PROJECT SUMMARY/ABSTRACT Microglia are the long-lived resident macrophages of the brain and spinal cord, which constantly survey the microenvironment to respond rapidly to injury and infection. Unlike neurons or other glia, microglia are transplantable, making them ideal candidates for targeted therapies to treat neurological diseases. A major barrier to creating and refining such treatments is an inability to genetically engineer microglia with lentiviruses. This proposal aims to identify and overcome microglia restriction factors to enable gene editing with lentiviruses and gain insights into microglia-mediated antiviral immunity. Aim 1 will test the hypothesis that cGAS/STING, a known anti-viral pathway, underlies microglial viral restriction using inhibitors and transgenic mouse microglia in vitro, with in vivo validation using a new microglia-specific Cre mouse. In Aim 2, I will perform an unbiased microglia-focused CRISPR knockout screen using newly created AAVs with microglia tropism to discover new anti-viral pathways that prevent lentiviral infection. Aim 2 demonstrates the first CRISPR screen in primary microglia. This Early Independence application offers a vision for my independent research program. It is based on a platform of microglia-based discovery tools I created over the last decade, and driven by my work as a pediatric neurologist who cares for children with neuroimmunological conditions including leukodystrophies and interferonopathies. It will provide new approaches to genetic targeting of microglia for neurotherapeutics, direct testing of microglia antiviral immunity, and will influence future directions related to the neurological sequelae of HIV latency and the pathogenesis of neuroimmunological disease.