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Updated: Apr 3, 2026
Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
Published on: February 10, 2023
MitoPerturb-Seq identifies gene-specific single-cell responses to mitochondrial DNA depletion and heteroplasmy
Stephen P Burr1,2, Kathryn Auckland1,2, Angelos Glynos1,2
1MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK.
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.
Insights
Mitochondrial DNA (mtDNA) mutations impact disease, influenced by copy number and heteroplasmy. New high-throughput screening reveals nuclear gene roles in controlling these mtDNA features, offering therapeutic insights.
Area of Science:
- Cell Biology
- Genetics
- Neuroscience
Background:
- Mitochondrial DNA (mtDNA) mutations contribute to various diseases, from rare mitochondrial disorders to common neurodegenerative conditions.
- The impact of mtDNA mutations is modulated by mtDNA copy number and heteroplasmy levels, which are tightly regulated by nuclear genes.
- Understanding the interplay between nuclear control and mtDNA maintenance is crucial for disease pathogenesis and therapeutic development.
Purpose of the Study:
- To investigate nuclear genes and pathways controlling mtDNA copy number and heteroplasmy using a high-throughput CRISPR-Cas9 screening approach.
- To elucidate the integrated stress response to mtDNA depletion and its underlying mechanisms.
- To identify disease-relevant mitochondrial-nuclear interactions for potential therapeutic targeting.
Main Methods:
- MitoPerturb-Seq: A CRISPR-Cas9-based, high-throughput single-cell screening method.
- Screening of mtDNA maintenance genes in mouse cells harboring a heteroplasmic mtDNA mutation (mt-Ta).
- Analysis of integrated stress response pathways, including ATF4, and cell proliferation rates.
Main Results:
- Identified common and perturbation-specific aspects of the integrated stress response to mtDNA depletion caused by knockout of Tfam, Opa1, and Polg.
- Demonstrated that these responses are only partially mediated by ATF4.
- Observed cell-cycle stage-independent slowing of cell proliferation.
Conclusions:
- MitoPerturb-Seq provides valuable experimental insights into mitochondrial-nuclear interactions relevant to disease.
- The study highlights the complex regulation of mtDNA copy number and heteroplasmy by nuclear factors.
- Findings may inform the development of therapies targeting cellular vulnerabilities in mitochondrial dysfunction.
