MitoPerturb-Seq identifies gene-specific single-cell responses to mitochondrial DNA depletion and heteroplasmy

Stephen P Burr; Kathryn Auckland; Angelos Glynos; Abhilesh Dhawanjewar; Cameron Ryall; Wei Wei; Antony Hynes-Allen; Malwina Prater; Matylda Sczaniecka-Clift; Julien Prudent; Patrick F Chinnery; Jelle van den Ameele

doi:10.1038/s41594-026-01779-7

MitoPerturb-Seq identifies gene-specific single-cell responses to mitochondrial DNA depletion and heteroplasmy

Nat Struct Mol Biol. 2026 Apr 1. doi: 10.1038/s41594-026-01779-7. Online ahead of print.

Authors

Stephen P Burr^#^{1

2}, Kathryn Auckland^#^{1

2}, Angelos Glynos^{1

2}, Abhilesh Dhawanjewar^{1

2}, Cameron Ryall^{1

2}, Wei Wei^{1

2}, Antony Hynes-Allen^{1

2}, Malwina Prater^{1

2

3}, Matylda Sczaniecka-Clift⁴, Julien Prudent¹, Patrick F Chinnery^{5

6}, Jelle van den Ameele^{7

8}

Affiliations

¹ MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK.
² Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK.
³ Altos Labs Cambridge Institute, Cambridge, UK.
⁴ MRC Laboratory of Molecular Biology, University of Cambridge, Cambridge, UK.
⁵ MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK. pfc25@cam.ac.uk.
⁶ Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK. pfc25@cam.ac.uk.
⁷ MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK. jv361@cam.ac.uk.
⁸ Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK. jv361@cam.ac.uk.

^# Contributed equally.

PMID: 41922875
DOI: 10.1038/s41594-026-01779-7

Abstract

Mitochondria contain their own genome, mitochondrial DNA (mtDNA), which is under strict control by the cell nucleus. mtDNA occurs in many copies per cell and mutations often only affect a proportion of them, giving rise to heteroplasmy. mtDNA copy number and heteroplasmy level together shape the tissue-specific impact of mtDNA mutations, eventually giving rise to both rare mitochondrial and common neurodegenerative diseases. Here, we use MitoPerturb-Seq for CRISPR-Cas9-based, high-throughput single-cell interrogation of the nuclear genes and pathways that sense and control mtDNA copy number and heteroplasmy. We screened a panel of mtDNA maintenance genes in mouse cells with a heteroplasmic mtDNA mt-Ta mutation. This revealed both common and perturbation-specific aspects of the integrated stress response to mtDNA depletion caused by Tfam, Opa1 and Polg knockout. These responses are only partially mediated by ATF4 and cause cell-cycle stage-independent slowing of cell proliferation. MitoPerturb-Seq, thus, provides experimental insight into disease-relevant mitochondrial-nuclear interactions and may inform development of therapies targeting cell-type- and tissue-specific vulnerabilities to mitochondrial dysfunction.