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

Mitochondria contain their own genome, the mitochondrial DNA (mtDNA), which is under strict control of the cell nucleus. mtDNA occurs in many copies in each cell, and mutations often only affect a proportion of them, giving rise to heteroplasmy. mtDNA copy number and heteroplasmy level together shape the cell- and tissue-specific impact of mtDNA mutations, ultimately giving rise to rare mitochondrial and common neurodegenerative diseases. However, little is known about how copy number and heteroplasmy interact within single cells, and how this is regulated by the nuclear genes and pathways that sense and control them. Here we describe MitoPerturb-Seq for CRISPR/Cas9-based high-throughput single-cell interrogation of the impact of nuclear gene perturbation on mtDNA copy number and heteroplasmy. We screened a panel of nuclear mtDNA maintenance genes in cells with heteroplasmic mtDNA mutations. This revealed both common and perturbation-specific aspects of the integrated stress-response to mtDNA depletion, that 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 novel therapies targeting cell- and tissue-specific vulnerabilities to mitochondrial dysfunction. Overall design: Mouse Embryonic Fibroblasts (MEFs) carrying a heteroplasmic mitochondrial (mt)DNA mutation in mt-Ta (m.5024C>T) and stably expressing Cas9 were transduced with a pooled lentiviral CRISPR library targeting 19 genes (three guides per gene) plus three non-targeting control gRNAs using the CROPseq method. 10 days post-transduction cells were flow sorted to select transduced cells based on expression of mRFP, and sorted cells were fixed and permeabilized before being submitted for 10X Genomics single-cell MultiOme ATAC and gene expression (GEX) sequencing. Following library preparation, CROPseq guide mRNA sequences were enriched from the GEX cDNA library using the TAP-seq protocol, and mtDNA sequences were enriched from the final ATACseq library by hybridization capture using a bespoke probeset from IDT