An Amplified Luminescent Proximity Homogeneous Assay-Implemented CRISPR/Cas13a System for Noncoding RNA Detection

Abstract

The CRISPR-Cas13a detection system is an RNA detection method developed in recent years, which precisely identifies the target RNA sequence to be detected by base complementary pairing. In this study, the homogeneous light‑initiated chemiluminescence system and CRISPR-Cas13a are integrated to develop a novel RNA detection system with higher sensitivity, the amplified luminescent proximity homogeneous assay (Alpha)-CRISPR RNA detection system. The RNA probe is functionalized with Alpha beads to form an innovative reporter. The Alpha RNA reporter exhibits enhanced luminescence efficiency compared to conventional RNA fluorescent probes, yielding superior sensitivity for the detection system. The results demonstrate that this method achieves precise RNA target detection within 1 h, halving the detection time compared to PCR. With a detection limit of 100 fM without requiring amplification steps, it exhibits 100-fold higher sensitivity than systems using conventional fluorescent probes. The platform successfully detects diverse RNA biomarkers, including long noncoding RNAs, microRNAs, and circular RNAs. Validation using clinical serum samples from healthy volunteers and patients with kidney disease confirms that the assay reliably quantifies lncRNA H19 across a range of concentrations, with results showing concordance with those obtained by RT-qPCR. The Alpha-CRISPR RNA detection system developed in this study integrates AlphaLISA technology with the CRISPR-Cas13a system, achieving significantly enhanced detection sensitivity. This synergistic integration expands the repertoire of CRISPR-Cas13a-based RNA detection methodologies, demonstrating substantial application potential in fundamental medical research and clinical diagnostics.