Rapid Nucleic Acid Detection Based on a CRISPR-Cas12a Coupled Fluorescence Sensing Platform

Rapid and accurate nucleic acid detection plays a pivotal role in pathogen screening, clinical diagnosis, and molecular biology research. However, conventional polymerase chain reaction (PCR)-based detection methods suffer from inherent limitations, making them difficult to fully meet the practical requirements of point-of-care testing (POCT). The CRISPR-Cas12a system has emerged as a promising platform for rapid next-generation nucleic acid detection, leveraging its exceptional target recognition specificity and collateral trans-cleavage activity against single-stranded DNA (ssDNA) reporters upon target activation, coupled with the high sensitivity and facile readout of fluorescence-based sensing strategies. This study aimed to develop an efficient, convenient, and highly sensitive CRISPR-Cas12a-coupled fluorescence sensing platform for rapid nucleic acid detection. By optimizing the ratio of Cas12a protein to specific crRNA, incorporating isothermal pre-amplification, and introducing a nanomaterial-based signal enhancement strategy, the proposed system enabled rapid target nucleic acid responses under a mild temperature condition of 37°C. Experimental results demonstrated that the developed sensing system exhibited outstanding specificity, effectively discriminating against single-base mismatches and minimizing nonspecific interference. Furthermore, the platform achieved an ultrahigh detection sensitivity down to 1.31 fM, while reducing the total assay time to less than 45 min.