Base editing of BnaFAD2 fine-tunes oleic acid content in allotetraploid rapeseed without compromising yield

Rapeseed (Brassica napus L.) is a major global oil crop, with its nutritional and functional value largely determined by the degree of fatty acid desaturation in seed oil. High-oleic rapeseed is primarily developed through loss-of-function mutations in the fatty acid desaturase 2 gene (BnaFAD2). However, BnaFAD2 is dose-sensitive, and complete gene inactivation often leads to negative phenotypes. To mitigate negative effects on plant growth, we generated BnaFAD2 amino acid variants using base editing. Here, we successfully developed BnSTEME, a dual-base editing system optimized for allotetraploid rapeseed, which targeted conserved domains of BnaFAD2.A5 and BnaFAD2.C5 via 15 sgRNAs. This system enabled simultaneous C-to-T and A-to-G conversions with a base-editing efficiency of 1.29%. BnSTEME together with our previously established cytosine base editor BnA3A1-PBE induced a total of 29 missense mutations in BnaFAD2, including 26 variant alleles not previously reported. By oleic acid content screening, we identified two key mutations—S229F (a strong allele) and D230N (a weak allele)—that significantly increased oleic acid content, yielding rapeseed lines with oleic acid content ranging from 71.08% to 84.22%. Field trials for the base-edited line with the highest oleic acid PST-Z3-15-56-1 (aaS229FccD230N, 84.22% C18:1) demonstrated agronomic stability without yield penalty, which outperformed the knockout line CR-aacc. This study establishes base editing as a powerful tool for fine-tuning dosage-sensitive traits in polyploid crops and provides high-oleic rapeseed germplasm for sustainable oilseed breeding.