Identification and application of cell-type-specific enhancers for the macaque brain

Genetic targeting methods for monitoring and manipulating neuronal activity are not widely used for studying the primate brain, largely owing to the lack of a cell-type-specific targeting method. Using single-cell RNA and ATAC sequencing of macaque brains combined with in vivo screening, we identified a large set of enhancers capable of driving targeted gene expression in specific cell types. AAV vectors driven by these enhancers successfully targeted layer-specific glutamatergic neurons, GABAergic interneuron subtypes, astrocytes, and oligodendrocytes with high specificity. Cross-species comparison revealed that some macaque enhancers are conserved and functional across species, but enhancers with layer-specific targeting in macaques did not label neurons in mice, highlighting evolutionary differences in cortical CREs. Targeting precision was further improved using a FLPo-dependent intersectional approach with two enhancers. These enhancer-AAVs were validated by monitoring and manipulating activity in macaque visual cortex, providing valuable tools to dissect primate neural circuit functions.