The excellent specificity and selectivity of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated nuclease (Cas) is determined by CRISPR RNA’s (crRNA’s) interchangeable spacer sequence, as well as the position and number of mismatches between target sequence and the crRNA sequence. Some diseases are characterized by epigenetic alterations rather than nucleotide changes, and are therefore unsuitable for CRISPR-assisted sensing methods. Here we demonstrate an in vitro diagnostic tool to discriminate single CpG site methylation in DNA by the use of methylation-sensitive restriction enzymes (MSREs) followed by Cas12a-assisted sensing. Non-methylated sequences are digested by MSREs, resulting in fragmentation of the target sequence that influences the R-loop formation between crRNA and target DNA. We show that fragment size, fragmentation position and number of fragments influence the subsequent collateral trans-cleavage activity towards single stranded DNA (ssDNA), enabling deducting the methylation position from the cleavage activity. Utilizing MSREs in combination with Cas12a, single CpG site methylation levels of a cancer gene are determined. The modularity of both Cas12a and MSREs provides a high level of versatility to the Cas12a–MSRE combined sensing method, which opens the possibility to easily and rapidly study single CpG methylation sites for disease detection.

成簇规则间隔短回文重复序列 (CRISPR)/相关核酸酶 (Cas) 的出色特异性和选择性取决于 CRISPR RNA (crRNA) 的可互换间隔序列，以及靶序列和 crRNA 序列之间错配的位置和数量。一些疾病的特征是表观遗传改变而不是核苷酸变化，因此不适合 CRISPR 辅助传感方法。在这里，我们展示了体外通过使用甲基化敏感限制酶 (MSRE) 和 Cas12a 辅助传感来区分 DNA 中单个 CpG 位点甲基化的诊断工具。非甲基化序列被 MSRE 消化，导致靶序列片段化，从而影响 crRNA 和靶 DNA 之间的 R 环形成。我们表明，片段大小、片段位置和片段数量会影响后续的侧枝反式-对单链DNA (ssDNA)的切割活性，能够从切割活性中扣除甲基化位置。将 MSRE 与 Cas12a 结合使用，可确定癌症基因的单个 CpG 位点甲基化水平。Cas12a 和 MSRE 的模块化为 Cas12a-MSRE 组合传感方法提供了高度的多功能性，这为轻松快速地研究单个 CpG 甲基化位点以进行疾病检测提供了可能性。