Amplification-free detection of N6-Methyladenosine (m6A) based on droplet digital CRISPR-Cas13a.

m6A (N6-methyladenosine), a prevalent RNA modification in eukaryotic mRNA involved in multiple biological processes, enables early cancer screening via its detection. m6A methylation detection is considered challenging, with most existing methods being limited by complex chemical transformations that incur high cost and procedural complexity. To overcome these challenges, a novel droplet-based digital CRISPR-Cas13a (Dd-Cas13a) approach was developed for the detection of m6A modifications in RNA. The method employs an m6A-specific antibody enrichment strategy to selectively enrich RNA molecules harboring m6A modifications, while RNA molecules lacking m6A remain unselected. Integration of the targeted recognition capability of the CRISPR/Cas13a system with droplet technology enables amplification-free detection of m6A-modified RNA at fM concentrations, with a significant improvement in sensitivity. The Dd-Cas13a detection platform enables visualization and quantitative analysis of methylation site proportions in cellular RNA, providing a novel approach for rapid and sensitive detection of RNA methylation, with significant potential for applications in early disease diagnosis and therapeutic efficacy assessment.