Researchers have developed what can be described as the first precision gene editing tool for mitochondrial DNA (mtDNA). This new tool is different from the well-known CRISPR/Cas9 method of gene editing and should make the study of mitochondrial biology and mitochondrial diseases easier.
This new discovery is a game changer
Prior to this, creating mouse models of mitochondrial disease for study and drug development has been very challenging; this transformative technology has the potential to make creating such models far easier.
The related study, which was published in the journal Nature, showcases the new mitochondrial gene editing tool [1]. Mutations within mtDNA can cause a variety of unusual and poorly understood conditions, and while CRISPR/Cas9 and related gene editing technologies have allowed researchers to edit DNA faster and easier than before, they only work on the DNA stored in the cell nucleus, the home of the majority of our genetic information.
CRISPR/Cas9 gene editing technologies cannot be used to edit mtDNA, as they cannot access it properly. CRISPR/Cas9 uses a small guide RNA to home the Cas9 enzyme in on a specific target spot on the genome, where it can then cut both strands of DNA. However, no one has figured out how to move the guide RNA into the mitochondria, meaning that it is not possible to edit mtDNA in the same way.
This new discovery came when the researchers were studying how bacteria fight each other using toxins, specifically a group of bacterial toxins known as deaminases, which can damage DNA and RNA bases by removing nitrogen-containing parts from them. Normally, these deaminases target single strands of DNA or RNA; however, they found that a particular cytidine deaminase enzyme, DddA, had no effect on single-stranded DNA or RNA.
After much testing with no success, they discovered that DddA did not work on single-strand DNA or RNA but in fact did work on double-strand DNA. It was assumed that deaminases only worked on single-strand DNA or RNA and that was their purpose in this context, so it was therefore a real surprise to find that DddA bucked this trend.
Taming the beast
The researchers realized that this unusual quirk might be useful for gene editing, in particular for editing mtDNA, as it could bypass the problem of getting the RNA guide inside the mitochondria.
However, it was not as simple as just using DddA directly, due to it being a naturally occurring bacterial toxin; if left to run amok, it could destroy DNA everywhere it encountered it. In order to use DddA to edit mtDNA, the researchers had to find a way to stop DddA from changing mtDNA until the desired target location was reached.
To do so, they split the protein into two inactive pieces; they used 3D imaging data to work out how to cut the protein into two parts in such a way that, when divided, they were harmless, but the rejoined protein was able to resume its original function.
The team joined each part of the DddA protein to customizable DNA-targeting proteins that do not require guide RNA to locate their target. These customizable DNA-targeting proteins are designed to bind to specific regions of DNA, which then brings the two parts of the DddA protein together again. This allowed DddA to work as a precision gene editing tool, preventing it from going on the rampage and destroying all nearby DNA.
They tested the approach to make changes to specific mitochondrial genes, which resulted in fully functional mitochondria, with the exception of the gene that they had edited to cause a defect.
The next step for the researchers will be to search for other bacterial deaminases that could potentially be developed into gene editors. The team hopes that such tools could allow for more effective modeling of mitochondrial diseases as well as assist the development of therapies to address them.
Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques. Because previously described cytidine deaminases operate on single-stranded nucleic acids, their use in base editing requires the unwinding of double-stranded DNA (dsDNA)—for example by a CRISPR–Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria. As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases. Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.
Conclusion
The arrival of a new tool that overcomes the limitations of CRISPR/Cas9 in the context of mitochondrial gene editing is a very welcome development indeed. Mitochondrial dysfunction is one of the hallmarks of aging, so having tools that could repair damaged mtDNA as well as address mitochondrial diseases has a huge amount of potential to be truly transformative, just as the discovery of CRISPR/Cas9 has been.
Literature
[1] Mok, B. Y., de Moraes, M. H., Zeng, J., Bosch, D. E., Kotrys, A. V., Raguram, A., … & Mougous, J. D. (2020). A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature, 1-7.
