Multiplex FAD2/FATB Editing Generates Ultra‐High‐Oleic, Low‐Saturate Soybean With Increased Seed Fatty Acid Content

Abstract

Oleic acid (18:1), saturated fatty acid (SFA), and polyunsaturated fatty acid (PUFA) levels are important traits for storage stability and edibility. In this study, we aimed to develop high‐oleic soybean ( Glycine max ) by simultaneously targeting fatty acid desaturase 2 ( FAD2 ) and fatty acyl‐ACP thioesterase B ( FATB ) gene families using CRISPR/Cas9. Considering the paleopolyploid genome of soybean, multiple sgRNAs were designed to target GmFAD2‐1 , GmFAD2‐2 , and GmFATB genes expressed during seed development. Compared with targeting GmFAD2‐1 alone (~83%), additional editing of GmFAD2‐2B, GmFAD2‐2C, and GmFATB1a increased the 18:1 fatty acid content to over 90%. Editing of GmFATB significantly reduced the SFA content by more than 40% compared with wild‐type (WT). Reduced absolute SFA content was also observed in GmFAD2‐only edited lines with decreased GmFATB expression. Notably, all evaluated high‐oleic genotypes in the Williams82‐background showed higher mean total fatty acid (TFA) content than the WT, both per unit seed mass and per seed. Correlation analysis of 805 individual seed profiles from Williams82‐background lines further characterized the relationship between 18:1 fatty acid composition and TFA content per unit seed mass. Gene expression analysis revealed no consistent increase in triacylglycerol (TAG) synthesis genes, whereas the expression of sugar‐dependent 1 ( SDP1 ) was reduced. In contrast, genes involved in phosphatidic acid (PA) metabolism, including diacylglycerol kinase ( DGK ) and phospholipase D ( PLD ), were partially upregulated. These transcriptional changes support a hypothesis that altered lipid turnover may contribute to the higher TFA phenotype. Collectively, this study defines effective multiplex target combinations for ultra‐high oleic, low‐saturated soybean.