THE PROPOSED RESEARCH ALIGNS WITH PROGRAM AREA #5: OTHER RESEARCH TOPICS DESIGNED TO SUPPORT THE PURPOSES OF THE BRAG PROGRAM. OUR PROPOSAL AIMS AT TESTING THE EFFICACY AND SPECIFICITY OF MULTIPLEX MUTAGENESIS IN CIS-REGULATORY REGIONS USING GENOME EDITING TO ENGINEER BROAD AND DURABLE DISEASE RESISTANCE AGAINST TWO FUNGAL PATHOGENS IN THE VEGETABLE CROP TOMATO. TO ACHIEVE THIS, WE WILL 1) EVALUATE THE EFFICIENCY OF GENOME EDITING AT INDUCING MULTIPLE TARGETED MUTATIONS IN AT LEAST FOUR SUSCEPTIBILITY GENES, 2) CHARACTERIZE TRAIT EFFICACY AND POTENTIAL PLEIOTROPIC EFFECTS IN AGRONOMIC PERFORMANCE AND 3) DETERMINE POTENTIAL OFF-TARGETS AND OTHER COMPLEX STRUCTURAL VARIATION THAT COULD ORIGINATE FROM MULTIPLEX GENOME EDITING. OUR RESULTS WILL PROVIDE AN EXTENSIVE DATASET TO EVALUATE THE EFFICACY AND SPECIFICITY OF MORE COMPLEX GENOME EDITING APPROACHES IN THE VEGETABLE CROP TOMATO. PHENOTYPIC AND GENOMIC DATA WILL BE USED FOR DETERMINING WHETHER MULTIPLEX EDITING IS A VIABLE OPTION TO DEPLOY BROAD AND DURABLE DISEASE RESISTANCE AND TO ASSESS ANY UNINTENDED OFF-TARGET AND PLEIOTROPIC EFFECTS THAT COULD AFFECT AGRONOMIC PERFORMANCE. THE DATA AND RESULTS GENERATED MAY BE USED TO DETERMINE INTENDED OR UNINTENDED EFFECTS BEYOND THOSE THAT MAY BE OBTAINED BY OTHER TECHNOLOGIES.