Multiplex genome editing targeting soybean with ultra-low anti-nutritive oligosaccharides

Soybean is the primary source of plant protein for humans.Owing to the indigestibility of the raffinose family of oligosaccharides(RFO),raffinose and stachyose are considered anti-nutritive factors in soybean seeds.Low-RFO soybean cultivars are generated by mutagenesis of RFO biosynthesis genes,but the carbohydrate profiles invite further modification to lower RFOs.This study employed a pooled multiplex genome editing approach to target four seed-specifically expressed genes mediating RFO biosynthesis,encoding three raffinose synthases(RS2,RS3,and RS4)and one stachyose synthase.In T1progeny,rs2/rs3 and rs4/sts homozygous double mutants and a rs2/rs3/rs4/sts quadruple mutant(rfo-4m)were characterized.The rs2/rs3 mutant showed reduced raffinose and stachyose contents,but the rs4/sts mutant showed only reduced stachyose in seeds.The RFO contents in the rfo-4m mutant were almost eliminated.Metabolomic analysis showed that the mutation of four RFO biosynthesis genes led to a shift of metabolic profile in the seeds,including the accumulation of several oligosaccharides-related metabolites.These mutants could contribute to precision breeding of soybean cultivars for soy food production.

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.

Expressing a human RNA demethylase as an assister improves gene-editing efficiency in plants

Dear Editor,Despite myriad successful applications of gene editing in plant functional genomics research and precision breeding,many challenges persist around the efficiency of gene-editing tools for many plant species.For instance,soybean(Glycine max)is a major crop providing oil and protein to human diets and feedstock,but its gene-editing efficiency remains relatively low(Bai et al.,2019).

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.

Genome editing toward biofortified soybean with minimal trade-off between low phytic acid and yield

Phytic acid(PA)in grain seeds reduces the bioavailability of nutrient elements in monogastric animals,and an important objective for crop seed biofortification is to decrease the seed PA content.Here,we employed CRISPR/Cas9 to generate a PA mutant population targeting PA biosynthesis and transport genes,including two multi-drug-resistant protein 5(MRP5)and three inositol pentose-phosphate kinases(IPK1).We characterized a variety of lines containing mutations on multiple IPK and MRP5 genes.The seed PA was more significantly decreased in higher-order mutant lines with multiplex mutations.However,such mutants also exhibited poor agronomic performance.In the population,we identified two lines carrying single mutations in ipk1b and ipk1c,respectively.These mutants exhibited moderately reduced PA content,and regular agronomic performance compared to the wild type.Our study indicates that moderately decreasing PA by targeting single GmIPK1 genes,rather than multiplex mutagenesis toward ultra-low PA,is an optimal strategy for low-PA soybean with a minimal trade-off in yield performance.