Fig. 5: USeqFISH profiling of pooled microRNA target sites in the AAV genome across neuronal subtypes in mouse cortical layers.

a, A pool of 13 variants (12 miRNA TSs in the 3′ UTR of the AAV genome and one control, ‘No TS’) was designed, packaged in AAV-PHP.eB and IV delivered to mice. We applied USeqFISH to the brain tissue harvested after 4 weeks of expression. b, Labeling of the cortex region by DAPI and the spatial location of cell types identified from clustering analysis based on endogenous gene expression. Two representative images of transgene expression (‘No TS’ and ‘433-3p’) are shown at the bottom. c, Endogenous (top, cividis color map) and viral gene expression profiles (enrichment: middle, viridis; log₂ fold change: bottom, coolwarm) across the cell type clusters identified. We identified 16 total clusters, including two L2/3 (red), three L4 (orange), one L5 (green), two L5/6 (sky blue), one L6a (blue), two L6b (purple), four inhibitory and one hippocampal neuron. Colored cell types are visualized in b.