MitoPerturb-Seq identifies common and gene-specific single-cell responses to mitochon-drial DNA depletion and heteroplasmy

Mitochondria contain their own genome, the mitochondrial DNA (mtDNA), which is under strict control of 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 heter-oplasmy level together shape tissue-specific impact of mtDNA mutations, eventually giving rise to rare mitochondrial and common neurodegenerative diseases. Here we describe 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 cells with heteroplasmic mtDNA mutations. This re-vealed both common and perturbation-specific aspects of the integrated stress-response to mtDNA depletion, which were only partially mediated by Atf4, and caused cell-cycle stage-independent slowing of cell-proliferation. MitoPerturb-Seq thus provides novel experimental insight into disease-relevant mito-nuclear interactions, ultimately informing development of therapies targeting cell- and tissue-specific vulnerabilities to mitochondrial dysfunction.