Mitochondrial DNA (mtDNA) mutations are associated with tumor progression and metabolic dysregulation, yet their spatiotemporal visualization and selective therapeutic modulation remain challenging owing to their low abundance and mitochondrial double-membrane barrier. We report a near-infrared (NIR) light-inducible CRISPR-Cas12a/tetrahedral DNA nanosystem for spatiotemporally resolved imaging and editing of mtDNA mutations. The nanosystem integrates upconversion nanoparticles as NIR to ultraviolet converters, photocleavable DNA linkers, and Cas12a/crRNA modules. NIR irradiation triggers linker cleavage, activates Cas12a-cleavage, and generates amplified fluorescence for mtDNA mutation detection with a detection limit of 0.83 pM. In living cells and tumor-bearing mice, the nanosystem enables high-spatiotemporal resolution imaging of mtDNA mutations while inducing DNA self-assembly and CRISPR-based mtDNA editing. These disrupt the mitochondrial membrane potential, increase the level of reactive oxygen species, and promote apoptosis, resulting in effective tumor suppression. This nanosystem enables the controllable imaging of mutant mtDNA and gene modulation, offering new opportunities for spatiotemporal cancer theranostics. trans
