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Adeno-associated viruses (AAVs) represent efficient and specific vectors for in vivo gene delivery. However, although there is a growing number of engineered AAV variants with potentially diverse tropisms, high-throughput, high-resolution assays to characterize their in vivo transduction profiles are lacking. Now, Jang et al. have developed a spatial transcriptomic method for AAV tropism profiling, called ultrasensitive sequential fluorescence in situ hybridization (USeqFISH). To demonstrate the ability of USeqFISH to profile systemic AAVs, a pool of six variants were analysed in the mouse brain. USeqFISH recapitulated known characteristics of each variant, such as overall transduction efficiency and major cell type tropism, and revealed distinct cell subtype tropisms across multiple brain regions. A new variant, AAV-PHP.AX, was found to efficiently and broadly transduce neuronal subtypes and astrocytes. In addition, the capability of USeqFISH to perform in situ profiling of pooled regulatory cargos of AAVs was demonstrated — the method profiled the regulatory effect of microRNA target sites inserted in the AAV genome on transgene expression, across neuronal subtypes in the mouse brain. USeqFISH is also applicable to other species — in situ AAV detection and integrative analysis of cell morphology and transcriptional profiles was performed in the non-human primate brain.