** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** THIS RESEARCH PROJECT ADDRESSES THE BRAG PROGRAM AREA OF INVESTIGATION AND MITIGATION OF UNINTENDED AND OFF-TARGET MODIFICATIONS BY GENOME EDITING TECHNOLOGIES. THE CYTOSINE BASE EDITORS (CBES), ENGINEERED FROM THE CRISPR-CAS SYSTEMS, ARE POISED TO REVOLUTIONIZE MODERN CROP BREEDING DUE TO THEIR PROMISING APPLICATIONS FOR TARGETED MUTAGENESIS, ALLELE REPLACEMENT, AND ENGINEERING OF QUANTITATIVE TRAITS. CBES CAN INTRODUCE PREMATURE STOP CODONS TO KNOCK OUT GENES AND EFFICIENTLY GENERATE HOMOZYGOUS MUTANTS WITHIN ONE GENERATION. HENCE, CBES HOLD GREAT PROMISE FOR MULTIPLEXED EDITING IN CROPS. IT HAS BEEN RECENTLY SHOWN THAT MULTIPLEXED EDITING BY CAS12A NUCLEASES COULD LEAD TO CHROMOSOMAL REARRANGEMENTS CAUSED BY CONCURRENT DNA DOUBLE-STRAND BREAKS (DSBS) IN PLANTS. UNLIKE CAS9 AND CAS12A NUCLEASES, CBES DERIVED FROM CAS9 AND CAS12A GENERALLY DO NOT PRODUCE DSBS IN THE PLANT GENOME. FOR EXAMPLE, CAS9-BASED CBES MAINLY NICK DNA, AND CAS12A-BASED CBES CANNOT CUT DNA. HENCE, IT IS ANTICIPATED THAT CBES ARE UNLIKELY TO GENERATE CHROMOSOMAL REARRANGEMENTS IN A MULTIPLEXED EDITING SETTING. TO INVESTIGATE THIS IMPORTANT ISSUE, WE WILL FIRST DEVELOP MULTIPLE IMPROVED CAS9-CBES AND CAS12A-CBES FOR HIGH-EFFICIENCY MULTIPLEXED EDITING IN RICE. THEN WE WILL CONDUCT GENOME-WIDE OFF-TARGET INVESTIGATIONS IN THE RICE PLANTS WITH MULTIPLEXED EDITS USING WHOLE-GENOME SEQUENCING. THE RELEVANCE OF THE PROPOSED PROJECT TO THE BRAG PROGRAM IS VERY SIGNIFICANT BECAUSE THE RESULTS WILL PROVIDE CRITICAL INFORMATION FOR THE GLOBAL REGULATORY AGENCIES TO COME UP WITH SCIENCE-BASED REGULATORY POLICIES AS WELL AS TO GUIDE REGULATORY DECISIONS ABOUT THE 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 THE USDA AND FDA.