Visualization and Quantification of Single‐Base m6A Methylation

N 6‐methyladenosine (m6A) has emerged as the most prevalent form of RNA modification found across various RNA classes. The detection and quantification of m6A RNA modifications under various physiological conditions are crucial for elucidating disease mechanisms and identifying potential therapeutic targets. However, visualizing intracellular m6A modifications at single‐nucleotide resolution remains a significant challenge. Existing methods based on high‐throughput sequencing or in vitro assays are not suitable for in situ m6A RNA imaging. In this work, we introduce the TadA8.20‐assisted N6‐methyladenosine RNA imaging at single‐nucleotide resolution (TARS) method for precise visualization and quantification of both A and m6A forms at specific RNA sites within single cells. Validation studies using TARS on MALAT1 lncRNA in HeLa cells and CCND1 mRNA in breast cancer cell lines demonstrated its high specificity and efficiency in mapping and quantifying m6A modifications at single‐nucleotide resolution. TARS represents a novel tool that advances m6A RNA modification research by offering accurate and detailed insights into m6A modifications at the single‐nucleotide level.