Abstract：
The precise determination of DNA methylation at specific sites is critical for the timely detection of cancer, as DNA methylation is closely associated with the initiation and progression of cancer. Herein, a novel ratiometric fluorescence method based on the methylation-sensitive restriction enzyme (MSRE), CRISPR/Cas12a, and catalytic hairpin assembly (CHA) amplification were developed to detect site-specific methylation with high sensitivity and specificity. In detail, AciI, one of the commonly used MSREs, was employed to distinguish the methylated target from nonmethylated targets. The CRISPR/Cas12a system was utilized to recognize the site-specific target. In this process, the protospacer adjacent motif and crRNA-dependent identification, the single-base resolution of Cas12a, can effectively ensure detection specificity. The trans-cleavage ability of Cas12a can convert one target into abundant activators and can then trigger the CHA reaction, leading to the accomplishment of cascaded signal amplification. Moreover, with the structural change of the hairpin probe during CHA, two labeled dyes can be spatially separated, generating a change of the Förster resonance energy transfer signal. In general, the proposed strategy of tandem CHA after the CRISPR/Cas12a reaction not only avoids the generation of false-positive signals caused by the target-similar nucleic acid but can also improve the sensitivity. The use of ratiometric fluorescence can eradicate environmental effects by self-calibration. Consequently, the proposed approach had a detection limit of 2.02 fM. This approach could distinguish between colorectal cancer and precancerous tissue, as well as between colorectal patients and healthy people. Therefore, the developed method can serve as an excellent site-specific methylation detection tool, which is promising for biological and disease studies.
摘要：
精确测定特定位点的DNA甲基化对于及时检测癌症至关重要。DNA甲基化与癌症的发生和发展密切相关。在这里，一种基于甲基化敏感限制酶（MSRE）的新型比率荧光法，CRISPR/Cas12a,和催化发夹组装（CHA）扩增被开发用于检测具有高灵敏度和特异性的位点特异性甲基化。详细来说,AciI,一种常用的MSRE，用于区分甲基化靶标和非甲基化靶标。CRISPR/Cas12a系统用于识别位点特异性靶标。在这个过程中,原型间隔区相邻基序和crRNA依赖性鉴定，Cas12a的单基分辨率，能有效保证检测的特异性。Cas12a的反式切割能力可以将一个靶标转化为丰富的激活剂，然后可以触发CHA反应，导致级联信号放大的完成。此外，随着CHA过程中发夹探针的结构变化，两种标记的染料可以在空间上分开，产生福斯特共振能量转移信号的变化。总的来说,所提出的CRISPR/Cas12a反应后的串联CHA策略不仅避免了由靶相似核酸引起的假阳性信号的产生,而且还可以提高灵敏度.比率荧光的使用可以通过自校准消除环境影响。因此,该方法的检出限为2.02fM。这种方法可以区分结直肠癌和癌前组织，以及结直肠患者和健康人之间。因此,开发的方法可以作为一个优秀的位点特异性甲基化检测工具，这对生物学和疾病研究很有希望。
