PAPAYA (CARICA PAPAYA L.) IS AN ECONOMICALLY IMPORTANT NUTRITIOUS FRUIT CROP, WIDELY GROWN IN THE TROPICS AND SUBTROPICS. BESIDES ITS USE AS FOOD, THE FRUIT IS ALSO USED IN THE COSMETICS, PHARMACEUTICAL AND FOOD PROCESSING INDUSTRIES. PAPAYA PLANTS OCCUR IN THREE SEX TYPES, BUT ITS SEX DETERMINING GENE (S) HAVE YET TO BE DEFINED. A RANGE OF VIRAL, FUNGAL AND OOMYCETE PATHOGENS SEVERELY THREATEN PAPAYA PRODUCTION. TO BETTER UTILIZE PAPAYA PRODUCTS AND REDUCE THE COST IN PRODUCTION ASSOCIATED WITH DISEASE CONTROLAND IDENTIFICATION OF HERMAPHRODITE PLANTS, FUNCTIONAL UNDERSTANDING OF PAPAYA GENES IS A PREREQUISITE,BUT HINDERED BY THE LACK OF AN EFFECTIVE GENE KNOCKOUT SYSTEM IN PAPAYA.CRISPR-MEDIATED GENE EDITING TECHNOLOGY HAS REVOLUTIONIZED FUNCTIONAL GENOMICS OF MANY ORGANISMS, AND IS A POWERFUL TOOL TO BREED TRANSGENE-FREE NEW VARIETIES WITH NUMEROUS DESIRABLE TRAITS. HOWEVER, A GENE EDITING SYSTEM HAS NOT BEEN ESTABLISHED IN PAPAYA. THIS PROJECT IS TO ESTABLISH METHODSFOR EFFECTIVEGENOME EDITING OF PAPAYA TO MEET THE DIVERSE NEEDS OF GENE FUNCTIONAL ANALYSES AND BREEDING.TO MAXIMIZE THE SUCCESS, WE WILL UTILIZE TWO CRISPR SYSTEMS (CAS9 AND CAS12) AND COMPLEMENTARY APPROACHES. SPECIFICALLY, WE WILL: 1) ESTABLISH AN EFFECTIVE AND ROUTINE PAPAYA GENE EDITING SYSTEM VIA AGROBACTERIUM-MEDIATED TRANSFORMATION, AND 2) DEVELOP DNA-FREE GENOME EDITING OF PAPAYA WITH PREASSEMBLED CRISPR-NUCLEASE RIBONUCLEOPROTEIN COMPLEXES.