WITH THE ADVANCES OF GENE EDITING TECHNOLOGIES, IT IS NOW POSSIBLE TO MODIFY A PLANT GENOME AT A DESIRED SITE, WHICH IS UNLIKE CONVENTIONAL GENETIC ENGINEERING WHERE A TRANSGENE IS INTRODUCED AND INTEGRATES RANDOMLY INTO A GENOME. THERE HAS BEEN A SWIFT INCREASE IN APPLICATIONS OF CRISPR-CAS SYSTEMS DURING THE PAST FEW YEARS IN PART DUE TO THE LONG-TERM DESIRE FOR A TECHNOLOGY THAT ALLOWS PRECISE MODIFICATION AND IS STRAIGHTFORWARD TO DESIGN. ANOTHER ATTRACTIVE FEATURE IS THAT THE CRISPR-CAS TRANSGENE CAN BE GENETICALLY SEGREGATED AWAY, LEAVING ONLY THE TARGETED MODIFICATION IN THE PROGENY. BASED ON THE RECENTLY PASSED SECURE RULE, USDA-APHIS WILL NOT REGULATE GENE EDITED CROPS IF THE INTRODUCED DNA CHANGES CAN BE FOUND IN NATURE OR DERIVED FROM CONVENTIONAL BREEDING METHODS. SCIENCE BASED REGULATION WILL GREATLY ENCOURAGE THE USE OF GENE EDITING TECHNOLOGIES FOR FAST-TRACK CROP BREEDING TO MAKE IMPROVED CROPS THAT ARE NUTRITIOUS, HIGH YIELD, AND RESISTANT TO BIOTIC AND ABIOTIC STRESSES. GENE EDITING HAS ALSO GREATLY STIMULATED INNOVATION IN PLANT SYNTHETIC BIOLOGY AND AGRICULTURE. IN THIS PROJECT, WE PROPOSE TO ASSESS THREE EMERGING CRISPR GENOME EDITING SYSTEMS FOR ANY UNINTENDED MODIFICATIONS AT OTHER LOCI IN THE GENOMES OF RECOVERED EDITED RICE LINES. WE SPECIFICALLY ADDRESS THE BRAG PROGRAM AREA "5G", WHERE IT CALLS FOR COMPARISON OF THE TYPES AND FREQUENCIES OF NUCLEIC ACID CHANGES INTRODUCED INTO IMPORTANT CROP GENOMES, VIA GENETIC ENGINEERING TECHNIQUES VERSUS OTHER PLANT BREEDING TECHNIQUES. THEREFORE, WE WILL SPECIFICALLY ASSESS FOR UNINTENDED OFF-TARGET EFFECTS IN THE GENOMES OF EDITED LINES RECOVERED FROM 1) CAS12B EDITING, 2) CAS12A BASED MULTIPLEXED EDITING, AND 3) CAS9 BASED PRIME EDITING. IN PARALLEL, WE WILL DO A COMPARISON WITH CONTROL LINES RECOVERED THROUGH THE PLANT REGENERATION AND TRANSFORMATION PHASES BECAUSE OF UNINTENDED SOMACLONAL VARIATIONS. 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 GENE EDITING IN CROPS AS WELL AS FACILITATE THE DECISION-MAKING PROCESS AT REGULATORY AGENCIES SUCH AS USDA AND FDA.