GlaI cleavage assistant isothermal exponential amplification coupling with CRISPR/Cas12a for ultrasensitive detection of CLDN11 methylation: A potential marker for lung adenocarcinoma

Abstract

DNA methylation is closely associated with cancer, and sensitive detection of DNA methylation is immensely significant for early cancer screening. In this study, for the first time, we identified CLDN11 methylation as a valuable marker for lung adenocarcinoma diagnosis and prognosis based on database analysis and experimental verification using clinical tissues and sera. Then, a flexible platform was proposed for ultrasensitive detection of CLDN11 methylation by integrating methylation-dependent restriction endonuclease GlaI cleavage with exponential amplification reaction (EXPAR) and CRISPR/Cas12a (GlaI–EXPAR–Cas12a platform). GlaI only recognizes and cleaves methylated target sites with excellent selectivity; the released fragments are primers for triggering EXPAR–CRISPR/Cas12a. Using Cas12a, false positives generated by EXPAR can be weakened to a negligible level. The proposed platform for detecting CLDN11 methylation achieved excellent performance, with a low limit of detection (1.25 × 10–15 M), and reliably identified CLDN11 methylation at an abundance of 0.1 % even in the presence of a large number of unmethylated fragments. More importantly, the proposed platform can be used for the quantification of human serum and genomic DNA, as well as to distinguish normal cells (HEK293) from cancer cells (H1299, A549 and H358). Through this integration platform, all 20 lung adenocarcinoma tissues exhibited significantly higher levels of CLDN11 methylation than a total of 16 paracancerous tissues. Taken together, the GlaI–EXPAR–Cas12a platform is ultrasensitive and convenient, and has perfect specificity. Further, it may function as a flexible platform for early screening of lung adenocarcinoma.