A novel methyl-dependent DNA endonuclease GlaI coupling with double cascaded strand displacement amplification and CRISPR/Cas12a for ultra-sensitive detection of DNA methylation.

Detection of methylation changes associated with oncogenic transformation is essential for early screening and treatment of cancer. Herein, we propose a novel DNA methylation detection assay based on the methyl-dependent DNA endonuclease GlaI coupling with double cascaded strand displacement amplification and CRISPR/Cas12a (GlaI-DC-SDA-CRISPR/Cas12a). The GlaI enables highly specific recognition and digestion of methylated target site (dsDNA) but leaves unmethylated target intact. Therefore, only methylated DNA can be digested to produce two free 3'-OH terminus for triggering the next SDA-CRISPR/Cas12a. Compared with the fluorescence response under single amplification template, DC-SDA with double amplification templates shows higher sensitivity. Benefiting from the high specificity of GlaI and the cascaded amplification effect of DC-SDA combined with CRISPR/Cas12a, the proposed method shows excellent performance for DNA methylation detection with low LOD (1.28 × 10-13 M), ultra-low background interference and wide detection range (2 × 10-13 to 4 × 10-11, 4 × 10-11 to 1 × 10-8 M). 0.1% of DNA methylation can be discriminated from the mixture with a mass of unmethylated DNA. Most importantly, the proposed assay can be applied to the actual detection of human serum and genomic DNA, as well as to distinguish normal cells from cancer cells. It can also quantify DNA methylation in genomic DNA (HCT116) with a LOD of 37.95 ng, indicating its great potential in early clinical cancer screening.