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

Functional RNAs often exhibit distinct subcellular localization, but studying circular RNA (circRNA) localization has been challenging due to their extensive sequence overlap with linear mRNAs. We developed amplicon-based circular RNA fluorescence in situ hybridization (abcFISH), a method that employs an optimized rolling circle amplification (RCA) strategy targeting back-splicing junction (BSJ) 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 circPOLR2A(9,10) expression related to a combinatorial effect of RNA-binding proteins; map the distinct spatial distribution patterns of multiple circRNAs in neurons and brain tissues; and show the functional interplay of Cdr1as and Cyrano co-localization. Furthermore, we applied abcFISH to validate the efficacy of therapeutic double-stranded circRNA aptamers (ds-cRNAs), simultaneously visualizing AAV-delivered ds-cRNAs and a consequent reduction in astrocyte infiltration around transduced cells. Collectively, abcFISH establishes a robust and user-friendly toolkit for deciphering circRNA localization and function in vivo.