Single-Molecule Counting of FTO in Human Breast Tissues Based on a Rolling Circle Transcription Amplification-Driven Clustered Regularly Interspaced Short Palindromic Repeat─Cas12a

N6-methyladenosine modification as an mRNA modification in mammalian cells is dynamically reversible, regulated by RNA demethylase [e.g., fat mass and obesity-associated protein (FTO)]. The abnormal expression of FTO is closely related to numerous diseases (e.g., various cancers and obesity). Herein, we demonstrate the single-molecule counting of FTO in human cancer cells and breast tissues based on a T7 RNA polymerase-mediated rolling circle transcription (RCT) amplification-driven clustered regularly interspaced short palindromic repeat (CRISPR)─Cas12a. When FTO is present, it demethylates the DNA substrate, initiating the DpnII-mediated cleavage reaction. After magnetic separation, the cleaved DNA fragments trigger the T7 RNA polymerase-mediated RCT amplification, activating CRISPR-/Cas12a-mediated cleavage of signal probes and releasing abundant FAM molecules that are simply counted via single-molecule detection. In this assay, only target FTO can generate CRISPR RNAs, efficiently improving detection specificity. Moreover, the integration of single-molecule detection with magnetic separation achieves zero background and effectively enhances detection sensitivity. This method can specifically and sensitively monitor FTO activity with a limit of detection of 1.20 × 10–13 M, and it may measure FTO at the single-cell level. Furthermore, it may accurately discriminate the FTO expression level in breast tissues between healthy persons and breast cancer patients and screen the FTO inhibitors as well, with great potential in clinical diagnosis and drug discovery.