A cross-species AAV platform for precise microglial interrogation.

Targeting microglia with adeno-associated viruses (AAVs) remains challenging because of inefficient transduction, off-target expression, and inconsistent payload performance. Here, we develop an integrated, Cre-independent AAV platform that combines a compact 446-bp microglia-specific regulatory element (MSRE), a high-yield AAV2/9.m capsid, microRNA-mediated neuronal de-targeting, and a microglia-tailored codon-optimization strategy. Following direct intracranial delivery, the optimized vector achieves robust, long-lasting transgene expression with greater than 90% microglial specificity across multiple mouse brain regions, including in an Alzheimer's disease model. With doxorubicin-assisted delivery, the platform also enables efficient and selective microglial targeting in rats, rhesus macaques, and human ex vivo brain slices. Codon optimization supports the expression of otherwise challenging functional payloads, enabling calcium imaging, chemogenetic manipulation, and CRISPR-Cas9-mediated genome editing. This cross-species toolkit provides a versatile resource for investigating microglial biology and developing microglia-directed therapeutic strategies.