CRISPR-Cas9 mediated targeted disruption of FAD2–2 microsomal omega-6 desaturase in soybean (Glycine max.L)
Recent innovation in the field of genome engineering encompasses numerous levels of plant genome engineering which attract the substantial excitement of plant biologist worldwide. RNA-guided CRISPR Cas9 system has appeared a promising tool in site-directed mutagenesis due to its innovative utilization in different branches of biology. CRISPR-Cas9 nuclease system have supersedes all previously existed strategies and their associated pitfalls encountered with site-specific mutagenesis.
Here we demonstrated an efficient sequence specific integration/mutation of FAD2–2 gene in soybean using CRISPR-Cas9 nuclease system. A single guided RNA sequence was designed with the help of a number of bioinformatics tools aimed to target distinct sites of FAD2–2 loci in soybean. The binary vector (pCas9-AtU6-sgRNA) has been successfully transformed into soybean cotyledon using Agrobacterium tumafacien. Taken together our findings complies soybean transgenic mutants subjected to targeted mutation were surprisingly detected in our target gene. Furthermore, the detection of Cas9 gene, BAR gene, and NOS terminator were carried out respectively. Southern blot analysis confirmed the stable transformation of Cas9 gene into soybean. Real time expression with qRT-PCR and Sanger sequencing analysis confirmed the efficient CRISPR-Cas9/sgRNA induced mutation within the target sequence of FAD2–2 loci. The integration of FAD2–2 target region in the form of substitution, deletions and insertions were achieved with notably high frequency and rare off-target mutagenesis.
High frequent mutation efficiency was recorded as 21% out of all transgenic soybean plants subjected to targeted mutagenesis. Furthermore, Near-infrared spectroscopy (NIR) indicates the entire fatty acid profiling obtained from the mutants seeds of soybean. A considerable modulation in oleic acid content up to (65.58%) whereas the least level of linoleic acid is (16.08%) were recorded. Based on these finding CRISPR-Cas9 system can possibly sum up recent development and future challenges in producing agronomically important crops.
KeywordsCRISPR-Cas9 Targeted mutagenesis FAD2–2 Oleic acid Soybean
Clustered regularly interspaced short palindromic repeat
Protospacer adjacent motif
Single guide RNA
Transcription Activator-Like Effectors Nucleases
Zinc Finger Nucleases
Genome editing is a critical and complex tool to study the functional genomics of plants in past decades. Amidst all previously adapted techniques such as virus-induced gene silencing, T-DNA/transposon insertion, antisense RNA, and RNA interference strategies CRISPR-Cas9 nuclease system have supersedes them all. Producing low-quality mutants, unwanted mutations, point mutation and several other pitfalls are associated with these classical methods. [1, 2]. These advances are good enough for carrying basic plant research however genetic crop improvement and constructing new mutant libraries require new tools for more precise genome editing, and regulation of gene expression. CRISPR-Cas9 system has remarkably captivated the title in 2011 as “methods of the year” in plant biology. This extensive system of type II clustered regularly interspaced short palindromic repeat CRISPR-associated protein 9 (Cas9) have been reported recently in a wide variety of plants including soybean , Arabidopsis , and rice [5, 6]. Underlying the principle of single nucleotide polymorphism, previously established ZFNs (Zinc Finger Nuclease) and TALENs (Transcription Activator-Like Effectors Nuclease) and CRISPR system paved the way for site directed mutagenesis. The advent of targeted mutation induced by CRISPR cas9 system has been proved very simple, efficient and predictable tool in functional genomics of many plants . The genetic improvement of agronomical traits such as yield, Rearrangement, disease resistance, adaptation against various stresses and controlling negative regulation of specific genes via induced target mutagenesis are the potential areas of future researches [8, 9].
Soybean (Glycine max (L.) is an essential legume crop which provides a rich source of dietary proteins, animal feed, sustainable agricultural products, vegetable oil, and direct human consumption. Recent advances have expanded soybean research towards the production of biodiesel from the feedstock of soybean . The utilization of soybean oils with high oleic acid content resulting in monounsaturated fatty acids could be useful in minimizing various health problems [11, 12, 13]. Researchers have been proved that Soybeans genome holds two similarly identical copies (FAD2–1 and FAD2–2) of microsomal 휔-6 desaturase . The primary expression of FAD2–1 was confirmed during seed developmental processing unlike FAD2–2 which was expressing in vegetative parts as wells [10, 11]. The conserved sequence homology of the FAD2–2 family reveals multiple isoforms such as FAD2–1A, FAD2–1B, FAD2–2B and FAD2–2C which are nearly related to their predecessor family by sharing 99% homology in amino acid sequence [14, 15, 16]. By these means, this confirmed the importance and pragmatic role of FAD2–2 family and their exploitation in the biosynthesis of peakoil in soybean [17, 18].
The authors aimed to use CRISPR-Cas9 gene editing technology to mutate the FAD2–2 gene in soybean in order to improve the seed oil profile We generated transgenic soybean lines with the CRISPR transgene, tested for the presence of mutations in the FAD2–2 locus, assessed for off-target effects and determined impact on fatty acid profiles. Furthermore, we provide the current advancement and applications of CRISPR-Cas9 induced mutation driven by an oligo SgRNA sequence in plants may overcome major limitations of crop genetic improvement.
CRISPR-Cas9 construction for target mutagenesis in soybean
Detection of positive transgenic soybean lines
In our study, we used BAR gene as a selectable marker to detect the presence of transgene (SgRNA-CRISPR-Cas9) in transgenic soybean plants (Fig. 2b). Besides this, we also detected the presence of NOS terminator gene in target transgenic lines of soybean (Fig. 2c). For this purpose genomic DNA was extracted using (NuClean plant Genomic DNA Kit, Beijing, China) from the selected transgenic lines of soybean and subjected next to PCR amplification with G°Taq Flexi DNA polymerase (Promega Corp, Madison, WI) using forward and reverse pairs of primers for both NOS terminator and BAR gene. (Additional file 1: Table S2).
A single enzyme digestion pattern with (HINDIII) enables the fractionation of BAR gene present in the transgenic soybean plants. Post hybridization all together 8 transformation events were detected with digoxigenin [DIG] 1-dUTP probe and DIG High Prime DNA Labeling chemicals separated on 0.8% agarose gel (Fig. 3). Various patterns of hybridization appear after immobilization of DNA onto nylon membranes (Amersham) showed a product of (552 bp) along with flanking sequence probes. This indicated the presence of our target exogenous DNA in several copies within mutant lines (Additional file 1: Figure S1).
Detection of targeted mutation in transgenic soybean
Mutation efficiency of FAD2–2 gene induced by CRISPR-Cas9 system
No. of examined plant
No. of plants with cas9 gene
No. of plants with mutations
Mutation efficiency %
Off-target analysis in soybean transgenic plants
Analysis of targeted gene mutations induced by CRISPR-Cas9 cassettes in transgenic soybean lines. Transgenic plants containing confirmed FAD2–2 mutagenesis were subjected to look for off target editing
No. of putative off target sites
No. of loci of putative off target sites
No. of examined loci
No. of plants examined
No. of off target mutation
Fatty acid profiling via NIR
The NIR analysis of complete fatty acid contents for FAD2–2 omega 6 destuarase mutants. Samples from P1-P8 represent the percentage composition of fatty acid contents in soybean transgenic lines
17.34h ± 0.20
45.08g ± 0.04
51.21e ± 0.11
49.27f ± 0.09
58.82c ± 0.37
54.05d ± 0.08
65.90a ± 0.33
59.12b ± 0.10
59.58b ± 0.22
Clustered regularly interspaced short palindromic repeat (CRISPR-Cas9). CRISPR-associated Cas9 nuclease can significantly induce targeted gene editing by cleaving target DNA sequence which is guided by synthetic sgRNA sequence incorporating double-strand break (DSB) on a target site . Small guide RNA is the first 20 nucleotide sequences complementary to one strand of the target DNA with an NGG motif at 3′-end (the protospacer adjacent motif, PAM) which scans a target genome for editing. The expression of sgRNAs is carried by U3/U6 promoters, which transcribed sgRNA with the help of RNA polymerase [23, 24]. To induce targeted genome editing in vitro efficiently requires an efficient and feasible delivery of a binary vector consist of (Cas9 and sgRNA expression cassettes) into various plants. The inherited stability of CRISPR-Cas9 based mutations was reported previously which can be transferred into next progeny. Here we have successfully demonstrated targeted genome editing in soybean using the CRISPR-Cas9 system, where most of the induced mutations were found as nucleotide substitution while some of them were nucleotide deletion and insertion (Fig. 4b). Our results were concordant with the previous findings . The mutation efficiencies in transgenic plants were 21% (Table 1).
Fatty acid desaturases enzymes are contained in large numbers in plant. These enzymes are mainly present in endoplasmic reticulum and chloroplast. The fatty acid desaturases 2 (FAD2; EC 18.104.22.168) is hydroponic transmembrane endoplasmic reticulum protein, controlling the cis double bond between c12 and c13 . The FAD2–2 gene was initially reported in Arabidopsis , then it was further identified in several crops such as sesame (Sesamum indicum) , corn (Zea mays), canola (Brassica napus) , olive (Olea europaea) , soybean (Glycine max) , sunflower (Helianthus annuus) , and cotton (Gossypium hirsutum) . Previously it has been stated that, the FAD2–2 gene is mainly responsible for the conversion of oleic acid into linoleic acid [29, 35]. Therefore, producing FAD2–2 mutant could decrease the conversion of oleic acid into linoleic acid. Moreover, the FAD2–2 gene expresses in all almost all plant tissues during oil biosynthesis but the expression level is relatively high in seeds . Similarly, we observed high expression level of FAD2-2 in stem and leaves comparing to seeds in both wild types (WT) and transgenic plant (T) (Fig. 5). However, there is a significant difference between the expression level of WT and T seeds tissue than that to other vegetative tissues, Where T plants showed relatively low expression level of FAD2–2 gene in seeds. The result is in line with the previously reported study by Zhang et al. . These finding further provide solace to the importance of FAD2–2 genes family in the biosynthesis of oil from edible seed plants. For instance, the oleic acid content is considered as the most important and effective molecule in enhancing the nutritional index and shelf life of soybean oil. It also play role in decreasing the risk of type diabetes . Though further study is required to produce double or triple mutant of FAD2–2 genes in order to increase the value of oleic acid contents and restrict their conversion into linoleic acid. As it was suggested by Wang et al. in their study of FAD2–2 genes family in peanut (Arachis hypogaea L.). In our findings, we found drastic diversity through NIR spectral data of overall lipid content of mutant plants seeds in comparison with wild soybean lines (Table 3). The maximum increase oleic acid content was recorded up to (65.90%, P6) while the lowest linoleic acid were found (16.08%, P6). These findings depict previous studies on FAD2–2 gene which encode ω-6 fatty acid desaturases catalyzes the conversion of oleic acid to linoleic acid in soybeans using antisense RNA technology [14, 39].
Additionally to tackle the most efficient and recent transformation method out of all available systems is a necessary step. In view of the fact that Agrobacterium-mediated transformation is proved the most widely used for potential applications of CRISPR-Cas9 which facilitate the efficient integration of T-DNAs consist of Cas9 and sgRNA expression cassettes in target plant . We have successfully transferred a binary vector (pCas9-atU6-sgRNA) into soybean cotyledon via Agrobacterium tumafaciens (EH105). The transgenic lines were verified with a reporter gene (BAR), NOS terminator and Cas-9 gene (Figs. 2 and 4). Our study signifies the exploitation of FAD2–2 gene using CRISPR-Cas9 induced mutation in soybean which results in a significant increase oleic acid content (Table 3). Our results concluded that CRISPR-Cas9 system can play a vital role in constructing a mutant library of soybean.
This work showed an efficient CRISPR-Cas9 gene editing technology to encounter FAD2–2 gene of soybean for targeted mutagenesis aimed to improve the fatty acid profiling of transgenic soybean lines. CRISPR transgene activity was detected by high throughput sequence analysis and southern blotting. The maximum induced rate of mutation was recorded 21% with high ratio of substitution. NIR analysis indicated the enhanced profiling of monounsaturated fatty acid content resulting in high oleic acid accumulation (65%).
Materials and methods
Seeds of Glycine max L. (JN38) was obtained from previously stored stock in our lab (Plant biotechnology centre, Jilin Agricultural University, China). Escherichia coli strain (DH5α), Agrobacterium tumefacien strain (EHA105) and CRISPR-Cas9 vector was purchased from Biogle Co. Ltd. Hangzhou. Restriction enzymes, DNA extraction kit and DNA ligases were purchased from (Takara Biotechnology, Company Beijing). DIG High Prime DNA Labeling and Detection Starter kit 1 (for color detection with NBT/BCIP, REF 11745832910) were purchased from (Takara Biotechnology Company Beijing).
Glycine max JN38 cultivar was selected for this study. After that, healthy seeds were selected and sterilized with 30 ml Cl2, (25mlNaocl, 5 ml HCl) for 16 h, which was further used for Agrobacterium-mediated transformation.
Prior to construction of our desired CRISPR Cas9 vector it is important to design our target synthetic sgRNA, for this purpose we extracted FASTA file of the complete Cds of (FAD2–2 microsomal omega-6 desaturase) gene having GenBank ACCESSION number (L43921) from NCBI. A BLASTN for (FAD2–2 microsomal omega-6 desaturase) sequence has been constructed against Physcomitrella paten in order to observe potential target sites within the coding regions. Physcomitrella paten is a model plant which is widely known for targeted gene knockouts and facilitating reverse genetics approaches.
The indication of percentage identity was recorded as 99.2% with best E. value in the exonic regions.. In line with previous studies a small guide RNA was designed at position 3:38046125, locus 15:+ 22245082 in the exonic region of FAD2–2 gene.
CRISPR-Cas9 (BGK041) plant gene knockout vector kit (Biogle Co. Ltd. Hangzhou, China) was used to construct the expression vector of the plant. The plasmid contain a codon-optimized Cas9 gene under the e35S promoter, sgRNA gene with U6 promoter from Soybean, NOS terminator for termination signal of the gene expression, and a reporter BAR gene (Fig. 1). The sgRNA scaffold (19-nt) target sequences were amplified by PCR using a pair of synthetic oligos according to manufacturers protocol (Additional file 1: Table S1). The annealing of oligos with CRISPR-Cas9 vector was followed according to manufacturer’s instructions and transformed into E.coli strain (DH5α).
Preparation of agrobacterium tumafecian strain
Agrobacterium tumefaciens strain EHA105 stock was prepared. The transformation of EHA105 with CRISPR-Cas9 vector was accomplished with heat and cold shock method, followed by 45 mins shaking in LB liquid medium containing antibiotic (kanamycin 1 ml/L) and incubated overnight at 28 °C. The half colony was selected for PCR using CRISPR-Cas9 specific primers (Additional file 1: Table S2) (Fig. 2a) after selecting positive clones the remaining half was re-cultured for infection in 5 ml LB/1 ml kanamycin for overnight shaking at 28 °C, 220 rpm.
Agrobacterium-mediated transformation of soybean
After 15 days incubation, the plants were observed and further transferred to selective (S2) media with herbicide treatment (glufosinate) for another period of 15 days (Fig. 6c). Finally, the explants which are glufosinate resistance were shifted to shooting media for 15 days (Fig. 6d) and then transferred to rooting media (Fig. 6e). Once root development was observed, double distilled water was added for 3 days, to make sure the damage less uprooting of plants. The uprooted plants further shifted to the sterilized soil for acclimatization (Fig. 6f, Additional file 1: Table S4).
Progeny analysis of transgenic plants
We confirmed the presence of CRISPR-Cas9 using NOS terminator, BAR gene and Cas9 gene by PCR analysis with specific primers (Additional file 1: Table S3). Detection with BAR gene the PCR profile includes, Master mix 12.5 μl, DNA 2 μl, R/F primers 1 μl, dd water 8.5 μl making the total volume 25 μl and was set to a control of 35 cycles of 94 °C for 30s, 58 °C for 30 s, and 72 °C for 45 s. However the Cas9 gene utilizes follows the PCR system as, Master Mix 12.5 μl, DNA 2 μl, both primers 1 μl, dd water 8.5 μl making the total volume 25 μl and was set to a control of 35 cycles of 94 °C for 30s, 64 °C for 30 s, and 72 °C for 50 s. Each PCR products were examined on 1.2% agrose gel.
Genomic DNA of the transgenic plants was extracted using a NuClean plant Genomic DNA Kit (Beijing, China) following the company protocol. In order to identify the mutation in positive soybean plants, we amplified the target gene through Polymerase Chain Reaction with gene-specific primers (Additional file 1: Table S3). The PCR system was followed as, Master mix 12.5 μl, DNA 2 μl, primers 1 μl, dd water 8.5 μl making the total volume 25 μl and was set to a control of 35 cycles of 94 °C for 30s, 56 °C for 30 s, and 72 °C for 50s. The purification of selective target bands was obtained with Axygen DNA Gel Extraction Kit (Beijing, China) and then cloned into a pMD18-T vector for sequencing. Latterly the sequencing results were analyzed for mutation through software (DNA MAN). Several transgenic plants were selected and cloned for the detection of targeted mutation that has been acquired, (Additional file 1: Figure S2).
Southern blot detection of transgenic plants
We selected positive plants for Southern blot analysis. Southern blot was performed with DIG High Prime DNA Labeling and Detection Starter kit 1 (for color detection with NBT/BCIP, REF 11745832910) with given manufacturer protocol. Genomic DNA was extracted from transgenic plants using a NuClean plant Genomic DNA Kit (beijing, China). DNA of transgenic plants was digested for overnight with hind111 and Tengo buffer and 1.2% gel was used for electrophoresis with a time interval of 2 h and then shakes in denatured solution for 2 h which was further placed in Amersham nylon membranes (Amersham) overnight. Marker BAR gene was extracted from transgenic DNA and used as a probe for detection. Afterward, 8 μl probe was mixed with 16 ml digoxigenin [DIG] 1-dUTP with DIG High Prime DNA Labeling chemicals. Hybridization was performed for 2 h at 42 °C and then further washed 2 times with 20 SSC. Furtherly, blocking and detection were performed according to the manufacturer’s protocol and then placed for detection at room temperature for 30 mints to 1 h.
During the present study, we have analyzed the Potential off target of FAD2–2 on CRISPR-P website (http://cbi.hzau.edu.cn/cgi-bin/CRISPR) containing almost 49 plant genomes datasets including (Glycine max genomic database). The first 19 potential off-target sites were examined on CRISPR-P website, then we searched these 19 potential off-target sequences and their loci in Glycine max genomic database and identified with PCR and sequence analysis.
We also investigate various others factors affecting the optimum or minimal off target mutation in target genome. To avoid minimum off target mutations the selection of SgRNA within exonic region with low GC content can influence the effect of off target mutagenesis. The FAD2–2-sgRNA2 potential off target sites was identified and analyzed with sequencing analysis.
Isolation of Total RNA and semi-quantitative RT-PCR analysis
The Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) from different tissues of transgenic and wild type soybean plantlets. Further, purification of RNA was achieved with treatment of RNase-free DNaseI (TaKaRa, beijing, China) to eliminate genomic DNA contamination according to the protocols recommended by the manufacturer. The first strand of cDNA was synthesized from 2.0 μg of total RNA using the M-MLV First Strand Kit (Invitrogen) and the cDNA products equivalent to 200 ng of total RNA were used as templates in a 25 μL PCR reaction system. All conditions were followed according to previously described method . Semi-quantitative RT-PCR analyses for gene expression were performed on a PCR instrument (S1000™ Thermal Cycler, BIO-RAD, Foster City, CA, USA), with the conditions of 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The GmActin gene (NM_001289231) was used as a positive internal control and the gene primers for the qRT-PCR were designed using conserved sequences of FAD2–2 gene. (Additional file 1: Table S1).
Fatty acid profiling via NIR
Soybean seeds were prepared for the near-infrared transmittance instrument (Infratec 1225) as described by . The Calibration samples are taken from the soybean transgenic mutants. Fatty acid profiling consist of (Palmitic acid, stearic acid, oleic acid, linoleic and linolenic acid) were selected for the prediction of NIRs in soybean seed. The results were significantly analyzed with statistical analysis by calculating their mean values.
The complete CDs of Glycine max FAD2–2 microsomal omega-6 desaturase in FASTA format were extracted from GenBank (L43921). This 1556 bp long fragment is then subjected to BLASTN against Physcomitrella patens genome using ENSEMBL PLANTS. To accomplish the potential Targeted sites for sgRNA within FAD2–2 sequence the filtered sequence is further subjected to CRISPR-P, and then selected the target genome of FAD2–2 (Glycine max L.) for which we were intended to design a highly specific targeted sgRNA sequence.
We are grateful to Prof. Wang Pi Wu for his kind guidance and providing lab equipment for this study.
This project was funded by the Natural science foundation of china under grant number (317710333).
Availability of data and materials
All the data which is generated and analyzed during our study are included within the article and additional files.
All authors have read and approved the manuscript and ensure that this is the case. Data analysis:, MT, NA2, DY and NW; Formal analysis, WN, PX and DY; Funding acquisition, WP; Investigation, WP; Methodology, NA2 and NW; Software, BX; Supervision, WP; Visualization, NA1, NA2 and BX; Writing – original draft, NA1 and RS; Writing – review & editing, NA1, NA2 and BX.
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