Strand displacement-enhanced CRISPR-Cas13a system for ultra-specific detection of RNA single nucleotide variation

RNA plays a critical role in biological systems, mediating genetic information transfer and regulating gene expression. However, RNA is susceptible to variations from endogenous and exogenous sources, with potentially profound biological consequences. The CRISPR-Cas13a system has emerged as a promising tool for RNA variation detection due to its cost-effectiveness, sensitivity, and user-friendly nature. Despite this, designing a simple, universal system with high discrimination factor (DF) for single-nucleotide variations remains a challenge. Here, we present the strand displacement-enhanced Cas13a single-nucleotide variation detection assay (SECND), a sensitive, universal, and easy-to-implement method with a high DF for RNA variations. Using SECND, we detected 5 types of single-nucleotide variations, achieving a maximum DF of 1083.2. We validated the assay's effectiveness on miRNA and SARS-CoV-2 genomic RNA simulants, incorporating a 4-way strand displacement mechanism to enhance detection limits to 10 pmol/L and 50 pmol/L, and to identify variations at frequencies as low as 0.01 % and 0.1 %. Additionally, we demonstrated SECND's utility in quantifying single-nucleotide variants of miR-200b and miR-200c in ovarian cancer and retinal glioma cells. This versatile tool not only advances RNA variation detection but also has significant implications for disease research, diagnostics, and viral classification, enhancing our understanding of the CRISPR-Cas13a system and its potential applications.