Ding Lihua, Cao Shengnan, Bai Lanxin, He Sitian, He Leiliang, Wang Yilin, Wu Yongjun, Yu Songcheng
College of Public Health, Zhengzhou University, Zhengzhou 450001, China.
College of Public Health, Zhengzhou University, Zhengzhou 450001, China; School of Nursing and Health, Zhengzhou University, Zhengzhou, China.
Spectrochim Acta A Mol Biomol Spectrosc. 2025 Mar 15;329:125520. doi: 10.1016/j.saa.2024.125520. Epub 2024 Dec 2.
The identification of DNA methylation at specific sites is crucial for the early detection of cancer since DNA methylation is intimately associated to the occurrence and development of cancer. Herein, two types of sensors that can detect site-specific DNA methylation were developed to meet practical requirements using methylation sensitive restriction endonuclease and CRISPR/Cas12a. To accomplish rapid detection of target, an AciI-mediated CRISPR/Cas12a assay was developed by coupling AciI to recognize DNA methylation with Cas12a to identify site-specific DNA. Since protospacer adjacent motif (PAM)-dependent endonuclease activity and trans-cleavage activity of Cas12a, it is possible to detect site-specific DNA methylation within 2 h with high specificity and acceptable sensitivity. To satisfy the needs of trace target detection, we developed an GlaI-strand displacement amplification (SDA) assisted CRISPR/Cas12a system. The system converts double-stranded methylated DNA to abundant single-stranded by GlaI and SDA. Then, the combination of SDA and CRISPR/Cas12a enable cascades amplification of signal. The approach can therefore be used to detect methylation at different specified sites, even those without PAM, and can increase sensitivity with a detection limit down to 8.19 fM. Importantly, the assay can distinguish between colorectal cancer and precancerous tissue, as well as identify colorectal patients and healthy people. This study provides a new avenue for the development of new biosensors for methylation analysis, and the two methods devised have the potential to meet the multiple requirements of site-specific methylation testing in various clinical settings.
特定位点DNA甲基化的识别对于癌症的早期检测至关重要，因为DNA甲基化与癌症的发生和发展密切相关。在此，利用甲基化敏感限制性内切酶和CRISPR/Cas12a开发了两种能够检测位点特异性DNA甲基化的传感器，以满足实际需求。为了实现对目标的快速检测，通过将识别DNA甲基化的AciI与识别位点特异性DNA的Cas12a偶联，开发了一种AciI介导的CRISPR/Cas12a检测方法。由于Cas12a的原间隔相邻基序（PAM）依赖性内切酶活性和反式切割活性，有可能在2小时内以高特异性和可接受的灵敏度检测位点特异性DNA甲基化。为了满足痕量目标检测的需求，我们开发了一种GlaI链置换扩增（SDA）辅助的CRISPR/Cas12a系统。该系统通过GlaI和SDA将双链甲基化DNA转化为大量单链。然后，SDA与CRISPR/Cas12a的结合实现信号的级联放大。因此，该方法可用于检测不同特定位点的甲基化，甚至是那些没有PAM的位点，并且可以将检测限降低至8.19 fM来提高灵敏度。重要的是，该检测方法可以区分结直肠癌和癌前组织，还能识别结直肠癌患者和健康人。本研究为开发用于甲基化分析的新型生物传感器提供了一条新途径，所设计的两种方法有可能满足各种临床环境中位点特异性甲基化检测的多种需求。
