Research progress of live-cell RNA imaging techniques.

RNA molecules play diverse roles in many physiological and pathological processes as they interact with various nucleic acids and proteins. The various biological processes of RNA are highly dynamic. Tracking RNA dynamics in living cells is crucial for a better understanding of the spatiotemporal control of gene expression and the regulatory roles of RNA. Genetically encoded RNA-tagging systems include MS2/MCP, PP7/PCP, boxB/λN22 and CRISPR-Cas. The MS2/MCP system is the most widely applied, and it has the advantages of stable binding and high signal-to-noise ratio, while the realization of RNA imaging requires gene editing of the target RNA, which may change the characteristics of the target RNA. Recently developed CRISPR-dCas13 system does not require RNA modification, but the uncertainty in CRISPR RNA (crRNA) efficiency and low signal-to-noise ratio are its limitations. Fluorescent dye-based RNA-tagging systems include molecular beacons and fluorophore-binding aptamers. The molecular beacons have high specificity and high signal-to-noise ratio; Mango and Peppers outperform the other RNA-tagging system in signal-to-noise, but they also need gene editing. Live-cell RNA imaging allows us to visualize critical steps of RNA activities, including transcription, splicing, transport, translation (for message RNA only) and subcellular localization. It will contribute to studying biological processes such as cell differentiation and the transcriptional regulation mechanism when cells adapt to the external environment, and it improves our understanding of the pathogenic mechanism of various diseases caused by abnormal RNA behavior and helps to find potential therapeutic targets. This review provides an overview of current progress of live-cell RNA imaging techniques and highlights their major strengths and limitations.