Precise Recognition and Quantification of Locus‐Specific <scp>DNA</scp> Methylation Using Engineered <scp>ROS1</scp>^†

Comprehensive Summary 5‐Methylcytosine (5mC) plays pivotal roles in numerous biological processes. To gain a deeper understanding of the biological functions of 5mC, it is essential to develop methods for its quantitative analysis. Herein, we engineered the repressor of silencing 1 (ROS1) protein to enhance its glycosylase/lyase activity towards 5mC, resulting in an engineered ROS1 (eROS1) protein that can effectively excise 5mC from DNA. Using eROS1, we developed a method termed engineered ROS1‐mediated quantitative (eRMQ) analysis, for the locus‐specific quantification of 5mC in genomic DNA. This method capitalizes on the ability of eROS1 to selectively cleave 5mC, which creates a one‐nucleotide gap. The presence of this gap hinders the extension of DNA polymerase, leading to a reduction in extension products that can be evaluated using real‐time quantitative PCR (qPCR). The limit of detection for the eRMQ method was as low as 1 fM. Using the eRMQ method, we achieved the quantitative analysis of 5mC at individual sites within genomic DNA and demonstrated a significant reduction in 5mC levels in lung cancer tissues compared to adjacent normal tissues. Taken together, this study introduces eRMQ method for the quantitative analysis of 5mC in DNA, offering a valuable tool for exploring epigenetic regulation in human diseases.