CRISPR/Cas9-targeted mutagenesis of Os8N3 in rice to confer resistance to Xanthomonas oryzae pv. oryzae
Genome editing tools are important for functional genomics research and biotechnology applications. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system for gene knockout has emerged as the most effective genome-editing tool. It has previously been reported that, in rice plants, knockdown of the Os8N3 gene resulted in enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo), while displaying abnormal pollen development.
The CRISPR/Cas9 system was employed to knockout rice Os8N3, in order to confer enhanced resistance to Xoo. Analysis of the genotypes and edited Os8N3 in T0, T1, T2, and T3 transgenic rice plants showed that the mutations were transmitted to subsequent generations, and homozygous mutants displayed significantly enhanced resistance to Xoo. Stable transmission of CRISPR/Cas9-mediated Os8N3 gene editing without the transferred DNA (T-DNA) was confirmed by segregation in the T1 generation. With respect to many investigated agronomic traits including pollen development, there was no significant difference between homozygous mutants and non-transgenic control plants under greenhouse growth conditions.
Data from this study indicate that the CRISPR/Cas9-mediated Os8N3 edition can be successfully employed for non-transgenic crop improvements.
KeywordsCRISPR/Cas9 Disease resistance Os8N3 Rice xa13 Xanthomonas oryzae pv. oryzae
Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease
Effector binding element
- TAL effector
Transcription activator-like effector
- Xoo PR6
Xanthomonas oryzae pv. oryzae Philippine Race 6
Rice (Oryza sativa L.) is one of the most important cereal crops in the world, directly feeding more people than any other crop. Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a prevalent and destructive rice disease that causes serious production loss worldwide (Zhang and Wang 2013). Enhancing rice plants’ resistance to Xoo is known to be an economical and effective approach for managing rice bacterial blight.
Xoo pathogenicity depends on a specific class of virulence factors, called transcription activator-like (TAL) effectors, which mimic plant transcriptional activators (Hutin et al. 2015; Blanvillain-Baufume et al. 2017). The TAL effectors target the host nucleus, where they bind to specific promoter elements of the plant genes and activate their expression, reprogramming the plant transcriptome (Schornack et al. 2013). The genomes of Xanthomonas strains typically contain highly variable numbers of TAL effectors between Asian Xoo (15–26), African Xoo (8–10), and North-American Xoo (0) (Erkes et al. 2017). The rice genes targeted by TAL effectors have been identified as host disease-susceptibility genes, acting as major susceptibility factors during rice and Xoo interactions. In some cases, DNA polymorphisms in the so-called TAL effector binding elements (EBEs), located at the promoter region of the susceptibility gene, lead to no development of the disease (Yang et al. 2006; Hutin et al. 2015). Rice Os8N3 (also known as OsSWEET11), which belongs to the Sugar Will Eventually be Exported Transporters (SWEET) family of sugar transporters, represents one of the susceptibility genes induced by TAL effectors (Yang et al. 2006; Chen 2014). The expression of Os8N3 is induced by strains of Xoo carrying pthXo1, which encodes the TAL effector PthXo1 (Yang et al. 2006; Yuan et al. 2009). PthXo1 from Xoo strain PXO99 directly activates Os8N3 through recognition of TAL EBEs located at the promoter region of Os8N3 (Romer et al. 2010). The recessive resistance gene xa13 occurs as a series of natural alleles of the susceptibility gene Os8N3 (Yang et al. 2006; Yuan et al. 2009). Although it has not been clearly demonstrated, Os8N3 is believed to remove toxic copper from xylem vessels where Xoo multiplies and spreads (Yuan et al. 2010), and make nutrients easily available to Xoo for its growth and virulence to cause disease (Chen et al. 2010; Chen et al. 2012).
Genome editing technologies enable precise modification of DNA sequences in vivo and promise a novel revolution in crop improvement (Sun et al. 2016; Feng et al. 2013). The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system has revolutionized genome editing and become widely popular because of its specificity, simplicity, and versatility. It allows targeted genome editing in organisms guided by a customizable small noncoding RNA called single guide RNA (sgRNA). Once susceptibility genes targeted by TAL effectors have been identified, the CRISPR/Cas9-mediated genome editing strategy can be employed to create a target mutation in the susceptibility genes. Although it was not edited by the CRISPR/Cas9, Os11N3 (also known as OsSWEET14), the susceptibility gene targeted by AvrXa7 and PthXo3, has been edited by Transcription Activator-Like Effector Nucleases (TALENs) to create bacterial blight-resistant rice through disrupting the EBE site in the promoter region (Li et al. 2012; Blanvillain-Baufume et al. 2017). It can also be applied to negative regulators of disease resistance that have been studied for the last decades (Grand et al. 2012; Wang et al. 2015; Chern et al. 2005). However, to date, only a few examples of improvement of disease resistance using the CRISPR/Cas9 approach have been reported (Wang et al. 2016; Pyott et al. 2016; Peng et al. 2017). For Os8N3, studies on its knockdown rice plants using the gene silencing system and promoter mutations reported that they showed enhanced resistance to Xoo while displaying abnormal pollen development (Yang et al. 2006; Chu et al. 2006). Recently, CRISPR/Cas9-mediated knockout of Os8N3 displayed decreased sucrose concentration in the embryo sacs and defective grain filling, suggesting that Os8N3 plays important role in sucrose transport during early stage of rice grain filling (Ma et al. 2017; Yang et al. 2018).
Here, the CRISPR/Cas9-target mutagenesis of Os8N3 in Kitaake, a Japonica rice cultivar, is reported. The homozygous mutant lines carrying edited Os8N3 displayed significantly enhanced resistance to Xoo with normal pollen development. It was possible to select resistant mutant lines not containing the transferred DNA (T-DNA) by segregation in the T1 generation.
Os8N3 in the rice cultivar Kitaake
CRISPR/Cas9 design for xa13/Os8N3 editing
In monocot plants, the rice U3 small nuclear RNA promoter (OsU3) is generally used to express sgRNA (Belhaj et al. 2013). Recently, the efficiency of mutations targeted by sgRNAs driven by different small nuclear RNA promoters including OsU3, OsU6a, OsU6b, and OsU6c, were compared in an Indica cultivar 93–11 (Ma et al. 2015b). OsU6a was slightly more efficient in driving genome editing than the other promoters. It has also been reported that U6 promoters derived from the target plants function better than heterologous U6 promoters (Sun et al. 2015). Therefore, it was decided to use the OsU6a promoter isolated from the Japonica cultivar Kitaake. The OsU6a promoter amplified from Kitaake contains five single-nucleotide substitutions and one 5-bp deletion compared with one from Indica cultivar 93–11 (Additional file 1: Figure S1). The Arabidopsis U6 promoter in the CRISPR/Cas9 vector, pHAtC (Kim et al. 2016), was replaced with the Kitaake OsU6a promoter, and the resulting OsU6a::pHAtC was used for rice CRISPR/Cas9-mediated target mutagenesis.
CRISPR/Cas9-mediated targeted mutagenesis of xa13/Os8N3
Transmission and segregation of CRISPR/Cas9-mediated target mutagenesis from T0, T1, T2, and T3 of the OsU6a xa13m/Kit transgenic plant. The recovered mutated alleles of the xa13/Os8N3 gene in the OsU6a xa13m/Kit transgenic plant are shown below the Kitaake sequence. Nucleotide sequences at the target sites are shown in black capital letters and black dashes. PAM motifs are underlined. Red capital letters indicate the inserted nucleotide. The genotype of the mutation is indicated at the right of each sequence. WT indicates the nucleotide sequences identical to the Os8N3 gene in Kitaake plants. “+” indicates the insertion of the indicated number of nucleotides. No transgene: PCR negative for Cas9 gene; Transgenic: PCR positive for Cas9 gene; S: susceptible to PXO99; R: resistant to PXO99; Not available: inoculation data are not available
Inheritance of Os8N3 mutations and enhanced resistance to Xoo
To determine if and how the CRISPR/Cas9-targeted mutagenesis of Os8N3 by OsU6a::xa13m-sgRNA/pHAtC was transmitted to the next generation, all OsU6a xa13m/Kit T0 transgenic plants were self-pollinated and the targeted Os8N3 of some T1 transgenic plants was directly sequenced and analyzed (Fig. 3b, Table 1, and Additional file 3: Figure S3). The homozygous mutated T0 line (4A) produced homozygous mutated T1 progeny (4A-1, 4A-2, and 4A-3) and did not display additional different mutations. There was no mutation observed in the sequenced T1 progenies of the WT 1A, and 2A lines. However, new targeted sequence changes were detected in the T1 progeny of the WT 3A line. Previously, sequencing results indicated a putative WT genotype of the targeted Os8N3 in the T0 3A line, whereas three (3A-2, 3A-4, and 3A-6) out of the five sequenced T1 progenies of the WT 3A line displayed a 1-bp insertion (Table 1): 3A-2 was homozygous; 3A-4 was bi-allelic; and 3A-6 was heterozygous.
Main agronomic traits in xa13 mutants
Analysis of the agronomic traits of T3 mutant lines
Plant height (cm)
Flag leaf length (cm)
Flag leaf width (mm)
No. of productive panicles
Panicle length (cm)
69.8 ± 4.1a
27.8 ± 4.9a
11.7 ± 0.9a
3.0 ± 0.0a
11.1 ± 2.1a
Progeny of 3A-6-1
65.6 ± 6.4a
26.9 ± 4.0a
12.7 ± 0.5a
3.0 ± 0.0a
12.1 ± 1.4a
Progeny of 4A-1-7
65.7 ± 7.9a
29.1 ± 4.8a
12.0 ± 0.4a
2.5 ± 1.1a
12.0 ± 2.2a
Selection of transgene-free mutant rice lines
To select rice plants harboring the mutation in Os8N3 but without the T-DNA of the OsU6a::xa13m-sgRNA/pHAtC construct, PCR and phenotypic analysis for the OsU6a xa13m/Kit T0, T1, and T2 plants was performed. Thirty-one segregating T1 plants were analyzed and six of them (19.35%) did not generate a Cas9-specific amplicon from the T-DNA (Fig. 3b). Similarly, PCR analysis also failed to detect the T-DNA in 11 out of the 65 segregating T2 plants (16.92%) derived from nine T1 plants (1A-1, 1A-2, 1A-5, 1A-8, 1A-16, 3A-6, 4A-1, 4A-3, and 4A-7) (Fig. 3c). Notably, the 4A-1 plant was a Cas9-free homozygous mutant harboring the desired xa13/Os8N3 modifications (Fig. 3b and Fig. 4, and Additional file 3: Figure S3 and Additional file 4: Figure S4). None of the seven T2 plants derived from the T1 mutant plant 4A-1 generated the Cas9-specific amplicon (Fig. 3c). Two (4A-1-6 and 4A-1-7) out of the seven carried a 1-bp insertion (+A) and displayed significantly enhanced resistance to PXO99 (Fig. 5), which has also been observed in their parent (4A-1) (Fig. 4). The T3 plant (4A-1-7-1) not generating the Cas9-specific amplicon carried the same Os8N3 modification observed in the T2 mutant plant 4A-1-7 (Fig. 3d and Additional file 4: Figure S4 and Additional file 5: Figure S5). These results indicate that T-DNA-free mutant plants carrying the desired gene modifications can be acquired through genetic segregation in T1, T2, and T3 generations.
The CRISPR/Cas9 system has been widely used to provide new avenues in crop improvements in rice, tomato, wheat, and maize (Xu et al. 2015; Feng et al. 2013; Wang et al. 2016; Ito et al. 2015; Wang et al. 2014; Zhou et al. 2014). In this study, OsU6a::pHAtC, which replaced the Arabidopsis U6 promoter in the pHAtC vector (Kim et al. 2016) with the OsU6a promoter of Kitaake, was constructed for rice CRISPR/Cas9-mediated target mutagenesis. Using the OsU6a::pHAtC, targeted mutagenesis in the recessive resistance gene, Os8N3, was generated.
One xa13 mutant line 4A (T0) from four independent transgenic OsU6a xa13m/Kit plants carrying OsU6a::xa13m-sgRNA/pHAtC was obtained. However, new targeted sequence changes were continuously detected in the transgenic OsU6a xa13m/Kit plants in subsequent generations. For example, two additional independent mutant lines (progenies of 3A and 1A-5) were identified in the T1 and T2 generations, respectively. Except for line 2A, which was lost in T1, all available lines in T2 were successfully mutated at the target sequence. Because the CRISPR/Cas9 system has been shown to be active in heterozygous and chimeric plants (Xu et al. 2015; Zhou et al. 2014), it is possible for the WT allele to be continuously modified in subsequent generations. Therefore, non-mutated transgenic plants, in which the OsU6a::xa13m-sgRNA/pHAtC construct remained active, continually cleaved the target site for generations, resulting in new mutations. Multiple mutations were also detected at the target site in the T2 mutant plant 3A-6-1. Because 3A-6 was heterozygous, the presence of a chimeric mutation may result from delayed cleavage in the primary embryogenic cell of 3A-6-1. This chimeric mutation by the CRISPR/Cas9 system is likely a common phenomenon and has been reported in many plant species including rice (Xu et al. 2015; Feng et al. 2013; Wang et al. 2016), Arabidopsis (Feng et al. 2014), and tomato (Ito et al. 2015).
Regarding all examined agronomic traits, there was no significant difference between T3 homozygous mutants and Kitaake plants under greenhouse growth conditions. The homozygous mutant plants had a similar height, flag leaf length and width, number of productive panicles, panicle length, and pollen viability to Kitaake plants. It has been previously reported that Os8N3 is expressed at a high level in panicles and anthers during pollen development (Chu et al. 2006; Yang et al. 2006). Consistent with these observations, although detailed molecular mechanisms have not been elucidated, Os8N3-silenced rice plants displayed reduced fertility, and most pollen grains were defective (Chu et al. 2006; Yang et al. 2006). Therefore, Os8N3, conferring disease resistance by expressional loss-of-function in rice, has been considered an essential constituent for pollen development. However, in this study, homozygous mutants in both Os8N3 alleles were generated, and the mutations were stably transmitted to later generations, T3. The homozygous T3 mutant plants had normal pollen development, and most pollen grains were well preserved, in comparison with ones from Kitaake plants.
Thus far, it has been believed that Os8N3 plays roles in both copper and sugar transport, indicating its complex function in copper/sugar metabolism and signaling (Chen et al. 2010; Chen 2014; Yuan et al. 2010). However, no one dissected the molecular connection between Xoo resistance by copper/sugar metabolism and pollen development. Among the different in vivo functions of xa13/Os8N3, knockout mutation, in particular, displayed enhanced resistance against Xoo without affecting pollen development. It is not yet understood why OsU6a xa13m/Kit mutant lines did not display the sterile phenotype previously observed in Os8N3-knockdown rice plants (Chu et al. 2006; Yang et al. 2006). Because frameshift mutations of Os8N3 in OsU6a xa13m/Kit lines are located at the very beginning of the Os8N3 polypeptide, it is very unlikely that the mutated polypeptide is functional. Lack of a functional Os8N3 protein in the mutant lines was also supported by a robust resistant phenotype of the homozygous mutant lines, but not heterozygous or Kitaake plants. Therefore, it is possible that there is a novel gene genetically compensating essential pollen development directly or indirectly in homozygous OsU6a xa13m/Kit mutant lines. Genetic compensation was recently proposed to explain increasing numbers of studies revealing phenotypic differences between knockouts and knockdowns in plants (Gao et al. 2015; Braun et al. 2008; Chen et al. 2014) and animals (Young et al. 2009; De Souza et al. 2006; Daude et al. 2012; McJunkin et al. 2011; Law and Sargent 2014; Evers et al. 2016; Karakas et al. 2007; Morgens et al. 2016; Kok et al. 2015; Rossi et al. 2015). For example, similar to Os8N3, there have been studies on Arabidopsis auxin-binding protein 1 (ABP1) that revealed phenotypic differences between knockouts and knockdowns (Gao et al. 2015; Braun et al. 2008; Chen et al. 2014). Inducible abp1 knockdown lines showed defects in shoot and root growth, cell remodeling, or clathrin-mediated endocytosis of PIN auxin efflux carriers (Braun et al. 2008; Paque et al. 2014; Robert et al. 2010). However, abp1 knockout mutants generated by CRISPR/Cas9 are indistinguishable from wild type plants at every developmental stage analyzed (Gao et al. 2015). Although one possible explanation for the difference is off-target effects of ABP1 antisense RNA, it is not yet understood how independent abp1 knockdown lines, which generate fundamentally different approaches for functional down-regulation of the ABP1 gene, display indistinguishable morphological defect phenotypes (Michalko et al. 2016). Recently, genetic compensation was studied in depth on zebrafish (Rossi et al. 2015). While knockdown of zebrafish EGF-like domain 7 (egfl7), an endothelial extracellular matrix gene, leads to severe vascular defects, most egfl7 mutants display no obvious defects (Rossi et al. 2015). Elastin microfibril interfacer (Emilin) genes were proposed as compensating genes in the edgl7 knockout mutants (Rossi et al. 2015). Supporting this hypothesis, Os8N3 mutants showed increased expressions of several SWEET genes such as OsSWEET3a, OsSWEET6b, OsSWEET13, and OsSWEET15 (Ma et al. 2017; Yang et al. 2018) and double mutants of Os8N3 and OsSWEET15 displayed much more wrinkled grain morphology, compared with single Os8N3 mutant (Yang et al. 2018). These reports suggest that some of SWEET genes are able to at least partially compensate for the lack of Os8N3. Currently, we are trying to identify candidate genes that compensate for xa13/Os8N3 in the pollen development pathway without affecting Xoo resistance in homozygous mutant lines.
In summary, the CRISPR/Cas9 system was highly efficient in generating Os8N3 gene editing in rice. Mutant lines harboring the desired modification in Os8N3 but without the T-DNA of the OsU6a::xa13m-sgRNA/pHAtC were obtained. T-DNA-free homozygous mutant lines displayed significantly enhanced resistance to Xoo and normal pollen development. This study provides a successful example of improving bacterial blast resistance using CRISPR/Cas9 technology.
Materials and methods
Plant and pathogen materials
Rice cultivar Kitaake (Oryza sativa L. ssp. Japonica) was generously provided by Prof. Pamela Ronald (University of California Davis, USA). Rice plants in this study were maintained in the greenhouse facility at Sejong University in Korea. Xoo strain PXO99 was used in this study. PXO99 was cultured in peptone sucrose agar media (PSA: peptone 10.0 g/L, sucrose 1.0 g/L, L-glutamic acid 1.0 g/L, and agar 16.0 g/L) containing 15.0 mg/L cephalexin at 28 °C for two days (Bai et al. 2000).
The Gateway™ destination vector, pHAtC binary vector (Kim et al. 2016), was used to construct OsU6a::pHAtC carrying the OsU6a promoter to express sgRNA. A 472-bp DNA fragment containing the OsU6a promoter (Ma et al. 2015c) was amplified from the genomic DNA of Kitaake using primers, EcoRI_OsU6a_F (5′-GGAATTCTTTTTTCCTGTAGTTTTCCCAC-3′) and XhoI_OsU6a_R (5′-GCTCGAGACACCTGCCTCCAATCCGGCAGCCAAGCCAGCACCC-3′). The PCR product was cloned into the pGEM®-T Easy Vector according to the manufacturer’s instructions (Promega, USA), and the insert was confirmed by Sanger sequencing. The OsU6a promoter was cut out from the pGEM®-T Easy Vector using EcoRI + XhoI and cloned into the pHAtC, generating an OsU6a::pHAtC vector.
Cloning of sgRNA expression vector
The OsU6a::xa13m-sgRNA/pHAtC vector expressing sgRNA for xa13/Os8N3 (xa13m-sgRNA) was constructed according to the method previously described (Kim et al. 2016). Briefly, the target sequence (xa13m) for Os8N3 editing of Kitaake was designed by the CRISPR-RGEN Tools website (http://rgenome.ibs.re.kr) (Park et al. 2015). The sgRNA templates (xa13m) for Os8N3 were annealed using two primers, 5′-GATTGCTTGTCCATGGCTAACCCGG-3′ and 5′- AAACCCGGGTTAGCCATGGACAAGC-3′, and cloned into AarI-digested OsU6a::pHAtC. Construction of the sgRNA expression vector, OsU6a::xa13m-sgRNA/pHAtC, and its flanking sequences were confirmed by Sanger sequencing.
Rice transformations were carried out as previously described (Chern et al. 2005). Agrobacterium tumefaciens strain LBA4404 was used to infect callus tissue induced from Kitaake seeds. Transformants carrying OsU6a::xa13m-sgRNA/pHAtC constructs were selected using hygromycin. Transgenic Kitaake plants overexpressing xa13m-sgRNA (OsU6a xa13m/Kit) were confirmed by PCR using Cas9-specific primers, Cas9_RT_F (5′-CGAGCTGACCAAGGTGAAGT-3′) and Cas9_RT_R (5′-CGTTGATAAGCTTGCGGCTC-3′).
For reverse transcription polymerase chain reaction (RT-PCR) analysis of Cas9 and sgxa13 transgenes, total RNA was extracted from fully expanded leaves of OsU6a xa13m/Kit plants using TRIzol reagent (Invitrogen, USA). First-strand cDNA was synthesized using quantified RNA (5 μg of total RNA). Expression of Cas9 was confirmed by RT-PCR using Cas9_RT_F and Cas9_RT_R. Meanwhile, the rEFla cDNA fragment was amplified as a control using specific primers, rEF1a1048F (5′-ACTGCCACACCTCCCACATTG-3′) and rEF1a1552R (5′-CAACAGTCGAAGGGCAATAATAAGTC-3′).
Identification of mutant transgenic plants
Rice genomic DNA was extracted from Kitaake leaves and transgenic OsU6a xa13m/Kit plants. All transgenic hygromycin-resistant T0 plants were analyzed by PCR using the Cas9-specific primers, Cas9_RT_F and Cas9_RT_R. Subsequently, the DNA fragment across the xa13 target site was amplified from the genomic DNAs of all PCR-positive plants using xa13-specific primers, xa13_cas9 60-79_F (5′-TCTGGCTAGTTTCTAGCTGG-3′) and xa13_cas9 nuclease_R (5′-TGCATGAGCTGAAGCTAGGG-3′). The PCR amplicons were then directly sequenced using primer xa13_cas9 60-79_F. The sequencing chromatograms with superimposed peaks of bi-allelic and heterozygous mutations were decoded using the Degenerate Sequence Decoding method (http://skl.scau.edu.cn/dsdecode/) (Liu et al. 2015; Ma et al. 2015a).
Xoo inoculation and determination of bacterial populations
For Xoo inoculation, Kitaake, XA21, and transgenic OsU6a xa13m/Kit plants were grown in a greenhouse normally until they reached the eight-week stage, unless otherwise stated. PXO99 was used to inoculate rice plants using the scissors dip method (Song et al. 1995; Chern et al. 2005). For lesion length measurements, at least three inoculated leaves were measured to calculate the average and standard deviation 12 days after inoculation (DAI). Representative leaves were photographed 12 DAI. For Xoo colony counts from inoculated leaves 0 and 12 DAI, 20 cm of leaf tissue from the top, including lesions and tissue showing no lesions, was ground up and resuspended in 10 ml water to harvest bacteria. The extract was diluted accordingly and plated out on PSA plates containing 15.0 mg/L cephalexin. Plates were incubated at 28 °C for two days, and then colony forming units (CFU) were counted. Statistical analysis was performed using Tukey’s HSD test.
Pollen viability tests
Pollen viability was evaluated as previously described (Chhun et al. 2007). Before flowering, six anthers from Kitaake and transgenic OsU6a xa13m/Kit plants were removed and crushed into a fine powder. Pollen grains were stained with 10 μl I2-KI solution (1% I2, 3% KI) and 1 μl of stained pollen grains was harvested to observe fertile and infertile pollen under a light microscope. Dark-stained pollen grains were considered viable and the percentage of pollen viability was calculated relative to the total pollen counted in five microscopic images. Seed viability represents the percentage of spikelets that set seed per total number. Statistical analysis was performed using Tukey’s HSD test.
We thank Hyesu Jo for technical assistance in generating transgenic plants.
YAK, HM and CJP conceived and designed the experiments. YAK and HM performed the experiments and analyzed the data. YAK, HM, and CJP wrote the manuscript. All authors read and approved the final manuscript.
This work was supported by the Basic Research Program of National Research Foundation of Korea (NRF-2017R1D1A1B03032215), funded by the Ministry of Science and ICT.
Ethics approval and consent to participate
Consent for publication
The manuscript has been approved by all authors.
The authors declare that they have no competing interests.
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