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[Preprint]. 2024 Oct 17:2024.10.15.618543.
doi: 10.1101/2024.10.15.618543.

Engineering mtDNA Deletions by Reconstituting End-Joining in Human Mitochondria

Yi Fu, Max Land, Ruobing Cui, Tamar Kavlashvili, Minsoo Kim, Toby Lieber, Keun Woo Ryu, Emily DeBitetto, Ignas Masilionis, Rahul Saha, Meril Takizawa, Daphne Baker, Marco Tigano, Ed Reznik, Roshan Sharma, Ronan Chaligne, Craig B Thompson, Dana Pe'er, Agnel Sfeir

Engineering mtDNA Deletions by Reconstituting End-Joining in Human Mitochondria

Yi Fu et al. bioRxiv. .

Update in

  • Engineering mtDNA deletions by reconstituting end joining in human mitochondria.
    Fu Y, Land M, Kavlashvili T, Cui R, Kim M, DeBitetto E, Lieber T, Ryu KW, Choi 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. Fu Y, et al. Cell. 2025 May 15;188(10):2778-2793.e21. doi: 10.1016/j.cell.2025.02.009. Epub 2025 Mar 10. Cell. 2025. PMID: 40068680

Abstract

Recent breakthroughs in the genetic manipulation of mitochondrial DNA (mtDNA) have enabled the precise introduction of base substitutions and the effective removal of genomes carrying harmful mutations. However, the reconstitution of mtDNA deletions responsible for severe mitochondrial myopathies and age-related diseases has not yet been achieved in human cells. Here, we developed a method to engineer specific mtDNA deletions in human cells by co-expressing end-joining (EJ) machinery and targeted endonucleases. As a proof-of-concept, we used mito-EJ and mito-ScaI to generate a panel of clonal cell lines harboring a ∼3.5 kb mtDNA deletion with the full spectrum of heteroplasmy. Investigating these isogenic cells revealed a critical threshold of ∼75% deleted genomes, beyond which cells exhibited depletion of OXPHOS proteins, severe metabolic disruption, and impaired growth in galactose-containing media. Single-cell multiomic analysis revealed two distinct patterns of nuclear gene deregulation in response to mtDNA deletion accumulation; one triggered at the deletion threshold and another progressively responding to increasing heteroplasmy. In summary, the co-expression of mito-EJ and programable nucleases provides a powerful tool to model disease-associated mtDNA deletions in different cell types. Establishing a panel of cell lines with a large-scale deletion at varying levels of heteroplasmy is a valuable resource for understanding the impact of mtDNA deletions on diseases and guiding the development of potential therapeutic strategies.

Highlights: Combining prokaryotic end-joining with targeted endonucleases generates specific mtDNA deletions in human cellsEngineering a panel of cell lines with a large-scale deletion that spans the full spectrum of heteroplasmy75% heteroplasmy is the threshold that triggers mitochondrial and cellular dysfunctionTwo distinct nuclear transcriptional programs in response to mtDNA deletions: threshold-triggered and heteroplasmy-sensing.

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