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1 Molecular Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
2 Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
3 Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
4 Computational Oncology, Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
5 Department of Pathology and Genomic Medicine, Thomas Jefferson University, Philadelphia, PA, USA.
6 Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Howard Hughes Medical Institute, New York, NY, USA.
7 Molecular Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA. Electronic address: sfeira@mskcc.org.
1 Molecular Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
2 Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
3 Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
4 Computational Oncology, Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
5 Department of Pathology and Genomic Medicine, Thomas Jefferson University, Philadelphia, PA, USA.
6 Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Howard Hughes Medical Institute, New York, NY, USA.
7 Molecular Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA. Electronic address: sfeira@mskcc.org.
Recent breakthroughs in the genetic manipulation of mitochondrial DNA (mtDNA) have enabled precise base substitutions and the efficient elimination of genomes carrying pathogenic mutations. However, reconstituting mtDNA deletions linked to mitochondrial myopathies remains challenging. Here, we engineered mtDNA deletions in human cells by co-expressing end-joining (EJ) machinery and targeted endonucleases. Using mitochondrial EJ (mito-EJ) and mito-ScaI, we generated a panel of clonal cell lines harboring a ∼3.5 kb mtDNA deletion across the full spectrum of heteroplasmy. Investigating these cells revealed a critical threshold of ∼75% deleted genomes, beyond which oxidative phosphorylation (OXPHOS) protein depletion, metabolic disruption, and impaired growth in galactose-containing media were observed. Single-cell multiomic profiling identified two distinct nuclear gene deregulation responses: one triggered at the deletion threshold and another progressively responding to heteroplasmy. Ultimately, we show that our method enables the modeling of disease-associated mtDNA deletions across cell types and could inform the development of targeted therapies.
Keywords:
DOGMA-seq; end joining; mitochondrial pathologies; mtDNA; mtDNA deletion.
Declaration of interests A.S. is a co-founder, consultant, and shareholder of Repare Therapeutics. C.B.T. is a member of the board of directors and a shareholder of Regeneron and Charles River Laboratories, and a founder of Agios Pharmaceuticals. D.P. is on the scientific advisory board of Insitro.
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, Reznik E, Sharma R, Chaligne R, Thompson CB, Pe'er D, Sfeir A.Fu Y, et al.bioRxiv [Preprint]. 2024 Oct 17:2024.10.15.618543. doi: 10.1101/2024.10.15.618543.bioRxiv. 2024.Update in: Cell. 2025 May 15;188(10):2778-2793.e21. doi: 10.1016/j.cell.2025.02.009.PMID: 39463974Free PMC article.Updated.Preprint.
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