technology and Research, or ASTAR, in Singapore.
That Aha moment when you discover something which was once thought difficult or impossible is truly one of the most rewarding experiences in research, says Mock.
But this mtDNA base editor could only change C to T, leaving the other base letters untouched.
In 2022, Kim and his colleagues created another base editor that changes A to G, a correction that could fix more than 40 of known pathogenic mtDNA mutations.
Kim's tool uses DDDA in conjunction with another engineered enzyme derived from Escherichia coli bacteria and, like the C2T base editor, relies on customized proteins instead of RNA to bind to specific locations on the DNA.
The team is now adapting the A to G base editor to target the mutations underlying mitochondrial diseases, including LHON Melas syndrome, which affects multiple organs, and Lee disease, a severe neurological disorder that causes seizures.
But to be more versatile in the lab and eventually useful in the clinic, researchers will need to develop precise editors that target an even broader set of mutations, says Sabine Fuchs, a paediatrician who specialises in mitochondrial diseases at University Medical Centre Utrecht in the Netherlands.
There's still only a particular subset of mutations we can correct, she says.
Researchers are also focused on preventing the base editors from making changes to DNA outside their target sequences, which will depend, in part, on a better understanding of how these editors orchestrate the switch.
Work is already underway to use these base editors to develop animal models of mitochondrial diseases.
In January, Xiao Shui Zhang, a biologist at Peking University in Beijing, and her colleagues tweaked existing base editors and used them to create two mouse models one of Li disease, in which the mutated animals have a reduced heart rate similar to humans with the disease, and one of LHON, in which the mice are blind.
A few months later, Liang Chen, a biologist at Linggang Laboratory in Shanghai, and his team used an editor to introduce a lead disease mutation into a different gene in rat embryos and developed a separate editor to reverse the mutation in the next generation.
The rats that were born with the mutation developed weak muscles and heart abnormalities.
When the change was corrected, in the offspring of the edited rats more than half of the healthy mtDNA was restored on average and their symptoms lessened.
Researchers are also excited about using the base editors to answer basic questions about mitochondrial biology.
Marais and his team, for example, are editing mtDNA to understand how it expresses long single RNA molecules that encode multiple proteins, which are rarely seen in nuclear DNA.
Nobody understands this process well, says Morais.
Moving mtDNA editing into the clinic faces many of the same hurdles that hinder CRISPR editing.
As well as making mtDNA editors safe and accurate enough in humans, researchers need to work out the best way to deliver them to the appropriate organs and cells, says Fuchs.
Mitochondrial diseases affect multiple systems in the body.
And although organs such as the liver are relatively accessible, the brain, heart and muscle are more difficult to reach.
She says.
These limitations haven't deterred biotechnology start-up firms from developing mtDNA editing tools to treat mitochondrial diseases. founded by Ecker in Rockville, Maryland, is focusing on building customizable base editors for single-letter mutations.
Others, including Precision Biosciences, a gene editing company in Durham, North Carolina, are developing nucleases that modify the degree of heteroplasmy.
Minchuk and Kim have also started biotech companies focused on mitochondrial therapies.
It took more than a decade from the debut of CRISPR to the first government-approved CRISPR therapy in 2023.
Called Cascavy, it treats sickle cell disease and a related blood disorder, beta thalassemia.
Getting mitochondrial DNA editors into the clinic is likely to take at least a decade, researchers say.
But the disease models that are under development – could benefit people who have mitochondrial diseases much sooner.
They're helping researchers to understand the basic biology of the disorders.
That will enable testing of more targeted treatments that don't require gene editing.
Muzhou expects to see many more mtDNA-based editors emerge, which will offer even more ways to tinker with the mitochondrial genomes.
It's been gratifying to see how our initial discovery in this area has grown into a field unto itself, he says.
