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

Abstract 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 2 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.