Proc Natl Acad Sci U S A. 2026 Sep 08. 123(36):
e2531151123
Zhengyang Zhang,
Liwei Zhang,
Peng An,
Xu Zhang,
Yi Xia,
Yunlu Kang,
Xiaoxia Chen,
Rongrong Hua,
Yinhua Zhu,
Yanling Hao,
Yuan Huang,
Yongting Luo,
Junjie Luo,
Guisheng Wang.
Simultaneously profiling mitochondrial DNA (mtDNA) heteroplasmy and phenotypic variability at the single-cell level remains a challenge due to the absence of integrated methods that map mitochondrial genotypes alongside their functional states. We introduce human single-cell mitochondrial phenotype-coupled mtDNA sequencing (scMPCDS), a platform that quantifies mtDNA mutations and heteroplasmy together with mitochondrial membrane potential and reactive oxygen species within individual cells. Unlike bulk sequencing or separate single-omics techniques, scMPCDS directly correlates mitochondrial genomic instability with functional outcomes. Using this approach, we demonstrate that DdCBE-mediated mtDNA editing induces cell-specific off-target mutations in the mitochondrial genome, which coincide with diverse phenotypic changes. Applying scMPCDS to HeLa cells and clear cell renal cell carcinoma tissues, we identify single-cell subpopulations exhibiting distinct mtDNA mutation burdens and altered bioenergetic profiles, implicating potential mitochondrial heterogeneity-driven tumor evolution. Overall, scMPCDS serves as a versatile tool to unravel mitochondrial genotype-phenotype relationships at the single-cell level in both normal and disease states, thereby advancing precise mitochondrial diagnostics and therapeutics.
Keywords: cancer; mitochondrial phenotype; mtDNA editing; mtDNA heteroplasmy; single cell