Microfluidic-free droplet-based CRISPR/Cas12a assay for semi-digital detection of DNA methylation

• A microfluidic-free droplet-based assay enables sensitive DNA methylation detection. • Restriction enzyme digestion, isothermal amplification and nuclease-based sensing are integrated in droplets. • Droplet compartmentalization enables detection of rare methylated DNA molecules. • The assay provides a rapid and accessible strategy for molecular diagnostics. DNA methylation is an important epigenetic biomarker for early cancer diagnosis; however, sensitive detection of rare methylated DNA molecules in complex biological samples remains technically challenging. Here, we report a microfluidic-free droplet-based CRISPR/Cas12a assay, termed ddMeRpaCas, for sensitive detection of methylated DNA targets. The platform integrates methylation-sensitive restriction digestion, recombinase polymerase amplification, and CRISPR/Cas12a fluorescence detection within vortex-generated droplets. Unmethylated DNA templates are selectively digested, whereas intact methylated targets are amplified and then partitioned into droplets containing the CRISPR/Cas12a detection components, where target recognition activates collateral cleavage and produces fluorescence signals. Droplet compartmentalization converts bulk fluorescence accumulation into discrete fluorescence-positive droplet events, enabling semi-digital readout and improving detection of rare methylated DNA molecules. Using methylated FOXD3 as a model biomarker, ddMeRpaCas achieved a detection sensitivity of 5 copies/μL and discriminated methylated DNA fractions as low as 0.01%. Droplet generation is achieved by simple vortex emulsification without microfluidic devices or specialized digital PCR instruments, and the workflow can be completed within 120 min. These results highlight a practical engineering strategy for droplet-based signal compartmentalization in CRISPR diagnostics, with potential applications in epigenetic analysis and molecular detection.