Single-cell mitochondrial genotyping of exonuclease-deficient DNA polymerase ? (POLGD274A) knock-in HEK293 cell lines
Exonuclease-deficient DNA polymerase ? (POLGD274A) lead to an hypermutated mitochondrial genomes and cause OXPHOS defect. Exonuclease-deficient DNA polymerase ? (POLGD274A) lead to an elevated mutational rate and hypermutated mitochondrial genomes, which cause OXPHOS defect. Here, leverage mitochondrial single-cell ATAC-seq (mtscATAC-seq) to estimate genome-wide mtDNA mutational burden in HEK293 cell lines with POLGD274A knock-in. Our method sensitively detects up to a a median of 472 variants per cell, corresponding to a 50-fold increase comared to control, with a predominance of replication-driven C>T transitions. We further examined how POLGD274A knock-in cells adapt to OXPHOS-enforcing metabolic stress in galactose. After 1 and 3 days in glucose or galactose, CTRL and KI36 were subjected to mtscATAC-seq to examine the mtDNA variants heteroplasmy dynamic. We found that pseudo-bulk heteroplasmy, single-cell VAF distributions, and mtDNA mutational burden were unchanged. Instead, mtDNA copy number rose, and subclone DAGs highlighted stress/inflammatory programs and increasing accessibility at GDF15, suggesting population-wide metabolic rewiring. Overall design: POLGD274A knock-in HEK293 cell lines, with (Knock-in clone A2, KIA2) or without (Knock-in clone 36, KI36) the introduction of a tetracycline-inducible mitochondrial restriction exonuclease mitoEagI, were previously described (PMID: 29712893). Cryopreserved cells were thawed cultured for a maximum of 2 weeks before single-cell sequencing to limit the accumulation of additional mtDNA mutations. For the Galactose treatment experiment, control and POLGD274A  knock-in clone 36 HEK293 cell lines were cuturled in glucose-free DMEM supplemented with 1 mM sodium pyruvate and with either 10 mM glucose (3 days) or 10 mM galactose (1 day or 3 days). Each cell line–treatment combination was labeled with dedicated TotalSeq-B hashtag antibodies, washed, and pooled at equal ratios. The pooled cells were subsequently processed using the mtscATAC-seq protocol.