Precise amplification-free detection of highly structured RNA with an enhanced SCas12a assay.

The CRISPR/Cas12a system has revolutionized molecular diagnostics, yet the direct detection of RNA, particularly those with complex structures, remains a significant challenge. Here, we present an updated SCas12a system, termed SCas12aV2, which enables precise, amplification-free detection of highly structured RNA molecules. By optimizing the length of scaffold RNA, targeting asymmetric structures, and utilizing dsDNA-ssDNA hybrid activators, we have significantly reduced steric hindrance in the detection system, thereby markedly enhancing both sensitivity and kinetics compared to traditional DNA activators. The SCas12aV2 assay achieves a detection limit of 246 aM for pooled activators and 10 pM for single-site targeting, demonstrating high specificity for single nucleotide polymorphisms (SNPs). It successfully identifies viable bacteria and SARS-CoV-2 infections in clinical samples. The assay is versatile and can be applied to various Cas12a orthologs, including thermostable CtCas12a. This work advances molecular diagnostics by improving the accuracy and efficiency of RNA detection.