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

Abstract The CRISPR/Cas12a system has revolutionized molecular diagnostics; however, its application in directly detecting complex structured RNA remains challenging. Recently, we have developed a RNA detection method called SCas12a, which exhibits high sensitivity and efficiency in detecting RNA molecules devoid of intricate secondary structures. Here, we present an enhanced SCas12a assay (SCas12aV2) that facilitates precise amplification-free detection of highly structured RNA molecules. Our approach reengineers the split Cas12a system by optimizing the scaffold RNA length and targeting asymmetric RNA structures, thereby minimizing steric hindrance. We observe that utilization of a dsDNA-ssDNA hybrid DNA activator significantly enhances both the sensitivity and kinetics compared to those achieved using traditional ssDNA or dsDNA activators. The SCas12aV2 assay demonstrates exceptional sensitivity, with a limit of detection reaching 246 aM for pooled activators and 10 pM for single-site targeting. It also exhibits high specificity for single nucleotide polymorphisms (SNPs) and successfully identifies viable bacterial populations and SARS-CoV-2 infections from clinical samples. The assay's versatility is further highlighted by its applicability to various Cas12a orthologs, including the thermostable CtCas12a. This work offers a significant advance in molecular diagnostics, enhancing the potential for accurate and efficient RNA detection.