One-pot CRISPR/Cas12a system enables direct RNA and DNA detection without thermal cycling
Investigators developed a reverse transcriptase and locked nucleic acid probe-mediated CRISPR/Cas12a positive feedback system designed for one-pot nucleic acid detection. The assay eliminates the need for pre-amplification and thermal cycling, achieving 0.5 attomolar sensitivity within 27 minutes, single-base resolution, and the ability to directly detect structured RNAs up to 985 nucleotides in length. Performance was validated in practical samples through the detection of lncRNA HULC and miR-21. The platform offers a streamlined, high-performance alternative to conventional CRISPR biosensing workflows, potentially simplifying molecular testing in rapid-response laboratory settings.
The original study
A One-Pot Reverse Transcriptase-Mediated, Pre-amplification-Free CRISPR/Cas12a Assay for Ultrasensitive Nucleic Acid Detection.
- Authors
- He R, Zhang C, Zhang J, Zhang K, Yin W, Qiao B, et al.
- Journal
- ACS sensors
- Type
- Journal Article
- PMID
- 42603302
Original 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.