SUMMARY Interactions between central nervous system (CNS)-resident cells are highly heterogeneous; astrocytes and microglia nourish and protect neurons, while inflammatory subsets drive demyelination and neurodegeneration in neurologic diseases. However, the molecular mechanisms that control CNS-resident immune cell interactions remain mostly unknown because methods for defining the specific cell types, pathways and molecules involved are limited. In this project we apply two novel approaches that we developed to study astrocyte-microglia interactions during inflammation: 1) an in vivo barcoded rabies tracing strategy that analyzes cell connections and transcriptomes with single cell resolution, and 2) a droplet-based platform for genome-wide, unbiased CRISPR/Cas9 screening of interacting cell pairs. These methods provide a unique opportunity to study pathways used by CNS-resident immune cells to communicate with each other and control inflammation and neurodegeneration. We propose to: SPECIFIC AIM 1: Define the transcriptomes of single cells in pro-and anti-inflammatory networks. We will simultaneously sequence connections and single cell transcriptomes of CNS cells in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis (MS). These studies will identify pro- and anti-inflammatory cellular networks and the molecular mechanisms that regulate disease-relevant cell-cell interactions within these networks. SPECIFIC AIM 2: Identify novel astrocyte-microglia interactions using a droplet-based platform for CRISPR/Cas9 forward genetic screens. We will perform unbiased genome-wide screens for genes that participate in microglia-astrocyte crosstalk. We will co-incubate and microfluidically sort millions of picoliter water-in-oil droplets containing single microglia harboring a CRISPR/Cas9 library mutation and single astrocytes carrying a fluorescent reporter. Independent droplets do not mix, providing a powerful platform for the identification of immune interactions mediated by cell surface and soluble molecules.