** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** A HOMING GENE DRIVE TARGETING A FEMALE-ESSENTIAL GENE IS POTENTIALLY A VERY EFFICIENT GENETIC MECHANISM FOR CONTROLLING POPULATIONS OF INSECT PESTS. HERE WE ARE TARGETING THREE FLIES: DROSOPHILA SUZUKII, AN INVASIVE PEST OF SOFT-SKINNED FRUITS; DROSOPHILA HYDEI, A PEST OF STORED SWEETPOTATO; AND COCHLIOMYIA HOMINIVORAX, A DEVASTATING OBLIGATE PARASITE OF LIVESTOCK. A NATIVE OF ASIA, DROSOPHILA SUZUKII, ALSO KNOWN AS SPOTTED WING DROSOPHILA, IS NOW WIDESPREAD IN THE USA. FEMALES HAVE A HIGHLY DEVELOPED EGG LAYING APPARATUS WHICH MEANS THEY CAN INFEST A WIDE RANGE OF HOST FRUIT BEFORE HARVEST. GROWERS ARE CURRENTLY APPLYING BROAD SPECTRUM INSECTICIDES MORE FREQUENTLY THAN IN THE PAST TO PROTECT FRUIT, BUT THE EFFECTIVENESS OF THE SPRAYS IS WEATHER DEPENDENT. RESISTANCE TO INSECTICIDES HAS BEEN REPORTED IN CALIFORNIA. NEW CONTROL TACTICS ARE NEEDED. IN NORTH CAROLINA, DROSOPHILA HYDEI HAS EMERGED AS A SERIOUS PEST IN FACILITIES THAT STORE SWEETPOTATOES. IN WARMER MONTHS, THE FLIES CAN BE PARTICULARLY NUMEROUS REQUIRING THE FACILITIES TO RELEASE INSECTICIDE SPRAYS MULTIPLE TIMES A DAY. RECENT RESEARCH BY THE CO-PD HULSETH AND COLLEAGUES AT NC STATE HAS SHOWN THAT D. HYDEI IS LIKELY TO BE AN IMPORTANT VECTOR FOR CERATOCYCTIS FIMBRIATA, THE CAUSAL AGENT OF SWEETPOTATO BLACK ROT. FEMALE NEW WORLD SCREWWORM (COCHLIOMYIA HOMINIVORAX) LAY THEIR EGGS IN OPEN WOUNDS OR A NATURAL ORIFICE. THE HATCHED LARVAE THEN FEED ON THE ANIMAL'S LIVING TISSUE. ANIMALS WITH SEVERE SCREWWORM INFESTATIONS MAY DIE IF UNTREATED. ONE OF THE GREATEST ACHIEVEMENT'S OF THE USDA IN THE 20TH CENTURY WAS THE DEVELOPMENT OF THE STERILE INSECT TECHNIQUE (SIT) FOR THE ERADICATION OF C. HOMINIVORAX FROM THE U.S.A INITIALLY AND SUBSEQUENTLY FROM MEXICO ALL OF CENTRAL AMERICA. TO PREVENT RE-INFESTATION FROM SOUTH AMERICA, STERILIZED FLIES WERE BEING CONSTANTLY RELEASED IN A BUFFER ZONE IN EASTERN PANAMA AND ALONG THE BORDER WITH COLOMBIA. HOWEVER, DUE TO THE RECENT OUTBREAK IN PANAMA, STERILE FLIES ARE CURRENTLY BEING RELEASED IN WESTERN PANAMA AND IN COSTA RICA. IT WOULD BE VERY EXPENSIVE AND CHALLENGING TO USE SIT TO SUPPRESS SCREWWORM IN COUNTRIES WHERE IT REMAINS ENDEMIC (E.G. BRAZIL, COLOMBIA, CUBA, JAMAICA).WE HAVE PREVIOUSLY MADE EFFICIENT D. SUZUKII SPLIT HOMING GENE DRIVE STRAINS THAT TARGET THE DOUBLESEX (DSX) GENE, WHICH IS ESSENTIAL FOR FEMALE DEVELOPMENT. IN A SPLIT GENE DRIVE THE CAS9 GENE IS LOCATED ON A DIFFERENT CHROMOSOME THAN THE DSX GENE. WE INSERTED A SINGLE GUIDE RNA (SGRNA) GENE INTO THE DSX FEMALE EXON ALONG WITH A RED FLUORESCENT PROTEIN GENE TO MAKE IT EASY TO IDENTIFY OFFSPRING THAT INHERITED THE DISRUPTED DSX GENE. WHEN CAS9 IS EXPRESSED IN THE GERMLINE ALONG WITH THE SGRNA, THE WILD TYPE DSX GENE IS CUT AND THE BROKEN. THE DISRUPTED DSX GENE IS THEN USED AS A TEMPLATE TO REPAIR THE BROKEN DNA. IN THIS WAY BOTH COPIES OF THE DSX GENE THEN HAVE THE FEMALE EXON WITH THE SGRNA GENE AND RED FLUORESCENT PROTEIN GENE. THIS PROCESS IS CALLED HOMING. WE MADE DRI,VE STRAINS THAT WERE HETEROZYGOUS FEMALE STERILE (I.E. DOMINANT) AND HOMOZYGOUS FEMALE STERILE (I.E. RECESSIVE). MATHEMATICAL MODELING SHOWED THAT THE DRIVE STRAINS SHOULD BE EQUALLY EFFECTIVE FOR POPULATION SUPPRESSION AT MUCH LOWER RELEASE RATIOS THAN USED FOR SIT (AND THUS LESS EXPENSIVE). FURTHER MODELING SHOWED THAT HAD WE MADE A FULLY AUTONOMOUS DRIVE, THE DOMINANT STERILE DRIVE WOULD BE LESS INVASIVE THAN THE RECESSIVE STERILE DRIVE. A FULLY AUTONOMOUS DRIVE HAS BOTH CAS9 AND SGRNA GENES INSERTED INTO THE DSX GENE. A LESS INVASIVE DRIVE WOULD BE CONSIDERED LOWER RISK FOR A FUTURE FIELD RELEASE. WE PROPOSE TO BUILD DOMINANT FEMALE STERILE HOMING GENE DRIVE STRAINS FOR D. SUZUKII AND D. HYDEI. GENE DRIVE EFFICIENCIES WILL BE MEASURED UNDER DIFFERENT ENVIRONMENTAL CONDITIONS. FOR D. SUZUKII, WE ALSO PROPOSE TO BUILD SPLIT HOMING GENE DRIVES THAT CARRY TWO OR MORE SGRNA GENES IN THE DSX GENE. IT IS PREDICTED THAT A GENE DRIVE THAT USES TWO OR MORE SGRNAS WOULD BE LESS LIKELY TO FAIL DUE TO THE EMERGENCE OF RESISTANCE TO DRIVE. AS WE ARE NOT ALLOWED TO WORK WITH C.HOMINIVORAX IN NORTH CAROLINA, WE PROPOSED TO DEVELOP SPLIT GENE DRIVE STRAINS IN COCHLIOMYIA MACELLARIA, A VERY CLOSE RELATIVE THAT IS NOT A PEST AND IS COMMONLY FOUND IN THE USA. IF THE DRIVE SYSTEMS ARE EFFECTIVE, WE WILL SEND THE GENE CONSTRUCTS TO OUR COLLABORATORS IN URUGUAY FOR EVALUATION IN C. HOMINIVORAX. URUGUAY HAS RECENTLY COMPLETED THE CONSTRUCTION OF A BIOSECURE FACILITY FOR SCREWWORM GENE DRIVE RESEARCH.LAB CAGE POPULATION SUPPRESSION EXPERIMENTS WILL BE PERFORMED WITH THE D. SUZUKII AND D. HYDEI DRIVE STRAINS. MATHEMATICAL MODELS WILL BE REFINED BASED ON THE GENE DRIVE AND COLLECTED LIFE HISTORY DATA. THE MODELS WILL GUIDE LAB CAGE SUPPRESSION STUDIES AND PLANNED FUTURE TRIALS IN LARGE FIELD CAGES AND SWEETPOTATO STORAGE FACILITIES.