A One-Pot Reverse Transcriptase-Mediated, Pre-amplification-Free CRISPR/Cas12a Assay for Ultrasensitive Nucleic Acid Detection

Abstract CRISPR/Cas12a holds great promise for biosensing and diagnostics, but conventional methods suffer from low catalytic efficiency, high background, and reliance on pre-amplification. Direct detection of structured RNAs also remains challenging. Herein, we report the development of a reverse transcriptase and LNA probe (LNA-p)-mediated CRISPR/Cas12a positive feedback system (RTLC) for highly efficient, one-pot detection of both DNA and RNA. Without pre-amplification or thermal cycling, the assay achieves a 0.5 aM detection sensitivity within 27 min, exhibits single-base resolution, and allows direct detection of RNAs up to 985 nt in length. Together, RTLC is successfully validated in practical samples by detecting lncRNA HULC and miR-21, offering a robust, versatile tool for high-performance nucleic acid diagnostics.