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 T_0 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 T_1 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 T_2 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.

Combination of two multiplex genome-edited soybean varieties enables customization of protein functional properties

Dear Editor,Soybean(Glycine max)is a major source of plant-based protein for people worldwide,providing a healthy,affordable,and environmentally friendly alternative to animal-based protein(Montgomery,2003).It has been estimated that there are currently over 12000 food products that contain soy protein(Montgomery,2003).Different food applications of soy protein require distinct properties that affect its function and sensory features.For instance,the sensory quality of soymilk is significantly affected by protein emulsibility,whereas tofu quality is determined by protein-gelling ability(Kinsella,1979).

Elimination of an unfavorable allele conferring pod shattering in an elite soybean cultivar by CRISPR/Cas9

Pod shattering can lead to devastating yield loss of soybean and has been a negatively selected trait in soybean domestication and breeding.Nevertheless,a significant portion of soybean cultivars are still pod shattering-susceptible,limiting their regional and climatic adaptabilities.Here we performed genetic diagnosis on the shattering-susceptible trait of a national registered cultivar,Huachun6(HC6),and found that HC6 carries the susceptible genotype of a candidate Pod dehiscence 1(PDH1)gene,which exists in a significant portion of soybean cultivars.We next performed genome editing on PDH1 gene by clustered regularly interspaced short palindromic repeats(CRISPR)-CRISPR-associated protein 9(Cas9).In T2 progenies,several transgene-free lines with pdh1 mutations were characterized without affecting major agronomic traits.The pdh1 mutation significantly improved the pod shattering resistance which is associated with aberrant lignin distribution in inner sclerenchyma.Our work demonstrated that precision breeding by genome editing on PDH1 holds great potential for precisely improving pod shattering resistance and adaptability of soybean cultivars.