**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** THE HIGH PATHOGENIC AVIAN INFLUENZA VIRUS, AN INFLUENZA A VIRUS, HAS BEEN A DEVASTATING ISSUE WORLDWIDE, INCLUDING THE OUTBREAK THAT HAS RESULTED IN THE LOSS OF OVER 40 MILLION BIRDS IN THE UNITED STATES IN 2022. ALTHOUGH THE ECONOMIC IMPACT OF THE CURRENT OUTBREAK STILL REMAINS UNDETERMINED, AN OUTBREAK COMES WITH MULTIPLE LAYERS OF ECONOMIC IMPACTS, I.E., THE RECENT 2014-15 OUTBREAK THAT RESULTED IN BILLION-DOLLAR LOSSES TO THE POULTRY INDUSTRY. THE 2014-15 OUTBREAK RESULTED IN THE DEATH OF MORE THAN 50 MILLION BIRDS IN THE U.S. WHICH COMPRISED 12% AND 8% OF THE ANNUAL LAYER AND TURKEY POPULATIONS, RESPECTIVELY. AS A RESULT, APPROXIMATELY 13% OF POULTRY EXPORTS DROPPED DURING THE FIRST HALF OF 2015 DUE TO THE EXPORT BAN ON U.S. PRODUCED POULTRY, WHICH AFFECTED AS MANY AS 233,000 POULTRY FARMS. DEPSITE THE HUGE IMPACTS AND ASTRONOMICAL BIRD LOSSES, THERE REMAINS A LACK OF STRATEGIES AGAINST THE AVIAN INFLUENZA VIRUS (AIV) BEYOND THE PASSIVE BIOSECURITY MEASURES SUCH AS SURVEILLANCE, QUARANTINE, AND PERIMETER DEPOPULATION. FURTHERMORE, THE EMERGENCE OF INFLUENZA A VIRUSES FROM ZOONOTIC RESERVOIRS POSES A GREAT THREAT TO HUMAN HEALTH AND CONTROLLING THE DISEASE IN THE ANIMAL SOURCE IS CRITICAL TO DECREASE RISK TO HUMANS AS INFLUENZA VIRUSES ARE IMPOSSIBLE TO ERADICATE.BASED ON THE CURRENT AND POTENTIAL THREAT OF AIV OUTBREAKS, IT IS URGENT TO DEVELOP BETTER AND MORE ACTIVE STRATEGIES TO LIMIT THE DAMAGE CAUSED BY THE VIRUS. PREVIOUS STUDIES HAVE MOSTLY FOCUSED ON IDENTIFYING AND UNDERSTANDING VIRAL HOST FACTORS RELATED TO HUMAN INFLUENZA A VIRUS (IAV) INFECTION, HOWEVER VERY FEW HAVE FOCUSED ON IDENTIFYING THESE FACTORS IN POULTRY. ON THE MAMMALIAN SIDE, MULTIPLE GENOME-WIDE SCREENING APPROACHES HAVE BEEN EMPLOYED TO IDENTIFY HOST FACTORS INVOLVED IN IAV INFECTION. ALTHOUGH SOME COMMON HITS AND PATHWAYS HAVE BEEN REVEALED, META-ANALYSES HAVE DEMONSTRATED VERY LITTLE OVERLAP IN IDENTIFIED IAV HOST FACTORS WHICH COULD BE DUE TO A NUMBER OF THINGS INCLUDING DIFFERENCESIN STRAINS USED, TIME POINTS ASSAYED AND FUNCTIONAL READOUTS CHOSEN. FURTHERMORE, HOST FACTORS IDENTIFIED IN MAMMALIAN MODELS HAVE VERY LITTLE OR ZERO TRANSLATION TO POULTRY DUE TO THE SMALLER REPERTOIRE OF IMMUNE GENES IN AVIAN SPECIES. THUS, ALTERNATIVE SCREENING STRATEGIES ARE NECESSARY TO UNCOVER HOST FACTORS AND PATHWAYS INVOLVED IN AIV INFECTION IN POULTRY AND THE RECENT ADVANCEMENTS IN CRISPR/CAS9 TECHNOLOGIES PROVIDE A NEW METHOD TO DO THIS.CRISPR SCREENS ARE A POWERFUL SOURCE OF BIOLOGICAL DISCOVERY, ENABLING THE UNBIASED INTERROGATION OF GENE FUNCTION IN A WIDE RANGE OF APPLICATIONS AND SPECIES. IN POOLED CRISPR SCREENS, VARIOUS ENCODED PERTURBATIONS ARE INTRODUCED INTO POOLS OF CELLS THROUGH LENTIVIRAL TRANSDUCTION WHICH ALLOWS US TO INTRODUCE ONE PERTURBATION IN A SINGLE CELL. THE PERTURBED CELLS PROLIFERATE/SURVIVE UNDER A BIOLOGICAL CHALLENGE SUCH AS VIRAL INFECTION. SUBSEQUENTLY, THE PERTURBATION-INDUCED EFFECTS ARE EVALUATED BY NEXT-GENERATION SEQUENCING- BASED IDENTIFI,CATION OF THE GUIDE RNA'S THAT SPECIFY EACH PERTURBATION RESULTING IN A PARTICULAR PHENOYTPE. THE TYPICAL OUTPUT OF SUCH SCREENS ARE RANKED LISTS OF GENES THAT CONFER SENSITIVITY OR RESISTANCE TO THE BIOLOGICAL CHALLENGE OF INTEREST, SUCH AS VIRAL INFECTION. IN ADDITION, IT IS NOW POSSIBLE TO STUDY THE MOLECULAR MECHANISM OF EACH PERTURBATION AT THE SINGLE-CELL LEVEL DUE TO THE DEVELOPMENT OF SINGLE-CELL-BASED TRANSCRIPTOMICS.APPLYING THESE NEW TECHNOLOGIES TO ANIN VITROAVIAN MODEL WILL ALLOW FOR THE STREAMLINED IDENTIFICATION OF THE CANDIDATE HOST FACTORS INVOLVED IN RESISTANCE TO AVIAN INFLUENZA VIRUS. WHILE THESE FACTORS COULD BE IDENTIFIED BY OTHER MOLECULAR BIOLOGY-BASED APPROACHES, SEARCHING FOR VIRAL RESISTANCE HOST FACTORS ONE BY ONE IS BOTH COSTLY AND TIMELY. WE WILL UTILIZE WHOLE-GEMONE KNOCKOUT CRISPR SCREENING TO IDENTIFY CANDIDATE GENE(S) INVOLVED IN AVIAN INFLUENZA RESISTANCE IN ANIN VITROAVIAN CELL CULTURE MODEL AND UTILIZE FUNCTIONAL GENOMICS TO ANNOTATE AND CHARACTERIZE IDENTIFIED HOST FACTORS THAT MAY PLAY A ROLE IN AVIAN INFLUENZA RESISTANCE. THESE EXPERIMENTS WILL PROVIDE FOUNDATIONAL KNOWLEDGE THAT WILL EXPAND OUR KNOWLEDGE IN POTENTIAL CANDIDATE GENE(S) THAT MAY PLAY A ROLE IN AVIAN INFLUENZA RESISTANCE IN THE CHICKEN. IDENTIFICATION OF CANDIDATE GENES AND UNDERSTANDING THEIR FUNCTIONS WILL HELP DEVELOP ANTIVIRAL STRATEGIES AND AID IN THE DEVELOPMENT OF INFLUENZA VIRUS RESILIENT GENOME EDITED CHICKENS.