DUE TO EASE OF USE, CRISPR-CAS9 HAS ADVANCED REMARKABLY IN THE PAST FEW YEARS AND HAS BECOME A UBIQUITOUS GENOME-EDITING METHOD BEING APPLIED TO AN INCREASING NUMBER OF SPECIES. WHILE SEVERAL SUCCESSFUL CASES OF CROP IMPROVEMENT USING VARIOUS GENOME- EDITING PLATFORMS INCLUDING CRISPR SYSTEMS AND TALENS HAVE BEEN REPORTED, CONCERNS OF GENOME WIDE OFF-TARGETS HAVE NOT BEEN THOROUGHLY ADDRESSED. TREMENDOUS PROGRESS HAS BEEN MADE IN INCREASING THE SPECIFICITY AND THUS REDUCING OFF-TARGETING BY ENGINEERING CAS9 VARIANTS SUCH AS THE HYPER-ACCURATE CAS9 VARIANT, AN ALTERNATIVE CRISPR-CPF1 SYSTEM ANALOGOUS TO CRISPR-CAS9, AND A NEW BASE EDITING APPROACH, WHICH DOESN'T REQUIRE DNA CLEAVAGE TO INCORPORATE SPECIFIC MUTATIONS. REGULATORY AGENCIES NEED TO EVALUATE THE PRECISION OF THESE NEW AND EMERGING GENE- EDITING REAGENTS. WE PROPOSE TO USE THE CURRENT AND EMERGING GENOME-EDITING REAGENTS FOR TARGETED MUTAGENESIS OF COMMERCIALLY IMPORTANT CANDIDATE GENES IN TWO DIFFERENT CROP SPECIES, POTATO AND CAMELINA, WHICH HAVE CONTRASTING GENOMIC ARCHITECTURE AND MODES OF REPRODUCTION. IN POTATOMECHANICALDAMAGE TOTUBERS DURING HARVEST AND POST-HARVEST PROCEDURES LEAD TO DISCOLORATION DUE TO ENZYMATIC BROWNING CAUSED BY THE CANDIDATE GENEPOTATO POLYPHENOL OXIDASE (PPO). PPO CATALYZES THE CONVERSION OF O-DIHYDROXYPHENOLS TO O-QUINONES. THE QUINONES POLYMERIZE AND REACT WITH AMINO ACIDS RESULTING IN THE FORMATION OF BLACK/BROWN PIGMENTATION. OUR SECOND CANDIDATE GENE, DELTA-12-DESATURASE (FAD2) CONVERTS OLEIC ACID (18:1) FATTY ACID TO 18:2. BY KNOCKING OUTFAD2 WE RETAIN HIGH OLEIC ACID WHICH IS DESIRABLE IN INDUSTRIAL OILS.WE WILL COMPARE THE GENOME WIDE OFF-TARGET EFFECTS CAUSED BY THESE REAGENTS USING WHOLE GENOME SEQUENCING AND DETERMINE THE TYPE AND FREQUENCY OF ON- AND OFF-TARGET VARIATION ATTRIBUTABLE BY GENOME-EDITING REGEANTS. THIS STUDY WOULD PROVIDE A COMPREHENSIVE RISK ASSESSMENT OF GENOME- EDITED CROPS WITH CURRENT TECHNOLOGIES. THIS PROPOSAL ADDRESSES THE BRAG FOCUS AREA OF COMPARISON OF THE TYPES AND FREQUENCIES OF NUCLEIC ACID CHANGES INTRODUCED INTO IMPORTANT CROP GENOMES, VIA GENETIC INSERTION/MODIFICATION TECHNIQUES VERSUS OTHER PLANT BREEDING TECHNIQUES - OFF TARGET EFFECTS OF GENOME-EDITING TECHNOLOGIES.