Synapse - Engineering mtDNA deletions by reconstituting end joining in human mitochondria

Engineering mtDNA deletions by reconstituting end joining in human mitochondria Journal Article

Authors:	Fu, Y.; Land, M.; Kavlashvili, T.; Cui, R.; Kim, M.; DeBitetto, E.; Lieber, T.; Ryu, K. W.; Choi, E.; Masilionis, I.; Saha, R.; Takizawa, M.; Baker, D.; Tigano, M.; Lareau, C. A.; Reznik, E.; Sharma, R.; Chaligne, R.; Thompson, C. B.; Pe'er, D.; Sfeir, A.
Article Title:	Engineering mtDNA deletions by reconstituting end joining in human mitochondria
Abstract:	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. © 2025 Elsevier Inc.
Keywords:	controlled study; unclassified drug; human cell; gene deletion; genetics; sequence deletion; microscopy; flow cytometry; metabolism; apoptosis; cell line; fluorescence in situ hybridization; genetic engineering; western blotting; real time polymerase chain reaction; endonuclease; mass fragmentography; dna, mitochondrial; mitochondria; oxygen consumption; dna extraction; mitochondrion; oxidative phosphorylation; rna extraction; mitochondrial dna; genetic parameters; southern blotting; genotyping; mitochondrial respiration; procedures; mtdna; galactose; genetic damage; spectrophotometry; high throughput sequencing; dna end-joining repair; dna end joining repair; induced pluripotent stem cell; sanger sequencing; humans; human; article; heteroplasmy; hek293t cell line; mitochondrial myopathy; human embryonic stem cell; end joining; mitochondrial transcription factor a; dogma-seq; mitochondrial pathologies; mtdna deletion; mitochondrial myopathies; mitochondrial genome maintenance exonuclease 1; arpe-19 cell line; mitochondrial dna deletion; mitochondrial end joining machinery; msk-srf001 human induced pluripotent stem cell; wa09 cell line
Journal Title:	Cell
Volume:	188
Issue:	10
ISSN:	0092-8674
Publisher:	Cell Press
Date Published:	2025-05-15
Start Page:	2778
End Page:	2793.e21
Language:	English
DOI:	10.1016/j.cell.2025.02.009
PUBMED:	40068680
PROVIDER:	scopus
PMCID:	PMC12085298
DOI/URL:	https://www.scopus.com/inward/record.uri?eid=2-s2.0-86000629860&doi=10.1016%2fj.cell.2025.02.009&partnerID=40&md5=633ca76f8bb93db333897c1d21a9fd8f
Notes:	Article -- Source: Scopus