CIRCLE-F/V: a dual-mode CRISPR-Cas13a cascade biosensor for ultrasensitive and visual detection of low-abundance EGFR mutations

Abstract Precise identification of low-frequency EGFR L858R mutations is crucial for targeted therapy and prognosis in non-small cell lung cancer (NSCLC). Here, we present CIRCLE-F/V (CRISPR-based Integrated Restriction-assisted Cyclic Loop-mediated Enhancement Fluorescence/Visual platform), a dual-mode CRISPR-Cas13a cascade biosensor enabling ultrasensitive and specific mutation detection through integrated fluorescence and visual readouts. The system combines restriction-assisted mutant enrichment with cyclic CRISPR signal amplification. Genomic DNA is selectively digested with MscI to eliminate wild-type alleles, followed by T7 promoter–mediated PCR amplification and in vitro transcription. The resulting RNA activates Cas13a-crRNA complexes, initiating both direct reporter cleavage and dumbbell-triggered cascade amplification (DTCA) -mediated secondary signal enhancement. Under optimized conditions, the fluorescence mode achieved a variant allele frequency (VAF) sensitivity of 0.01%, with an estimated genomic DNA detection limit of ~ 3.6 fM in the integrated workflow and an RNA detection limit of ~ 2.16 pM. By replacing the fluorescent reporter with a FAM–biotin probe, the visual mode via lateral flow strips was realized with a VAF sensitivity of 0.01%. Clinical validation demonstrated complete concordance with next-generation sequencing (NGS), accurately classifying as high-, low-, and negative mutation samples. By integrating selective allele enrichment, cyclic amplification, and dual-mode readout, CIRCLE-F/V represents a sensitive, versatile, and clinically adaptable platform for rapid EGFR genotyping and early NSCLC screening.