DNA methylation represents a pivotal epigenetic biomarker for cancer, and precise profiling of carcinogenesis-associated methylation is essential for early diagnosis and prognostic evaluation. Herein, we report a novel label-free, sensitive DNA methylation biosensor by integrating GlaI-assisted double cascade strand displacement amplification with G-triplex-facilitated CRISPR/Cas12a (G-DCSDA/Cas12a). Instead of conventional fluorescent-quenched (FQ) probes, this assay employed G-triplex/Thioflavin T as a simple and efficient signal reporter for CRISPR/Cas12a. The methylation-specific endonuclease GlaI selectively digested methylated DNA to release free 3'-OH ends, which initiated the subsequent DCSDA and triggered Cas12a activation. The double-template cascade amplification system delivers significantly improved sensitivity in comparison with the single-template strategy. Upon activation, the trans-cleavage activity of Cas12a rapidly disrupted G-triplex/Thioflavin T complexes, generating a distinct fluorescence response. By combining the high specificity of GlaI, efficient signal amplification of DCSDA, and robust collateral cleavage of Cas12a, the G-DCSDA/Cas12a platform achieved ultrahigh sensitivity and selectivity, enabling detection of methylation levels as low as 0.005% in a background of excessive unmethylated DNA. Furthermore, this strategy was successfully integrated into a lateral flow assay (LFA), enabling visual and point-of-care testing (POCT) of DNA methylation. Importantly, the developed biosensor achieved sensitive detection of genomic DNA methylation in real samples and accurately discriminates cancer cells from normal cells, as well as between colorectal cancer tissue and adjacent normal tissue. These results highlighted the significant potential of the G-DCSDA/Cas12a platform for clinical early cancer diagnosis.
