Viral pathogens depend on the specialized microenvironments of their host for optimal replication and transmission. As obligate intracellular parasites, viruses, including influenza, rely on host proteins or "dependency factors" to successfully infect cells, replicate, and infect new cells. To interfere with the replicative cycle of viral pathogens, the host has evolved molecular defenses, herein referred to as "restriction factors." Inhibition of host dependency factors or the activation of host restriction factors can severely limit virus replication and constitute promising avenues for the development of next-generation therapeutics. We reasoned that exploiting emerging gene-editing methods would enable the unbiased and comprehensive identification of host-pathogen interactions important for viral replication and transmission. The overarching goal of this study is to understand, in-depth, how influenza viruses hijack the machinery of host cells to perpetuate itself. Achieving this goal is of utmost importance if we are to remediate the global economic and health impacts elicited by influenza virus. Towards this goal, we have developed the following two objectives:Objective 1. Establish a physiologically relevant swine cell-line permissive for influenza infection and Cas9-based gene editing. In the laboratory, canine kidney cells (MDCK) are routinely used to propagate Influenza virus. Although MDCK cells are permissive for influenza infection and replication to high titers, swine cells represent a physiological relevant cell type given that swine herds worldwide are susceptible to swine influenza virus. To date, however, there is a severe shortage of swine cell lines that are permissive for influenza virus infection. We have recently harvested primary respiratory cells from piglets and we will attempt to immortalize them by introducing either the telomerase gene or simian virus large T antigen (SV40). An immortalized cell line derived from swine respiratory cells will allow us to determine whether they are permissive for influenza infection and replication.Objective 2. Identify swine host genes required for influenza infection, replication, and pathogenesis using a CRISPR-Cas9 pooled library. The feasibility of identifying influenza dependency and restriction factors in swine cells will increase when using immortalized cells. Importantly, establishing a swine immortalized cell line is not an absolute requirement. With the advent of synthetic CRISPR-Cas9 ribonucleoproteins (crRNPs), it is possible to perform CRISPR-Cas9 based gene-editing directly in primary cells, albeit at an increased cost. An immortalized swine cell line derived from lung epithelium, MK1-OSU, is currently on hand. This cell lines needs to be modified to express Cas9.