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Soybean (Glycine max (L.) Merr.) oil is an important source of vegetable oil for supporting the human diet. However, the high level of polyunsaturated fatty acids in natural soybean oil renders the oil unstable and thus susceptible to the development of unpalatable flavors and trans fatty acids. Therefore, reducing the content of polyunsaturated fatty acids and increasing the content of monounsaturated fatty acids is a longstanding and important target for soybean breeding. However, soybean varieties with a high oleic acid content are rare in soybean germplasm resources, which introduces substantial difficulties in the cultivation of high-oleic-acid soybeans. In this study, CRISPR/Cas9-mediated gene-editing technology was used to create targeted knockout of the soybean fatty acid desaturase encoding genes GmFAD2-1A and GmFAD2-1B that contribute to the formation of polyunsaturated fatty acids. We obtained fad2-1a, fad2-1b, and fad2-1a/fad2-1b homozygous mutants using two sgRNAs. We found that the oleic acid content increased from 11% to 40-50% in the fad2-1a and fad2-1b mutants and to 85% in the fad2-1a/fad2-1b mutants. We also generated transgene-free double mutants that conferred higher oleic acid, and the fad2-1a/fad2-1b mutant had no adverse phenotyping compared with the wild type. Our study provided new materials for the selection and breeding of high-oleic-acid soybean varieties. [ABSTRACT FROM AUTHOR]