Generation of seed lipoxygenase-free soybean using CRISPR-Cas9

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)-CRISPR-associated 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 transgene- and lipoxygenase-free mutants could be used for soybean beany flavor reduction without restriction by regulatory frameworks governing transgenic organisms.

Full text

Generation of seed lipoxygenase-free soybean
using CRISPR-Cas9☆
Jie Wang
a,b
, Huaqin Kuang
b
, Zhihui Zhang
a,b
, Yongqing Yang
c
, Long Yan
d
,
Mengchen Zhang
d
, Shikui Song
b
, Yuefeng Guan
b,
⁎
a
College of Resources and Environment, Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Fujian Agriculture and
Forestry University, Fuzhou 350002, Fujian, China
b
FAFU-UCR Joint Center for Horticultural Plant Biology and Metabolomics, Haixia Institute of Science and Technology, Fujian Agriculture and
Forestry University, Fuzhou 350002, Fujian, China
c
Root Biology Center, College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
d
The 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
ARTICLE INFO ABSTRACT
Article history:
Received 30 April 2019
Received in revised form 12 June
2019
Accepted 11 September 2019
Available online xxxx
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)-CRISPR-
associated 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 sgRNAs and mutations. Among them, GmLox-28 and GmLox-60 were
gmlox1gmlox2gmlox3 triple mutants and GmLox-40 was a gmlox1gmlox2 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 transgene-
and lipoxygenase-free mutants could be used for soybean beany flavor reduction without
restriction by regulatory frameworks governing transgenic organisms.
© 2019 Crop Science Society of China and Institute of Crop Science, CAAS. Production and
hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.
0/).
THE CROP JOURNAL XX (XXXX) XXX
☆Peer review under responsibility of Crop Science Society of China and Institute of Crop Science, CAAS.
⁎Corresponding author.
E-mail address: guan@fafu.edu.cn. (Y. Guan).
Peer review under responsibility of Crop Science Society of China and Institute of Crop Science, CAAS.
https://doi.org/10.1016/j.cj.2019.08.008
Available online at www.sciencedirect.com
ScienceDirect
CJ-00429; No of Pages 8
Please cite this article as: J. Wang, H. Kuang, Z. Zhang, et al., Generation of seed lipoxygenase-free soybean using CRISPR-Cas9, The
Crop Journal, https://doi.org/10.1016/j.cj.2019.08.008

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 time-
consuming 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 CRISPR-
Cas9 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), lipoxygenase-
free 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]wasused.
The general procedure was as follows: first, two single
sgRNA CRISPR-Cas9 vectors were constructed separately
using sgRNA-GmLox1/2and sgRNA-GmLox3,andthen
Agrobacterium strains GV3101 containing each vector and
having similar optical density were mixed together. Finally,
the mixed Agrobacterium solution was transformed into the
soybeancultivarWTviaA. 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
performedusingtotalRNAextractedfromcotyledonsamples
(5 days after sowing) of WT and four T
1
plants 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.
2THE CROP JOURNAL XX (XXXX) XXX
Please cite this articleas: J. Wang, H. Kuang, Z. Zhang, et al., Generationof seed lipoxygenase-free soybean using CRISPR-Cas9, The
Crop Journal, https://doi.org/10.1016/j.cj.2019.08.008

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
−1
pH 9.0 sodium borate buffer,
1.5 mL 0.2 mol L
−1
pH 6.0 sodium phosphate buffer, and 1.5 mL
0.2 mol L
−1
pH 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
−1
pH 9.0 sodium borate
buffer, 12.5 μmol L
−1
methylene blue, 1.375 mmol L
−1
sodium
linoleate substrate). For detection of LOX2, 0.5 mL LS2 was
added to 1.0 mL substrate solution (0.125 mol L
−1
pH 6.0
sodium phosphate buffer, 12.5 μmol L
−1
methylene blue,
1.375 mmol L
−1
sodium linoleate substrate, 25 mmol L
−1
DTT,
12.5% acetone). For detection of LOX3, 0.5 mL LS3 was added to
1.0 mL substrate solution (0.125 mol L
−1
pH 6.6 sodium
phosphate buffer, 1.375 mmol L
−1
sodium 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 T
1
plant 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
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.
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in the second exon of these two genes, and sgRNA-
GmLox3, targeting GmLox3 in its third exon, were designed
(Fig. 1).
Stable transformation of soybean cotyledons yielded 76 T
0
plants. DNA was extracted from leaf tissue of these plants to
detect transgene presence, sgRNA distribution, and the type
Fig. 2 –Sequences of wild type and mutation types at target sites of GmLox1,GmLox2, and GmLox3, induced by CRISPR-Cas9
technology, in the T
0
soybean plants. The triple lipoxygenase mutants GmLox-28 and GmLox-60 are marked by red circles.
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Fig. 3 –Lipoxygenase activity and sequences of wild type and mutant types at target sites of GmLox1,GmLox2, and GmLox3,in
T
1
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 T
1
plants.
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and frequency of mutations generated. Based on sgRNA
specific PCR (SSP) amplification, 60 T
0
positive transgenic
plants were identified, of which 27 contained sgRNA-GmLox1/
2vector only, 22 contained sgRNA-GmLox3 vector only, and 11
contained both vectors (Table S2). Sanger sequencing showed
that 22 T
0
positive transgenic plants carried mutations in at
least one target site. Eleven, 14, and 10 positive transgenic T
0
plants 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.
3.2. Targeted mutations and lipoxygenase activity in T
1
generation
To characterize the sgRNA distribution and mutations in
the target site in T
1
plants, the genotypes of some T
1
plants
were examined. Three T
0
plants 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 self-
pollinated T
0
plant were grown in a growth chamber and a
total of 26 T
1
plants (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 T
0
plants could be
transmitted to their progeny: most T
1
plants of lines GmLox-
40 (except GmLox-40-2) contained sgRNA-GmLox1/2vector
only, all T
1
plants of lines GmLox-28 except GmLox-28-3, -4,
-8, and -9, and all T
1
plants of GmLox-60 contained both
vectors (Table S3). Further, all T
1
plants of lines GmLox-28 and
GmLox-60 showed heterozygous or homozygous targeted
mutations within all three GmLox genes, and all T
1
plants of
line GmLox-40 showed heterozygous or homozygous targeted
mutations within GmLox1 and GmLox2 (Fig. S1, Table S3). A
total of 12 T
1
plants (2 GmLox-28, 3 GmLox-60, and 7 GmLox-40),
9T
1
plants (2 GmLox-28, 3 GmLox-60, and 4 GmLox-40), and 11
T
1
plants (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 T
1
plants, T
2
seeds 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 T
1
plants 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). T
1
plants 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 T
2
seeds 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
T
2
plants
To obtain transgene- and lipoxygenase-free soybean plants,
four lipoxygenase-free T
1
plants (GmLox-28-4 and -8; GmLox-60-1
and -5) were selected for further analysis. About 30 seeds
collected from each self-pollinated T
1
plantweregrownina
growth chamber and 84 T
2
plants (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 sgRNA-
specific 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 T
2
plants (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
T
2
generations.
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, CRISPR-
Cas9 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 CRISPR-
Cas9 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 T
2
progenies. This result showed
that it is also feasible to obtain transgene-free lines in a
pooled CRISPR-Cas9 population.
6THE CROP JOURNAL XX (XXXX) XXX
Please cite this articleas: J. Wang, H. Kuang, Z. Zhang, et al., Generationof seed lipoxygenase-free soybean using CRISPR-Cas9, The
Crop Journal, https://doi.org/10.1016/j.cj.2019.08.008

Lipoxygenases (LOXs) are members of non-heme iron-
containing 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.
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8THE CROP JOURNAL XX (XXXX) XXX
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Crop Journal, https://doi.org/10.1016/j.cj.2019.08.008