ADVANCING GENETICALLY ENGINEERED PLANTS FROM THE LAB TO THE FIELD AND EVEN THE EVENTUAL LARGE-SCALE PRODUCTION, CAN BE HAMPERED BY THE REGULATORY PROCESS BECAUSE OF THE COSTS INCURRED DURING THE LENGTHY PROCESS. THE NECESSARY REGULATORY COSTS, WHICH ALSO INCLUDE THOSE RELATED TO EXTENSIVE TESTING, MAKE IT LESS LIKELY THAT INNOVATIVE APPROACHES FOR CROP IMPROVEMENT DEVELOPED BY THE PUBLIC SECTOR AND SMALL BIOTECHNOLOGY COMPANIES WILL BE REALIZED. AS A RESULT, ANY NEW GENETICALLY MODIFIED (GM) CROPS ARE MORE LIKELY TO BE PRODUCED BY LARGE MULTINATIONAL COMPANIES, AND EVEN THEN, IT IS MAINLY LIMITED TO CROPS SUCH AS MAIZE AND SOYBEAN THAT HAVE LARGE GLOBAL MARKET VALUES. THE CRISPR (CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS)-CAS9 BASE EDITING SYSTEMS ARE POISED TO REVOLUTIONIZE MODERN CROP BREEDING DUE TO THEIR PROMISING APPLICATIONS FOR GENERATING TRANSGENE-FREE CROPS WITH QUANTITATIVE TRAITS INTRODUCED AT BASE PRECISION AT THE DNA LEVEL. BASE EDITING TECHNOLOGY PROVIDESAN EFFICIENT AND INEXPENSIVE WAY FORWARD FOR CROP BREEDING COMPARED TO CONVENTIONAL BREEDING METHODS. THIS PROJECT IS TO ADDRESS AN IMPORTANT KNOWLEDGE GAP, WHICH IS THE SAFETY OF PLANT BASE EDITING SYSTEMS. WE WILL FIRST IMPROVE THE EXISTING CRISPR-CAS9 C TO T AND A TO G BASE EDITING SYSTEMS FOR HIGHER EFFICIENCY AND ALTERED EDITING WINDOWS. NEXT, WE WILL GENERATE BASE-EDITED LINES OF TWO IMPORTANT FOOD CROPS, RICE AND TOMATO, AND USE WHOLE-GENOME SEQUENCING TO ASSESS UNINTENDED OFF-TARGET EFFECTS. IN PARALLEL, WE WILL ALSO SEQUENCE CONTROL LINES RECOVERED THROUGH THE PLANT REGENERATION AND TRANSFORMATION PHASES TO DETERMINE THE INCIDENCE OF UNINTENDED AFFECTS THAT CAN RESULT FROM SOMACLONAL VARIATION. THE RELEVANCE OF THE PROPOSED PROJECT TO THE BRAG PROGRAM IS VERY SIGNIFICANT BECAUSE OUR GOALS ARE CLOSELY ALIGNED WITH THE MISSION, IN THAT THE RESULTS WILL PROVIDE INFORMATION ACCESSIBLE BY FEDERAL REGULATORY AGENCIES TO CONSIDER DURING EVALUATION FOR INTRODUCTION OF CRISPR-CAS GENERATED ORGANISMS INTO THE ENVIRONMENT. THE KNOWLEDGE GENERATED FROM OUR WORK WILL GREATLY AID PLANT SCIENTISTS PRACTICING BASE EDITING TECHNOLOGY AS WELL AS FACILITATE THE DECISION-MAKING PROCESS AT REGULATORY AGENCIES SUCH AS USDA AND FDA. THIS RESEARCH PROJECT IS TO ADDRESS THE BRAG PROGRAM'S AREA 5G, WHERE IT CALLS FOR COMPARISON OF THE TYPES AND FREQUENCIES OF NUCLEIC ACID CHANGES INTRODUCED INTO IMPORTANT CROP GENOMES, VIA GENETIC ENGINEERING TECHNOLOGIES VERSUS OTHER PLANT BREEDING TECHNIQUES.