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48 reviewsUnderstanding the diverse dynamic behaviors of individual RNA molecules in single cells requires visualizing them at high resolution in real time. However, single-molecule live-cell imaging of unmodifed endogenous RNA has not yet been achieved in a generalizable manner. Here, we present single-molecule live-cell fuorescence in situ hybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR–Csm complex with multiplexed guide RNAs for direct and efcient visualization of single RNA molecules in a range of cell types, including primary cells. Using smLiveFISH, we track individual native NOTCH2 and MAP1B transcripts in living cells and identify two distinct localization mechanisms including the cotranslational translocation of NOTCH2 mRNA at the endoplasmic reticulum and directional transport of MAP1B mRNA toward the cell periphery. This method has the potential to unlock principles governing the spatiotemporal organization of native transcripts in health and disease.
