Herpes simplex virus 1 (HSV-1) is a common virus that in rare cases invades the central nervous system (CNS), causing a devastating and potentially lethal viral encephalitis. Herpes simplex encephalitis (HSE) is the most common sporadic viral encephalitis in Western countries and despite antiviral therapy survivors often suffer from severe neurological deficits. The pathogenesis of HSE remains unclear, however susceptibility to HSV-1 in the CNS can occur due to a lack of cell-intrinsic viral resistance, which is caused by mutations in TLR3, UNC93B1, TRIF, TRAF3, and TBK1 impairing TLR3- and UNC93b-dependant IFN-α/β signaling. Despite the identification of these monogenic traits leading to impaired immunity to HSV-1 in the CNS, these genetic defects are only present in the minority of patients (~5%). We hypothesize that HSE as a complication of primary herpes simplex infection may result from other, as of yet unknown, single-gene inborn errors of immunity. Here we propose hPSC-derived neurons may serve as a tool not just for the study of candidate genes emerging from human genetic studies but may represent an alternative strategy to identify novel HSE-causing candidate genes. In this proposal, we will build on our exciting in vitro platform of generating highly defined and scalable hPSC-derived cortical neurons as a disease model for HSE, and we will combine this with a whole genome CRISPR/Cas9 screen to uncover novel HSV-1 susceptibility genes. In Aim 1 we propose a genome wide, pooled, loss-of-function screen based on our ability to generate hPSC-derived cortical neurons at large scale and high purity in a cell line with inducible expression of Cas9 containing a genome wide CRISPR library to identify novel candidates that trigger viral susceptibility to HSV-1 infection. In Aim 2 we will validate those candidate genes in a secondary screen, by generating single gene KO hPSC lines for functional assays, and by in silico analysis of validated candidate hits using a unique dataset of HSE patient whole-exome sequencing data. The unbiased discovery of genes responsible for the cell-intrinsic immunity to HSV-1 will open up new avenues for investigation and could dramatically increase our knowledge on the pathogenesis of HSE, knowledge that may aid in developing novel therapeutic strategies in addition to current antiviral therapy.