SORGHUM IS A MAJOR RESOURCE EFFICIENT CROP WITH IMMENSE POTENTIAL FOR EXPANDED PRODUCTION AS A GLUTEN-FREE SOURCE OF CALORIES AND PROTEIN IN U.S. AND WORLDWIDE. HOWEVER, THE DOMINANT ACCUMULATION OF KAFIRIN STORAGE PROTEINS IN THE KERNELS RESULTS IN GRAIN WITH LOW LEVELS OF LYSINE AND PROTEIN WITH POOR DIGESTIBILITY. KAFIRINS ARE DEVOID OF LYSINE, AN ESSENTIAL AMINO ACID IN THE DIETS OF HUMANS AND MONO-GASTRIC LIVESTOCK. THE POOR PROTEIN QUALITY AND PROTEIN AVAILABILITY SIGNIFICANTLY LIMIT THE CURRENT VALUE OF SORGHUM AS A PROTEIN SOURCE FOR HUMANS AND LIVESTOCK. TO SOLVE BOTH OF THESE LIMITATIONS, AND ALLOW BETTER REALIZATION OF SORGHUM'S AGRONOMIC ADVANTAGES, THE PI APPLIED CRISPR/CAS9, A STATE-OF-THE-ART GENE EDITING APPROACH, IN WHICH THE WHOLE, 20 MEMBER K1C ALPHA-KAFIRIN STORAGE PROTEIN GENE FAMILY WAS TARGETED FOR SUPPRESSION USING A SINGLE GUIDE RNA. CRISPR/CAS9 HAS THE GAME-CHANGING ADVANTAGE OF BEING ABLE TO PRODUCE NON-TRANSGENIC END-PRODUCTS WHICH ALLOWS RAPID UTILIZATION OF THE PRODUCTS FOR HUMAN AND ANIMAL NUTRITION. THE WORK ALREADY DEMONSTRATED PROOF-OF-CONCEPT BY GENERATING SORGHUM LINES WITH REDUCED KAFIRINS, AND INCREASED NON-KAFIRINS THROUGH THE PROCESS OF PROTEOME REBALANCING. THIS RESULTED IN LINES WITH INCREASED LYSINE, PROTEIN DIGESTIBILITY AND TOTAL PROTEIN IN THE GRAIN.THE PROJECT INVOLVES CROSSING THE KAFIRIN GENE EDITED LINES TO SORGHUM LINES WITH AND WITHOUT THE WAXY, HIGH AMYLOPECTIN TRAIT CONFERRED BY A MUTATION IN THE WAXY1 GENE ENCODING GRANULE BOUND STARCH SYNTHASE. THERE IS EVIDENCE THAT BOTH THE HIGH PROTEIN DIGESTIBILITY TRAIT AND THE HIGH AMYLOPECTIN TRAIT, WHICH CAN INCREASE STARCH SOLUBILITY, CAN ALONE AND ADDITIVELY IMPROVE THE DOUGH-MAKING CHARACTERISTICS OF SORGHUM FLOUR. THE WORK WILL CHARACTERIZE THE VARIOUS PARAMETERS OF DOUGH QUALITY OF THE HIGH AMYLOPECTIN LINES. THE NEXT PHASE OF THE PROJECT WILL REMOVE THE TRANSGENE, CURTAILING FURTHER KAFIRIN EDITING AND LEAVING BEHIND JUST THE BENEFICIAL REDUCED KAFIRIN PHENOTYPE. GENOMIC DNA SEQUENCING AND RNA-SEQUENCING WILL BE USED TO CHARACTERIZE THE STABLE KAFIRIN EDITS AND DETERMINE THEIR EFFECTS ON EXPRESSION OF INDIVIDUAL K1C KAFIRIN FAMILY GENES.RNA-SEQUENCING WILL ALSO BE USED TO ASSESS THE NATURE AND REGULATION OF THE INCREASE IN THE NON-KAFIRIN GENES WHICH LEAD TO PROTEOME REBALANCING EFFECT AND IMPROVED GRAIN NUTRITIONAL QUALITY. OTHER METHODS WILL BE USED TO ASSESS IF EPIGENETIC FACTORS SUCH AS GENOME METHYLATION AND CHROMATIN ACCESSIBILITY ARE INVOLVED IN GENE EXPRESSION CHANGES THAT LEAD TO PROTEOME REBALANCING. THESE DATA WILL BE RELEVANT TO MORE GENERAL STUDIES AIMED AT DISSECTING NITROGEN UTILIZATION DURING SEED STORAGE PROTEIN ACQUISITION.AFTER TRANSGENE-FREE LINES WITH OPTIMAL KAFIRIN EDITS ARE IDENTIFIED, FURTHER SELECTIONS WILL BE MADE USING LARGE-SCALE FIELD TRIALS WHICH WILL LIKELY CONFIRM THAT ALTERING THE SEED PROTEOME BALANCE DOES NOT HAVE ADVERSE PLANT PERFORMANCE OR YIELD EFFECTS. THE FINAL PHASE WILL BE TO CONFIRM SMALL-SCALE PREVIOUS IN VITRO RESULTS IN ONE EDITED LINE, WITH AND WITHOUT INTROGRESSION OF THE WAXY TRAIT, BY CONDUCTING MONO-GASTRIC ANIMAL PROTEIN DIGESTIBILITY ASSAYS AND FEEDING STUDIES USING PIGS AS THE MODEL SYSTEM. WHILE CONCEPTUALLY STRAIGHTFORWARD, THIS WORK WILL TAKE SOME TIME. THE PROJECT WILL EFFECT A PLAN FOR ACHIEVING THE GOALS AND DELIVERING A FINISHED PRODUCT WITHIN A THREE-YEAR TIMESCALE.