CRISPR/Cas13a-enhanced FRET-PAINT (CFP) platform for highly sensitive detection of schistosome-derived miRNA

• A new miRNA detector combines CRISPR/Cas13a with single-molecule FRET-PAINT for ultra-sensitivity. • Background noise is dramatically reduced and detection limits reach ∼103.58 aM. • Platform is adaptable for multiplexed, clinically robust miRNA diagnostics in real samples. MicroRNAs (miRNAs) are vital biomarkers for early diagnosis of numerous diseases, yet their low abundance and high sequence homology pose significant challenges to conventional detection methods. To address these limitations, we present a novel ultrasensitive and highly specific miRNA detection platform that integrates the CRISPR/Cas13a system with single-molecule FRET-PAINT imaging. By leveraging a precisely localized donor–acceptor FRET mechanism and transient probe binding, our method achieves an approximately 80% reduction in background noise compared to DNA-PAINT, reaching a detection limit as low as 103.58 aM. We further enhance specificity by introducing intentional mismatches in crRNA design, enabling robust discrimination among highly homologous sequences and rapid reconfiguration for new targets. The platform exhibits excellent anti-interference performance in unprocessed serum, with recovery rates ranging from 96.9% to 109.6%. Validation using nine clinical samples demonstrates 100% diagnostic accuracy. This innovative platform provides a reliable technological foundation for the early screening and precise diagnosis of miRNA-related diseases. CRISPR/Cas13a-Enhanced FRET-PAINT (CFP) Platform for Highly Sensitive Detection of Schistosome -Derived miRNA .