Generation of seed lipoxygenase-free soybean using CRISPR-Cas9

Beany flavor induced by three lipoxygenases（LOXs, including LOX1, LOX2, and LOX3）restricts human consumption of soybean. It is desirable to generate lipoxygenase-free new mutant lines to improve the eating quality of soybean oil and protein products. In this study, a pooled clustered regularly interspaced short palindromic repeats（CRISPR）-CRISPRassociated protein 9（Cas9） strategy targeting three GmLox genes（GmLox1, GmLox2, and GmLox3） was applied and 60 T0 positive transgenic plants were generated, carrying combinations of sg RNAs and mutations. Among them, GmLox-28 and GmLox-60 were gmlox1 gmlox2 gmlox3 triple mutants and GmLox-40 was a gmlox1 gmlox2 double mutant.Sequencing of T1 mutant plants derived from GmLox-28, GmLox-60, and GmLox-40 showed that mutation in the GmLox gene was inherited by the next generation. Colorimetric assay revealed that plants carrying different combinations of mutations lost the corresponding lipoxygenase activities. Transgene-free mutants were obtained by screening the T2 generation of lipoxygenase-free mutant lines（GmLox-28 and GmLox-60）. These transgeneand lipoxygenase-free mutants could be used for soybean beany flavor reduction without restriction by regulatory frameworks governing transgenic organisms.