Renal clearable CRISPR nanosensor targeting mitochondrial DNA mutation for noninvasive monitoring of tumor progression and metastasis

Mitochondrial DNA (mtDNA) mutations are emerging as important molecular features of tumorigenesis. Liquid biopsies, involving analysis of cell-free mtDNA, enable early cancer detection but suffer from low sensitivity due to scarce analytes. Here, we developed a CRISPR/Cas12a-mediated urinary biomarker, termed CasUber, for in vivo monitoring of tumor progression and metastasis. Our results demonstrate that CasUber can deliver a CRISPR detection system into tumor cell mitochondria, leverage the single-nucleotide variant recognition ability and trans-cleavage activity of Cas12a to convert tumor-specific mtDNA mutations into renal-clearable fluorescent biomarkers, and exocytosed along with the natural efflux pathway of damaged mtDNA. As a result, CasUber enables discrimination of ultrasmall tumor lesions (~1 cubic millimeter) and detection of lung tumor nodules earlier than bioluminescence imaging in a blood-lung metastasis model. This renal clearable nanosensor allows in situ recognition of specific gene mutation to generate amplified signals, overcoming the limitation of low mtDNA abundance and enabling noninvasive and ultrasensitive monitoring of tumor progression and metastasis via a simple urine test.