In a paper published in Cell, a research group led by Agnel Sfeir at Memorial Sloan Kettering Cancer Center showed that gene editing can be used to engineer specific mtDNA mutations.

Mitochondria are unusual cell organelles in that they contain their own mitochondrial (mt)DNA, and like other DNA, mtDNA can develop mutations that result in diseases. Some mutations involve the deletion of parts of the coding sequences. Fortunately, cells typically have more than 1,000 mitochondria, and in many cases, a minimum number of healthy mitochondria are sufficient to avoid disease. Nevertheless, mitochondrial diseases can be highly debilitating.

Advances in genome engineering and editing (e.g., TALENS, CRISPR, zinc finger nucleases) have raised the possibility of reconstituting point mutations repairing defective mtDNA to curing the diseases. However, mtDNA deletions in human cells have been challenging. Recently, a research team at Memorial Sloan Kettering Cancer Center, led by Agnel Sfeir, succeeded in engineering mtDNA deletions with an innovative approach by expressing bacterial DNA end joining enzymes in human cells.

The Sfeir team developed a group of human cell lines with deletions to the mtDNA of varying extents (Fu et al., 2025). Cells with deletions in more than 75% of the mitochondria had affected mitochondrial function. With these cells, the team found two patterns of effects on nuclear genes. One was activated at the deletion threshold. The other was in response to increasing levels of mutations.

More importantly, this combination of tools facilitates modeling of disease-associated mtDNA deletions in different cell types. In addition, the panel of cell lines with the different levels of deletions will greatly benefit the research community and help in developing therapeutics.

A Discussion with Agnel Sfeir

What are the next steps in your research?

Our research focuses on elucidating the molecular pathways that govern the propagation versus elimination of mtDNA deletions. Understanding these mechanisms is crucial for identifying factors influencing mtDNA integrity and stability.

Additionally, we aim to investigate how cells, particularly neurons and muscle cells, respond to mtDNA deletions. These cell types are highly dependent on mitochondrial function and are disproportionately affected by mtDNA mutations, leading to severe pathologies. By studying their adaptive or maladaptive responses, we hope to uncover potential therapeutic targets that could mitigate the impact of mtDNA deletions in mitochondrial diseases.

You note that you can use this system to examine stem cells. It sounds like that is a great way to look at looks of cell types, especially muscle and brain. Maybe even iPS cells could be used.

Yes, that’s exactly what we are in the process of doing. IPS cells provide a powerful strategy for studying a wide range of cell types, including muscle and neuronal cells, which are particularly affected by mitochondrial dysfunction. By differentiating iPS cells into specific lineages, we can systematically investigate how mtDNA deletions impact various cell types and really model disease-relevant phenotypes. This approach also allows for high-throughput screening of potential therapeutic interventions in a controlled and physiologically relevant system.

The ability to switch the carbon source between galactose and glucose is a real advantage to your system.

Absolutely. The ability to switch between glucose and galactose as carbon sources is a key advantage of our system. Heteroplasmy refers to the proportion of mutated mtDNA within a cell, and when it surpasses 75%, cells typically exhibit severe respiratory defects and abnormal morphology. However, the RPE1 cells we used primarily rely on glycolysis, allowing them to proliferate normally in glucose-containing media—even with heteroplasmy levels exceeding 75%. The defect became evident only when glucose was replaced with galactose, forcing the cells to rely on oxidative phosphorylation. This metabolic shift was crucial in unmasking the mtDNA deletion-associated defects in galactose-containing media, whereas culturing cells in glucose allowed us to obtain cells with ~100% mtDNA deletion. In fact, to date, no cellular models of mtDNA deletions have been able to achieve near-homoplasmy.

You discuss the application of your method for the study of disease, but I assume it might also be used to explore mitochondrial regulation as well.

While our method is highly applicable to studying disease, it also provides a powerful tool for exploring mitochondrial regulation. By analyzing the dynamic behavior of intact and deleted mtDNA, we can gain valuable insights into key mitochondrial quality control mechanisms, such as mitophagy and selective replication. Specifically, our cellular system allows us to examine how cells manage mutant mtDNA through preferential degradation of dysfunctional mitochondria via mitophagy, or the selective replication of intact mitochondrial genomes. This, in turn, enables us to understand the mechanisms that maintain mitochondrial homeostasis under normal conditions and how they are altered in the presence of mtDNA deletions.

Moreover, our approach provides a window into the biochemical and metabolic adaptations that occur in response to mitochondrial stress. By assessing how cells adjust their energy metabolism, stress signaling pathways, and mitochondrial dynamics, we can uncover the requirements necessary for cellular survival and adaptation. This is particularly crucial for understanding the threshold at which mitochondrial dysfunction becomes pathological and identifying potential targets for therapeutic intervention.

Your work and that of others attempts to correct mutations in mtDNA. Are there any genetic diseases of mitochondria that involve genes in the host genome that are specific to mitochondria?

Nuclear-encoded mitochondrial genes play a crucial role in mitochondrial function, and their mutations can lead to severe disorders. Several mitochondrial diseases arise from mutations in nuclear-encoded genes that regulate mitochondrial DNA replication and transcription. For example, POLG mutations impair mtDNA replication, leading to disorders such as Alpers syndrome, and defects in TFAM reduce mtDNA copy number and transcription.

How did you get interested in mitochondria?

I am a chromosome biologist with a deep fascination for how cells repair DNA breaks to maintain genome integrity. During my training and early career, I focused on nuclear DNA repair pathways and how their deregulation contributes to cancer progression. However, after starting my lab, I felt the need for a new intellectual challenge—an opportunity to step outside my comfort zone and explore an entirely different aspect of biology (something I believe all scientists should do at least once in their careers).

mtDNA had always intrigued me, and I saw a unique opportunity to apply my expertise in nuclear DNA repair to studying this independent genome within a distinct organelle. To immerse myself in the field, I dedicated two months solely to reading the mtDNA literature, identifying key questions, outlining a research plan, and recruiting a student and a post-doc to pursue these challenges. Despite its unique regulatory mechanisms, mtDNA still adheres to fundamental principles of genome maintenance, making it an exciting system to study. Today, half of my lab is dedicated to studying mtDNA biology, and with every discovery we make our curiosity deepens. The same curiosity that led me to work on mtDNA in the first place continues to drive our research forward.

 

Reference

Fu Y, Land M, Cui R, Kavlashvili T, Kim M, Lieber T, Ryu KW, DeBitetto E, Masilionis I, Saha R, Takizawa M,  Baker D, Tigano M, Lareau CA, Reznik E, Sharma R, Chaligne R, Thompson CB, Pe’er D, Sfeir A (2025) Engineering mtDNA deletions by reconstituting end-joining in human mitochondria. Cell

https://www.sciencedirect.com/science/article/pii/S0092867425001941?dgcid=author

Mitochondrial mutationsFu et al. (2025) used nucleases and an end-joining procedure to produce mutations in mitochondrial (mt) DNA. The mitochondria were then introduced into cells, and cell lines were produced with different numbers of mutated mitochondria. The resulting cell lines provide excellent models for testing the effects of the mutated mtDNA in diseases.