Project Summary Powassan virus (POWV) is a tick-borne flavivirus that can be transmitted to humans and causes symptoms including fever, vomiting, headache, and generalized weakness. The disease can progress to meningoencephalitis, resulting in altered mental status, seizures, aphasia, and paresis amongst other signs. Survivors of symptomatic disease are often left with residual sequelae and there is no specific treatment available. This study will identify host factors that POWV requires for its replication, focusing on human factors for which inhibitory drugs can potentially be developed. In the first aim, CRISPR/Cas9 will be used to knock out genes for factors previously identified as required for replication of two other flaviviruses and for which drugs could be developed. The knockout cells will be challenged with POWV and replication assessed. For those genes with identified drug interactions, wildtype cells will be treated with various doses of drugs targeting the factors and replication assessed to determine if the drugs inhibit POWV replication. In the second aim a CRISPR/Cas9 knockout library based on the druggable human genome will be generated and used to screen for potentially druggable host factors required by POWV for replication and cell killing. These factors would be targets for drug development in future studies.