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

Abstract

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.

Keywords: brain; calcium imaging; cell type; enhancer; labeling; optogenetics; primate; snATAC-seq; specificity.