Direct single-nucleotide resolution sequencing of DNA 5-methylcytosine using engineered DNA methyltransferase-mediated CMD-seq

5-Methylcytosine (5mC) is a crucial epigenetic modification in the mammalian genome, primarily occurring at CG dinucleotides. Accurate localization of 5mC is essential for understanding its functional significance. In this study, we discovered a novel DNA methyltransferase, designated M.MedI, from the bacterium <i>Mycoplasmopsis edwardii</i>. M.MedI exhibits carboxymethylation activity towards cytosines in CG sites in DNA. We further engineered a variant of M.MedI by mutating its critical active site residue 377 asparagine (N) to lysine (K), resulting in M.MedI-N377K. This engineered M.MedI-N377K enzyme demonstrated superior carboxymethylation activity towards cytosines in CG sites in both unmethylated and hemi-methylated DNA. Utilizing the newly identified M.MedI-N377K methyltransferase, we developed a novel method, engineered DNA methyltransferase-mediated carboxymethylation deamination sequencing (CMD-seq), for the stoichiometric detection of 5mC in DNA at single-nucleotide resolution. In CMD-seq, M.MedI-N377K efficiently transfers a carboxymethyl group to cytosines in CG sites in the presence of carboxy-<i>S</i>-adenosyl-l-methionine (caSAM), generating 5-carboxymethylcytosine (5camC). Subsequent treatment with the deaminase A3A deaminates 5mC to form thymine (T), which pairs with adenine (A) and is read as T, while 5camC remains unchanged, pairing with guanine (G) and being read as cytosine (C) during sequencing. We successfully applied CMD-seq to quantify 5mC sites in the promoters of tumor suppressor genes <i>RASSF1A</i> and <i>SHOX2</i> in human lung cancer tissue and adjacent normal tissue. The quantification results were highly comparable to those obtained using traditional bisulfite sequencing. Overall, CMD-seq provides a valuable tool for bisulfite-free, single-nucleotide resolution, and quantitative detection of 5mC in limited DNA samples.