THIS PROPOSAL IS GOING TO ADDRESS PROGRAM AREA 7: CROSSCUTTING PROGRAMS AND PROGRAM AREA PRIORITY (A): AGRICULTURAL INNOVATION THROUGH GENE EDITING OF THE USDA AFRI STANDARD RESEARCH PROGRAM. A PROBLEM IN THE USE OF GENE EDITING FOR CROP IMPROVEMENT IS THE EXISTENCE OF TECHNOLOGICAL BARRIERS THAT IMPEDE THE USE OF GENE EDITING IN SOME AGRICULTURALLY IMPORTANT CROPS. WE INTEND TO DEVELOP NOVEL TRANSFORMATION METHODS THAT DO NOT REQUIRE THE USE OF PLANT PEST SEQUENCES IN PLANTS AND DEVELOP GENE EDITING TECHNOLOGIES THAT DO NOT REQUIRE TRANSFORMATION. WE SEEK TO USE NANOPARTICLES TO DELIVER CAS9/GRNAS INTO TWO MINOR CROPS - TOMATO AND HEMP - FOR GENE EDITING. PHENOTYPIC AND MOLECULAR ANALYSIS WILL BE USED TO CONFIRM THAT THE EXPECTED PHENOTYPE IS DIRECTLY CAUSED BY THE EDITED GENES. THE OFF-TARGET EFFICIENCY OF THE CRISPR-CAS9 SYSTEM WILL BE EXAMINED BY NEXT-GENERATION SEQUENCING. GREENHOUSE EXPERIMENTS WILL BE USED TO TEST THE PHENOTYPIC STABILITY OF THE GENE EDITED MUTANTS. THE EXPECTED OUTCOME OF THIS PROJECT IS THE ESTABLISHMENT OF NOVEL GENE EDITING TECHNOLOGIES THAT WILL NOT REQUIRE THE USE OF PLANT PEST SEQUENCES OR PLANT TRANSFORMATION AND REGENERATION. THEY COULD BE ADAPTED TO OTHER SEXUALLY PROPAGATED, DICOTYLEDONOUS HORTICULTURAL CROPS FOR IMPROVEMENTS IN ANY TRAITS THAT NEED TO BE IMPROVED FOR SUSTAINABILITY. THEREFORE, THE RESEARCH DIRECTLY ADDRESSES KEY BARRIERS TO THE DEVELOPMENT OF NOVEL GENE EDITING TECHNOLOGY IN OTHER ECONOMICALLY IMPORTANT HORTICULTURAL CROPS WHICH ARE TRANSFORMATION RECALCITRANT AND CURRENTLY NOT AMENABLE TO GENE EDITING.