Detection of N4-Acetylcytosine in Escherichia coli Using APOBEC3A-Mediated Deamination Sequencing

N4-acetylcytosine (4acC) is a naturally occurring DNA modification; however, its presence and distribution characteristics in prokaryotes remain unclear. In this study, based on the differential deamination activity of APOBEC3A (A3A) toward cytosine (C) and 4acC, we developed a single-base-resolution sequencing method termed 4acC-A3A-seq (4acC-APOBEC3A-sequencing). Following validation using template strands and optimization of reaction conditions, we applied this method to the genome of Escherichia coli (E. coli) and successfully identified 2271 high-confidence candidate 4acC sites, generating the first single-base-resolution genome-wide map of 4acC in a prokaryote. This study provided a key technological approach and foundational data for further exploration of the biological functions and evolutionary conservation of 4acC at single-base resolution.