CARROT (DAUCUS CAROTA) IS A ROOT VEGETABLE OFTEN CLAIMED TO BE THE PERFECT HEALTH FOOD. IT IS TASTY AND HIGHLY NUTRITIOUS, CONTAINING HIGH LEVELS OF &BETA;-CAROTENE, FIBER, POTASSIUM, ANTIOXIDANTS, VITAMIN K1 AND VITAMIN B6. CARROTS ARE FOUND IN DIFFERENT COLORS, SUCH AS YELLOW, WHITE, ORANGE, RED AND PURPLE. CARROTS ARE AMONG THE TOP 10 VEGETABLES, BASED ON GLOBAL PRODUCTION RECORDS OF PRIMARY VEGETABLES BASED ON FAO 2017 (HTTP://WWW.FAO.ORG/STATISTICS). INTERESTINGLY, CARROT PRODUCTION PER CAPITA IN THE AMERICAS IS ONLY A THIRD AND A QUARTER TO THAT OF OCEANIA AND EUROPE, RESPECTIVELY. IT SUGGESTS AMPLE ROOM FOR PRODUCTION IMPROVEMENT AS AMERICANS ARE PROJECTED TO EAT A HEALTHIER DIET, RICH IN NUTRITIOUS VEGETABLES. CARROT IS A RELATIVELY RECENTLY DOMESTICATED VEGETABLE CROP. RECENT SEQUENCING OF A CARROT REFERENCE GENOME HAS PAVED THE WAY FOR GENETIC IMPROVEMENTS OF CARROT GERMPLASMS. AS A PROMISING BREEDING TOOL, GENE EDITING IS POISED TO REVOLUTIONIZE CARROT BREEDING. CONSEQUENTLY, THE DEVELOPMENT OF ROBUST CARROT GENOME EDITING PLATFORMS HAS BECOME URGENT. HENCE, IT IS IMPORTANT TO DEVELOP A NEW CARROT GENOME EDITING PLATFORM THAT IS TRANSGENE-FREE. IT WILL NOT ONLY MITIGATE THE REGULATORY BURDEN, BUT ALSO MAKE IT MORE APPEALING TO CONSUMERS AS WE ANTICIPATE THAT GENOME EDITING WILL HELP ACCELERATE THE DEVELOPMENT OF CARROT GERMPLASMS THAT BENEFIT CONSUMERS. WHILE THE GENOME EDITING TECHNOLOGY IS PROMISED TO ACCELERATE CROP BREEDING, ITS DEMONSTRATION AND OPTIMIZATION ARE LAGGING IN BREEDING PROGRAMS IN THE PUBLIC SECTOR. THIS IS ESPECIALLY TRUE FOR AGRICULTURALLY IMPORTANT MINOR CROPS SUCH AS CARROT. IN THIS PROJECT, A TRANSGENE-FREE GENOME EDITING PLATFORM WILL BE ESTABLISHED IN CARROT. BY APPLYING HIGHLY EFFICIENT CRISPR-CAS12A TOOLS TO CARROT THROUGH A TRANSGENE-FREE RNP DELIVERY METHOD, TWO COMPLEMENTARY GENOME EDITING SYSTEMS WILL BE TESTED AND ESTABLISHED IN CARROTS, ONE FOR HIGH-EFFICIENCY TARGETED MUTAGENESIS AND THE OTHER FOR PRECISE GENE REPLACEMENT. GIVEN THE BROAD APPLICABILITY OF THE GENOME EDITING SYSTEMS TO BE DEVELOPED, THE TECHNOLOGIES TO BE DEVELOPED IN THIS PROJECT ARE POTENTIALLY TRANSFORMATIVE AS THEY CAN BE APPLIED TO MANY OTHER MINOR CROPS. THIS PROJECT SPECIFICALLY ADDRESSES THE FOLLOWING TWO CRITERIA OF THE PROGRAM: 1) TRANSFORMATION METHODS THAT DO NOT REQUIRE THE USE OF PLANT PEST SEQUENCES IN PLANTS OR THE DEVELOPMENT OF GENE EDITING TECHNOLOGIES THAT DO NOT REQUIRE TRANSFORMATION; 2) IF TRANSFORMATION TECHNOLOGIES NEED TO BE DEVELOPED, THEY MUST BE COUPLED WITH THE DEVELOPMENT OF GENE EDITING TECHNOLOGIES. BY DEMONSTRATING THESE NEW TRANSGENE-FREE GENOMES EDITING PLATFORMS AND SHARING THE TOOLS AND PROTOCOLS WIDELY TO OTHER RESEARCHERS AND BREEDERS, THE SUCCESS OF THIS PROJECT WILL ENCOURAGE ADOPTION OF THESE BREEDING TECHNOLOGIES IN PUBLIC BREEDING SECTOR, SO THAT THE GOAL SET BY USDA TO INCREASE US AGRICULTURE PRODUCTIVITY BY 40% WHILE CUTTING THE ENVIRONMENTAL FOOTPRINT IN HALF BY 2050 CAN BE MET.