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

ABSTRACT: 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 (

SUBMITTER: Zhang J 

PROVIDER: S-EPMC11880562 | biostudies-literature | 2025 Mar

REPOSITORIES: biostudies-literature