** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** TARGETED GENETIC MODIFICATIONS HAVE THE POTENTIAL TO CREATE NOVEL AGRICULTURAL PRODUCTS THAT ARE FREE OF FOREIGN DNA. MODIFICATIONS THAT INVOLVE A SINGLE MUTATION ARE EXEMPT FROM THE REGULATORY PROCESS APPLIED TO TRADITIONAL BIOENGINEERED CROPS. HOWEVER, MULTIPLE GENE COPIES OR MEMBERS OF A GENE FAMILY CONTRIBUTE TO MANY AGRONOMICALLY IMPORTANT PLANT PHENOTYPES. THEREFORE, A SINGLE MUTATION IS INSUFFICIENT TO INDUCE THE DESIRED PHENOTYPE. FOR FEDERAL REGULATORY AGENCIES TO MAKE SCIENCE-BASED DECISIONS, IT IS NECESSARY TO DETERMINE IF MODIFICATIONS OF MULTIPLE GENE COPIES OR GENE FAMILY MEMBERS POSE AN INCREASED RISK TO THE ENVIRONMENT OR HUMAN HEALTH. WE WILL INVESTIGATE THE POTENTIAL FOR UNINTENDED CHANGES DUE TO CRISPR/CAS9 MODIFICATION OF FIVE AGRONOMICALLY IMPORTANT GENE FAMILIES IN SOYBEAN WHERE MULTIPLE GENE COPIES MUST BE MODIFIED TO INDUCE A DESIRED PHENOTYPE. WE WILL USE LONG-READ DNA SEQUENCING TO CHARACTERIZE THE FULL GENOMIC COMPLEMENT OF THE FIVE GENE FAMILIES IN TWO SOYBEAN VARIETIES. CRISPR/CAS9 EDITS GENERATE DOUBLE-STRANDED BREAKS IN DNA THAT CAN INDUCE CHROMOSOMAL STRUCTURAL VARIANTS. THUS, WE WILL USE LONG-READ SEQUENCING TO DETECT STRUCTURAL AND NUCLEOTIDE SEQUENCE VARIANTS POTENTIALLY INDUCED BY CRISPR/CAS9 EDITS OF MULTIPLE LINKED AND DISPERSED COPIES OF GENE FAMILY MEMBERS. VISUAL COMPARISONS AND HYPERSPECTRAL ANALYSIS WILL BE USED TO IDENTIFY ANY UNINTENDED PHENOTYPIC CHANGES IN MODIFIED PLANTS. OUR DATA WILL INCREASE UNDERSTANDING OF THE ENVIRONMENTAL RISKS ASSOCIATED WITH CRISPR/CAS9-INDUCED CHANGES IN AN IMPORTANT CROP PLANT AND PROVIDE A CONTRAST WITH VARIATION INDUCED BY UNREGULATED PRACTICES SUCH AS CONVENTIONAL BREEDING AND MUTAGENESIS.