abcFISH enables multiplexed, single-molecule visualization of circular RNA spatial heterogeneity

Understanding the subcellular localization of RNAs is critical for their functions, but visualizing circular RNAs (circRNAs) has been hindered by their sequence overlap with linear counterparts. Here we developed amplicon-based circRNA fluorescence in situ hybridization (abcFISH), which uses an optimized rolling-circle amplification strategy targeting back-splicing junction sites for robust, high specificity, single-molecule circRNA imaging. abcFISH enables quantitative, multiplexed imaging in cells and tissues, allowing us to reveal alternative circularization patterns from the single locus, uncover cell-type-specific circRNA expression linked to combinatorial effects of RNA-binding proteins, map distinct spatial distribution patterns of circRNAs in neurons and brain tissues, and show functional colocalization of circRNA Cdr1as and long noncoding RNA Cyrano. We further applied abcFISH to validate therapeutic double-stranded circRNA aptamers, simultaneously visualizing AAV-delivered double-stranded circRNA aptamers and the resulting reduction in astrocyte infiltration around transduced cells. Collectively, abcFISH provides a robust, user-friendly toolkit for deciphering circRNA localization and function in vivo.