A CRISPR/Cas9-regulated dual-ring topological allosteric probe for detection of the EGFR L858R resistance mutation in CTCs.

A single-nucleotide polymorphism (SNP) is a point mutation occurring at a defined genomic locus, and its precise and rapid detection in circulating tumor cells (CTCs) is essential for early diagnosis and therapeutic monitoring of non-small cell lung cancer (NSCLC). In this study, a CRISPR/Cas9-regulated dual-ring topological allosteric probe was developed for ultrasensitive and specific detection of the EGFR L858R mutation. The recognition ring selectively hybridizes with the target sequence and is cleaved by the Cas9-sgRNA complex, triggering the release of the reporter ring. The released reporter ring then serves as a template for rolling circle amplification (RCA), generating products that hybridize with dual-labeled fluorescent probes to yield measurable signals. This assay clearly distinguished L858R from the wild-type sequence and detected mutation frequencies as low as 1.0% with high specificity against other common EGFR variants. Its robustness was further validated using clinical blood samples, enabling sensitive detection of low-abundance L858R mutations. These results demonstrate that the integration of programmable target recognition, efficient signal amplification, and fluorescence readout provides a promising platform for SNP analysis in liquid biopsy, supporting precision diagnosis and treatment monitoring in NSCLC.