Generation of seed lipoxygenase-free soybean using CRISPR-Cas9

Jie Wang,Huaqin Kuang, Zhihui Zhang, Yongqing Yang, Long Yan,Menghen Zhang, Shikui Song,Yuefeng Guan,*

aCollege of Resources and Environment, Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian,China

bFAFU-UCR Joint Center for Horticultural Plant Biology and Metabolomics,Haixia Institute of Science and Technology,Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian,China

cRoot Biology Center,College of Resources and Environment, Fujian Agriculture and Forestry University,Fuzhou 350002,Fujian,China

dThe Key Laboratory of Crop Genetics and Breeding of Hebei, Institute of Cereal and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences,Shijiazhuang 050035,Hebei,China

ABSTRACT 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 sgRNAs and mutations. Among them, GmLox-28 and GmLox-60 were gmlox1gmlox2gmlox3 triple mutants and GmLox-40 was a gmlox1gmlox2 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.

1. Introduction

Soybean (Glycine max [L.] Merr.), is a globally important crop providing human dietary protein, vegetable oil, and animal feed. However, lipoxygenases (LOXs), which are present in mature soybean seeds, can catalyze the oxidation of unsaturated fatty acids such as linoleic and linolenic acids to produce conjugated unsaturated fatty acid hydroperoxides, which are converted to volatile compounds associated with unpleasant beany flavor [1-4]. The beany flavor of soybean seed products restricts human consumption of soybean [5]. In the food industry, treatments such as heat, microwave processing, and organic solvent extraction have been used to eliminate the beany flavor from soybean products (oil, soymilk, tofu, etc.), increasing the cost of soybean production and processing [6]. Breeding lipoxygenase-free soybean varieties is a promising strategy for eliminating beany flavor in soybean products without a cost penalty.

Mature soybean seeds contain mainly three lipoxygenase isozymes,LOX1,LOX2,and LOX3,encoded by Glyma.13g347600 (GmLox1), Glyma.13g347500 (GmLox2), and Glyma.15g026300 (GmLox3), respectively [7,8]. These isozymes are involved in the formation of beany flavor, LOX2 being the main isozyme responsible [6,9-11]. Natural or artificial mutants for single, double, and triple lipoxygenase isozymes have been identified [12-15] and a series of soybean varieties lacking lipoxygenase have been developed using these mutant lines [16-21]. Conventionally, backcrossing or selfing and rounds of selection over several generations, a timeconsuming and laborious process, are required to introgress mutations into elite soybean cultivars during the breeding of lipoxygenase-free soybean varieties.

The newly developed clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) technology presents new opportunities to rapidly and cost-effectively create new varieties [22-25]. The CRISPR-Cas9 system has become the most widely used technology for genome editing and has been applied in many crops including rice, maize, wheat, barley, cotton, tobacco, and sorghum [22-28]. In soybean, the successful application of the CRISPRCas9 system for mutating the genes GmFT2a, FAD2-2, and GmSPL9 has been reported [29-31], to modify flowering time, seed oil profile, and plant architecture, respectively. This achievement suggests that genetic improvement of soybean agronomical traits using the CRISPR-Cas9 system is feasible.

Here, we report the development of targeted mutagenesis of three GmLox genes (GmLox1, GmLox2, and GmLox3) in soybean, using a pooled CRISPR-Cas9 system [32]. Lipoxygenase-free soybean lines were characterized in the progenies, showing the feasibility of generation of new germplasms by this method.

2.Materials and methods

2.1. Plant material and growth

One of the main soybean cultivars of south China,Huachun 6 (WT), was used for transformation. Wild-type(WT, as a control; GmLox1, 2, 3-harboring), lipoxygenasefree cultivar Wuxing 4 (WX4, as a control; GmLox1, 2, 3-free), and mutant plants were cultivated in a greenhouse (60%-80% relative humidity) under cycles of 14/10 h with 27/25 °C (day/night).

2.2. Single-guide RNA (sgRNA) design, CRISPR-Cas9 expression vector construction, and soybean transformation

0.09pt?>For sgRNA design, guide RNA spacer sequences were computationally identified based on Wm82.a2 genomic sequences. SgRNA fragments were produced by annealing complementary oligonucleotides and ligating to BsaI-digested pGES201 plasmids with a T4 DNA ligation kit (Takara, Dalian, China) according to the manufacturer's instructions. After E. coli transformation, positive clones were identified by colony PCR and Sanger sequencing of the extracted plasmids. For mutagenesis of these three GmLox genes simultaneously, a pooled CRISPR-Cas9 knockout strategy described previously [32,33] was used. The general procedure was as follows: first, two single sgRNA CRISPR-Cas9 vectors were constructed separately using sgRNA-GmLox1/2 and sgRNA-GmLox3, and then Agrobacterium strains GV3101 containing each vector and having similar optical density were mixed together. Finally, the mixed Agrobacterium solution was transformed into the soybean cultivar WT via A. tumefaciens-mediated transformation. Soybean transformation was performed as described previously [34].

2.3. Mutation screening by sequencing analysis

Gene editing of target regions was assessed by PCR and sequencing. PCR primers were designed to amplify specifically the target regions (Table S1). The PCR products were purified for Sanger sequencing to detect potential mutations. Different types of gene editing were identified via sequence peaks and alignment to the reference sequences as previously described [32].

2.4. Quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR)

To measure the expression of GmLox genes in WT plants and GmLox mutants, Q-RT-PCR of three GmLox genes was performed using total RNA extracted from cotyledon samples (5 days after sowing) of WT and four T1plants each from lines GmLox-28, GmLox-40, and GmLox-60. Total soybean cotyledon RNA was extracted using the E.Z.N.A. RNA Extraction Kit (Omega Bio-Tek, Norcross, Georgia, USA) according to the manufacturer's protocol. The PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa Biotech, Kyoto, Japan) was used for RT, and first-strand cDNA was amplified according to the instructions for the SYBR Premix Ex Taq II ROX Plus Kit (TaKaRa Biotech, Kyoto, Japan). A G. max TEFS1 (Glyma.17G186600, encoding the elongation factor EF-1a) gene-specific primer was used as control to normalize the expression data. Three biological replicates were used for each sample. The primer sequences are presented in Table S1.

2.5. Detection of seed lipoxygenases with a colorimetric assay method

A colorimetric assay method for determination of lipoxygenase activity was applied as previously described[35,36], with minor modifications. Briefly, dry seed samples were separately ground into powder. For each sample,lipoxygenase solutions 1, 2, and 3 (LS1,LS2,and LS3;used for the detection of LOX1, LOX2, and LOX3, respectively) were extracted in 1.5 mL 0.2 mol L?1pH 9.0 sodium borate buffer,1.5 mL 0.2 mol L?1pH 6.0 sodium phosphate buffer,and 1.5 mL 0.2 mol L?1pH 6.6 sodium phosphate buffer from respectively 15, 30, and 15 mg of soybean seed powder, and the clear supernatant was collected after centrifugation (12,000 rpm,5 min, 4 °C). For detection of LOX1, 0.5 mL LS1 was added to 1.0 mL substrate solution(0.125 mol L?1pH 9.0 sodium borate buffer, 12.5 μmol L?1methylene blue, 1.375 mmol L?1sodium linoleate substrate). For detection of LOX2, 0.5 mL LS2 was added to 1.0 mL substrate solution (0.125 mol L?1pH 6.0 sodium phosphate buffer, 12.5 μmol L?1methylene blue,1.375 mmol L?1sodium linoleate substrate,25 mmol L?1DTT,12.5%acetone).For detection of LOX3,0.5 mL LS3 was added to 1.0 mL substrate solution (0.125 mol L?1pH 6.6 sodium phosphate buffer,1.375 mmol L?1sodium linoleate substrate,12.5% β-carotene at 50% saturation). After mixing, each reaction was incubated in a transparent tube for 15 min and the solution color was recorded (clear or blue for LOX1 and LOX2, clear or yellow for LOX3). Their absorbances at 660 nm(for measurement of LOX1 and LOX2) and 452 nm (for measurement of LOX3) were measured with a spectrophotometer (UV-1600;Shimadzu,Kyoto,Japan).

2.6. Phenotypic measurement of soybean seeds

Seeds from each T1plant or WT were randomly divided into three equal parts (treated as three biological replicates) for seed composition analysis.Seed protein and oil content were measured using a MATRIX-I Fourier-transform near-infrared reflectance spectroscope (FT-NIRS) (Bruker Optics, Bremen,Germany).

3. Results

3.1. Pooled CRISPR-Cas9 knockout of three GmLox genes in soybean

According to sequence similarity with reference to the full soybean genome assembly (http://www.phytozome.net/soybean), sgRNA-GmLox1/2, targeting GmLox1 and GmLox2 in the second exon of these two genes, and sgRNAGmLox3, targeting GmLox3 in its third exon, were designed(Fig. 1).

Fig.1- Schematic figure of gene structures and target sites in three GmLox genes.(A,B) Gene structures of GmLox1(A)and GmLox2(B)with the same target site“sgRNA-GmLox1/2”.(C)Gene structure of GmLox3 with the target site“sgRNA-GmLox3”.Nucleotides marked by black or red lines represent the target sites or the protospacer adjacent motif(PAM)sequences,respectively.

Stable transformation of soybean cotyledons yielded 76 T0plants. DNA was extracted from leaf tissue of these plants to detect transgene presence, sgRNA distribution, and the typeand frequency of mutations generated. Based on sgRNA specific PCR (SSP) amplification, 60 T0positive transgenic plants were identified,of which 27 contained sgRNA-GmLox1/2 vector only,22 contained sgRNA-GmLox3 vector only,and 11 contained both vectors(Table S2).Sanger sequencing showed that 22 T0positive transgenic plants carried mutations in at least one target site. Eleven, 14, and 10 positive transgenic T0plants carried heterozygous mutations of GmLox1, GmLox2,and GmLox3, respectively (Fig. 2, Table S2). Single or double lipoxygenase mutants were identified: GmLox-1 is a single lipoxygenase mutant (with heterozygous mutations at the target site of GmLox3) and GmLox-40 is a double lipoxygenase mutant (with heterozygous mutations at the target site of GmLox1 and GmLox2). GmLox-28 and GmLox-60 both harbored heterozygous mutations at the target sites of GmLox1,GmLox2,and GmLox3 (Fig. 2, Table S2). Thus, GmLox-28 and GmLox-60 were triple lipoxygenase mutants.

Fig.2- Sequences of wild type and mutation types at target sites of GmLox1,GmLox2,and GmLox3,induced by CRISPR-Cas9 technology,in the T0 soybean plants.The triple lipoxygenase mutants GmLox-28 and GmLox-60 are marked by red circles.

Fig.3-Lipoxygenase activity and sequences of wild type and mutant types at target sites of GmLox1,GmLox2,and GmLox3,in T1 soybean plants.Color reaction(A,D,and G)and absorbance value(B,E,and H)were used for detection of the enzyme activity of LOX1,LOX2,and LOX3,respectively.Detailed sequences at the target site of GmLox1(C),GmLox2(F),and GmLox1(I)were used for the identification of targeted mutations in T1 plants.

3.2. Targeted mutations and lipoxygenase activity in T1 generation

To characterize the sgRNA distribution and mutations in the target site in T1plants, the genotypes of some T1plants were examined. Three T0plants were selected for further analysis, including two triple lipoxygenase mutants, GmLox-28 and GmLox-60, and a double lipoxygenase mutant, GmLox-40 (Fig. 2, Table S2). Ten seeds collected from each selfpollinated T0plant were grown in a growth chamber and a total of 26 T1plants (9, 9, and 8 of lines GmLox-28, GmLox-60,and GmLox-40, respectively) were used to examine the genotypes at the target sites of these three GmLox genes. The T-DNA of the sgRNA/Cas9 vectors in T0plants could be transmitted to their progeny: most T1plants of lines GmLox-40 (except GmLox-40-2) contained sgRNA-GmLox1/2 vector only, all T1plants of lines GmLox-28 except GmLox-28-3, -4,-8, and -9, and all T1plants of GmLox-60 contained both vectors(Table S3).Further,all T1plants of lines GmLox-28 and GmLox-60 showed heterozygous or homozygous targeted mutations within all three GmLox genes, and all T1plants of line GmLox-40 showed heterozygous or homozygous targeted mutations within GmLox1 and GmLox2 (Fig. S1, Table S3). A total of 12 T1plants(2 GmLox-28,3 GmLox-60,and 7 GmLox-40),9 T1plants (2 GmLox-28, 3 GmLox-60, and 4 GmLox-40), and 11 T1plants (9 GmLox-28 and 2 GmLox-60) showed homozygous targeted mutations within GmLox1, GmLox2 and GmLox3,respectively (Table S3). Simultaneously, all three types of mutations were found at target site GmLox1 (3-, 4-, and 8-bp deletions),GmLox2(4-,6-,and 8-bp deletions),and GmLox3(4-and 8-bp deletions and 1-bp insertion) (Fig. S1, Table S3).Among them, GmLox-28-8 and GmLox-28-9 carried two homozygous mutations of all three GmLox genes (Table S3).The expression of the edited GmLox genes in GmLox-28,GmLox-40,and GmLox-60 lines was measured.The expression of most edited genes was reduced(GmLox1 in 9 of 12 mutants,GmLox2 in 10 of 12 mutants, and GmLox3 in all 8 mutants;P ＜0.05)(Fig.S2).

To determine the presence or absence of lipoxygenase activity in soybean T1plants, T2seeds collected from GmLox-28,GmLox-40,and GmLox-60 were used for colorimetric assay.WT(wild-type;GmLox1,2,3-harboring)and WX4(GmLox1,2,3-free) seeds were used as respectively positive and negative controls. In consistency with the knockout of the three GmLox genes, all T1plants of line GmLox-28 and line GmLox-60 maintained the color of the substrate solution, indicating that they were free of LOX1 (the solution remained blue), LOX2 (the solution remained blue), and LOX3 (the solution remained yellow) activities (Fig. 3A, D, G). T1plants of line GmLox-40 were negative for LOX1 and LOX2 but positive for LOX3 (Fig. 3A, D, G). The absorbances of these samples supported these results (Fig. 3B, E, H). When T2seeds collected from lines GmLox-28, GmLox-40, and GmLox-60 were subjected to seed composition analysis, no differences (P > 0.05, n =8)in seed oil or protein content were observed between WT and Gmlox mutants (Fig. S3).

3.3. Generation of transgene- and lipoxygenase-free soybean T2 plants

To obtain transgene- and lipoxygenase-free soybean plants, four lipoxygenase-free T1plants (GmLox-28-4 and -8; GmLox-60-1 and -5) were selected for further analysis. About 30 seeds collected from each self-pollinated T1plant were grown in a growth chamber and 84 T2plants (23, 18, 27, and 16 of lines GmLox-28-4, GmLox-28-8,GmLox-60-1,and GmLox-60-5, respectively) were used to screen for transgene- and lipoxygenase-free soybean plants. CRISPR-Cas9 and sgRNAspecific primers were used to detect transgene presence (Table S1). Two plants from line GmLox-60-1, one from line GmLox-28-4, one from line GmLox-28-8, and one from line GmLox-60-5 were found to be free of transgenes (Fig. S4, Table S4). In addition, seeds collected from eight randomly selected T2plants (two each from lines GmLox-28-4, GmLox-28-8, GmLox-60-1, and GmLox-60-5) were free of LOX1, LOX2, and LOX3 activities (Fig. S5). This result showed that the lipoxygenase-free trait could be inherited by the T2generations.

4. Discussion

In this study, we generated LOX-free soybean germplasms using a pooled CRISPR-Cas9 system.Triple mutants of lox loci may be obtained within two generations. Moreover, the lox loci can be knocked out in an elite cultivar background (such as Huachun 6 in this study), based on existing agricultural traits. In addition, transgenes in CRISPR-Cas9-edited plants could be eliminated by selfing or backcrossing.Thus,CRISPRCas9 technology provides a practical method to rapidly and cost-effectively create new LOX-free varieties.

Compared with the multiplex CRISPR-Cas9 system that carries multiple sgRNAs on a single vector[31],pooled CRISPRCas9 requires integration of multiple T-DNAs in a single line to generate multiplex mutants [32,33]. Such a strategy facilitates the characterization of combinations of desired mutations after a single transformation [32,33]. However, it might pose concerns about increased difficulty of identifying transgene-free lines. In this study, we identified five transgene-free plants in T2progenies. This result showed that it is also feasible to obtain transgene-free lines in a pooled CRISPR-Cas9 population.

Lipoxygenases (LOXs) are members of non-heme ironcontaining proteins that are widely distributed in plants [3]. LOXs may play roles in plant physiological processes, such as plant growth and development, responses to biotic and abiotic stresses, and mobilization of storage lipids during germination [37-41]. The lipoxygenase-free mutants await functional studies to characterize their agronomic characters, seed germination, and resistance to biotic and abiotic stresses.

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2019.08.008.

Declaration of competing interest

Authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by funds from the National Key Research and Development Program of China(2016YFD0100700)to Y.G.