HOPS ARE SPECIALTY CROP IN THE US PRIZED FOR THEIR VALUE IN BREWING BEER. DUE TO THE EXPLOSIVE GROWTH OF THE CRAFT BEER MARKET THROUGHOUT THE LAST DECADE, DEMAND FOR HOPS AND OVERALL PRODUCTION HAVE BEEN STEADILY INCREASING. ALTHOUGH ONCE A REGIONAL CROP GROWN ALMOST EXCLUSIVELY IN THE PACIFIC NORTHWEST, OTHER MINOR PRODUCTION AREAS ARE EMERGING THROUGHOUT THE US AS INTEREST IN LOCALLY GROWN AGRICULTURAL PRODUCTS HAS RISEN. PLANT DISEASES ARE A MAJOR CHALLENGE FACED BY HOP GROWERS. POWDERY MILDEW, A FUNGUS, IS PARTICULARLY PROBLEMATIC IN THE MAJOR HOP GROWING AREAS. MILLIONS OF DOLLARS IN REVENUE ARE LOST ANNUALLY TO DECREASED HOPYARD PRODUCTIVITY AND INCREASED LABOR COSTS DUE TO MANAGEMENT OF THIS DISEASE. PESTICIDE APPLICATIONS AND PRACTICES SUCH AS EARLY PRUNING CAN HELP LIMIT THE SPREAD OF POWDERY MILDEW, BUT THE MOST RELIABLE WAY OF CONTROLLING THE DISEASE IS BY PLANTING HOP VARIETIES THAT HAVE BUILT-IN GENETIC RESISTANCE TO IT. THUS, POWDERY MILDEW RESISTANCE IS A HIGHLY PRIORITIZED TRAIT IN BREEDING OF NEW HOP VARIETIES. THE BEST-STUDIED SOURCE OF GENETIC RESISTANCE TO POWDERY MILDEW STRAINS PREVALENT IN THE PACIFIC NORTHWEST IS REFERRED TO AS R6. THE EXACT GENE THAT CAUSES R6 RESISTANCE IS NOT KNOWN, BUT PREVIOUS RESEARCH USING GENETIC MAPPING HAS DETERMINED ITS LOCATION TO BE WITHIN A SMALL REGION OF ONE OF THE CHROMOSOMES IN THE HOP GENOME.GENETIC ENGINEERING COULD BE A VERY USEFUL TOOL IN NARROWING DOWN THE IDENTITY OF THE GENE THAT CAUSES R6 RESISTANCE IN HOP. HOWEVER, NO PROCEDURES FOR GENETIC ENGINEERING OF AMERICAN HOP VARIETIES, INCLUDING ONES THAT HAVE THE R6 TRAIT, HAVE BEEN PUBLISHED IN THE SCIENTIFIC LITERATURE UP TO THIS POINT. THEREFORE, THE FIRST OBJECTIVE OF THIS PROJECT IS TO DEVELOP A PROCEDURE FOR GENETIC ENGINEERING IN AN AMERICAN HOP CULTIVAR BY TESTING SEVERAL HOP VARIETIES WHILE ADJUSTING MULTIPLE DIFFERENT VARIABLES IN THE GENETIC ENGINEERING PROCESS. ONE NEW TECHNIQUE WITHIN GENETIC ENGINEERING, REFERRED TO AS GENE EDITING, USES A SET OF MOLECULAR MACHINERY CALLED CRISPR/CAS9 TO TURN A SPECIFIC GENE ON, OFF OR OTHERWISE ALTER IT BY MAKING MINOR CHANGES TO ITS DNA SEQUENCE. FOR THE SECOND OBJECTIVE, WE PLAN TO USE CRISPR/CAS9 TO TURN OFF SEVERAL GENES WITHIN THE REGION WHERE THE R6 GENE IS KNOWN TO BE LOCATED IN THE HOP GENOME. THE THIRD OBJECTIVE INVOLVES EXPOSING PLANTS THAT HAVE BEEN EDITED IN THIS WAY TO THE POWDERY MILDEW FUNGUS, WHICH WILL ALLOW US TO DETERMINE THE SPECIFIC GENE RESPONSIBLE FOR R6. THE FOURTH AND FINAL OBJECTIVE INVOLVES EXPLORING A TECHNIQUE THAT WILL HELP TO DEVELOP GENE EDITED PLANTS IN WHICH THE CRSIPR/CAS9 MACHINERY HAS BEEN REMOVED FROM THE PLANT'S CELLS AFTER IT HAS ALREADY DONE ITS EDITING JOB.IF THIS PROJECT IS SUCCESSFUL, IT WILL HELP TO IMPROVE HOP BREEDING FOR POWDERY MILDEW RESISTANCE WHICH WOULD LEAD TO REDUCED EXPENSES FOR HOP GROWERS AND ULTIMATELY FOR BREWERS AND BEER CONSUMERS. FURTHERMORE, THE PROCEDURES DEVELOPED HERE WOULD PAVE THE WAY FOR FUTURE RESEARCH IN HOP GENETICS AND WOULD OPEN A ROUTE FOR GENE EDITING TO BE USED DIRECTLY AS A BREEDING TECHNIQUE TO MORE QUICKLY IMPROVE HOP CULTIVARS.