THE DEVELOPMENT OF CRISPR-CAS9 AND RELATED TECHNOLOGIES FOR EDITING MAMMALIAN CELLS AND EMBRYOS HAS RESULTED IN UNPRECEDENTED OPPORTUNITIES FOR IMPROVING LIVESTOCK GENETICS. TO TAKE FULL ADVANTAGE OF GENE EDITING TECHNOLOGIES APPLIED TO ANIMALS SUCH AS INTROGRESSION OF ALLELES FOR IMPROVED GENETIC ADVANCE MULTIPLE EDITS ON THE SAME INDIVIDUAL ARE OFTEN REQUIRED WHICH CURRENTLY CONSTITUTES A TECHNOLOGICAL BARRIER FOR FOOD ANIMAL SPECIES. WE HAVE RECENTLY BEEN ABLE TO ISOLATE AND MAINTAIN STABLE BOVINE EMBRYONIC STEM CELLS (BESC) FULFILLING AN UNMET NEED FOR LIVESTOCK SPECIES THAT HAD BEEN PURSUED FOR THE LAST 30 YEARS. WE HAVE FURTHER OPTIMIZED THE CULTURE CONDITIONS FOR BOVINE ESCS AND EXTENDED THE TECHNOLOGY TO SHEEP AND GOAT SPECIES. ESCS REPRESENT AN IDEAL PLATFORM FOR INTRODUCING MULTIPLE GENETIC MODIFICATIONS AS THEY CAN BE CULTURED INDEFINITELY MAINTAIN GENOMIC STABILITY AND ARE AMENABLE TO SCNT/CLONING FOR PRODUCING ESC-DERIVED OFFSPRING. THE GOAL OF THE PROPOSED PROJECT IS TO DEVELOP A PLATFORM FOR EFFICIENT MULTIPLEX GENOME EDITING IN CATTLE GOAT AND SHEEP. WE WILL PURSUE THE FOLLOWING SPECIFIC OBJECTIVES: 1) TO OPTIMIZE CRISPR-CAS9 DELIVERY AND GENOME EDITING REAGENTS FOR EFFICIENT GENE DISRUPTION ALLELE INTROGRESSION AND TARGETED GENE INSERTION IN BOVINE OVINE AND CAPRINE ESCS; AND 2) TO OPTIMIZE CONDITIONS FOR MULTIPLEXED GENE EDITING IN CATTLE ESCS. OUTCOMES OF THIS PROPOSAL WILL INCLUDE STEM CELL-BASED PLATFORMS FOR RAPID AND EFFICIENT INTRODUCTION OF MULTIPLE GENOMIC MODIFICATIONS IN THREE IMPORTANT FOOD ANIMAL SPECIES.