CRISPR-Cas9 base editing approaches to discover ubiquitination events promoting flavivirus infection

PROJECT SUMMARY Viruses extensively remodel the cells that they infect. Ubiquitination networks are frequent targets of viral manipulation, but compared to other modifications such as phosphorylation, our general understanding of functional ubiquitination events is limited. In this project, we will apply CRISPR-Cas9 base-editing approaches to assess ubiquitination site function in a systematic, unbiased manner in the context of flavivirus infection. We have previously quantified ubiquitination responses to West Nile virus (WNV) infection, identifying hundreds of modulated ubiquitination targets with unknown functions. In Specific Aim 1, we will systematically introduce mutations at these ubiquitinated residues and assess the impact on WNV infection in a pooled base-editing screening format. In Specific Aim 2, we will apply base editing to perturb ubiquitination enzyme active sites for enzymes that physically interact with flavivirus NS5 proteins. Successful completion of these aims will discover novel pathways that promote flavivirus infection and will provide CRISPR-Cas9 base-editing resources tailored towards research questions in virology and ubiqutination functions.