作为一种主要的表观遗传修饰，DNA甲基化参与多种细胞功能，成为一种有前途的疾病诊断与监测生物标志物。在此，我们开发了一种甲基化敏感的转录增强单分子生物传感器，用于检测人类细胞和组织中的DNA甲基化。在这个生物传感器中，一个理性设计的转录机器被分成两个部分，包括启动转录的启动子序列（探针-P）和RNA合成的模板序列（探针-T）。 特定的DNA甲基化存在会通过探针-P和探针-T的序列特异性连接形成全长转录机器，从而启动大量ssRNA转录的合成。产生的ssRNA可以激活CRISPR / Cas12a催化荧光素和猝灭剂双标记信号探针的循环裂解，导致荧光素信号的恢复，可以通过单分子检测进行定量。利用分裂转录机器的高保真连接和转录和CRISPR / Cas12a裂解介导的双信号放大的高效性，这种单分子生物传感器实现了低检测限制为337 aM和高选择性。此外，它可以区分0.01％的甲基化水平，并甚至可以精确检测单个细胞和临床样品中的基因组DN甲基化，为表观遗传学研究和临床诊断提供了强有力的工具。 版权所有© 2023 Elsevier B.V.。保留所有权利。

As a major epigenetic modification, DNA methylation participates in diverse cellular functions and emerges as a promising biomarker for disease diagnosis and monitoring. Herein, we developed a methylation-sensitive transcription-enhanced single-molecule biosensor to detect DNA methylation in human cells and tissues. In this biosensor, a rationally designed transcription machine is split into two parts including a promoter sequence (probe-P) for initiating transcription and a template sequence (probe-T) for RNA synthesis. The presence of specific DNA methylation leads to the formation of full-length transcription machine through sequence-specific ligation of probe-P and probe-T, initiating the synthesis of abundant ssRNA transcripts. The resultant ssRNAs can activate CRISPR/Cas12a to catalyze cyclic cleavage of fluorophore- and quencher-dual labeled signal probes, resulting in the recovery of the fluorophore signal that can be quantified by single-molecule detection. Taking advantages of the high-fidelity ligation of split transcription machine and the high efficiency of transcription- and CRISPR/Cas12a cleavage-mediated dual signal amplification, this single-molecule biosensor achieves a low detection limit of 337 aM and high selectivity. Moreover, it can distinguish 0.01% methylation level, and even accurately detect genomic DNA methylation in single cell and clinical samples, providing a powerful tool for epigenetic researches and clinical diagnostics.Copyright © 2023 Elsevier B.V. All rights reserved.