Amplification-Free Detection of Highly Structured RNA Molecules Using SCas12aV2

The CRISPR/Cas12a system has revolutionized molecular diagnostics; however, conventional Cas12a-based methods for RNA detection typically require transcription and pre-amplification steps. Our group has recently developed a diagnostic technique known as the SCas12a assay, which combines Cas12a with a split crRNA, achieving amplification-free detection of miRNA. However, this method still encounters challenges in accurately quantifying long RNA molecules with complex secondary structures. Here, we report an enhanced version termed SCas12aV2 (split-crRNA Cas12a version 2 system), which enables direct detection of RNA molecules without sequence limitation while demonstrating high specificity in single-nucleotide polymorphism (SNP) applications. We describe the general procedure for preparing the SCas12a system and its application in detecting RNA targets from clinical samples. Key features • The SCas12aV2 assay enables efficient detection of long-chain RNA molecules with complex secondary structures. • PAM-distal sites can be effectively distinguished at the SNP detection level. • The entire experimental procedure can be completed in less than one hour.