The CRISPR/Cas9 technology, renowned for its ability to induce precise genetic alterations in various crop species, has encountered challenges in its application to grain legume crops such as pigeonpea and groundnut. Despite attempts at gene editing in groundnut, the low rates of transformation and editing have impeded its widespread adoption in producing genetically modified plants. This study seeks to establish an effective and stable CRISPR/Cas9 system in pigeonpea and groundnut through Agrobacterium-mediated transformation, with a focus on targeting the phytoene desaturase (PDS) gene. The PDS gene is pivotal in carotenoid biosynthesis, and its disruption leads to albino phenotypes and dwarfism. Two constructs (one each for pigeonpea and groundnut) were developed for the PDS gene, and transformation was carried out using different explants (leaf petiolar tissue for pigeonpea and cotyledonary nodes for groundnut). By adjusting the composition of the growth media and refining Agrobacterium infection techniques, transformation efficiencies of 15.2% in pigeonpea and 20% in groundnut were achieved. Mutation in PDS resulted in albino phenotype, with editing efficiencies ranging from 4–6%. Sequence analysis uncovered a nucleotide deletion (A) in pigeonpea and an A insertion in groundnut, leading to a premature stop codon and, thereby, an albino phenotype. This research offers a significant foundation for the swift assessment and enhancement of CRISPR/Cas9-based genome editing technologies in legume crops.
Research Article
CRISPR/Cas9-mediated mutagenesis of Phytoene desaturase in pigeonpea and groundnut
https://doi.org/10.21203/rs.3.rs-3914711/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 13 Mar, 2024
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CRISPR/Cas9
Gene editing
Groundnut
Pigeonpea
Phytoene desaturase (PDS)
As the human population is anticipated to rise, there is a need for agricultural production to expand to fulfill growing requirements for food, feed, and fiber, while ensuring minimal adverse effects on the environment (Hickey et al. 2019; Janssens et al. 2020). Agricultural crop production faces additional challenges due to climate change scenarios, including elevated global mean temperatures, shifts in atmospheric CO2 concentration, and a heightened occurrence of extreme weather events with increased frequency and intensity (Kulshreshtha and Wheaton 2018). At the same time, these alterations will impact the proliferation and intensity of both biotic and abiotic stresses, presenting a challenge to food security. Pigeonpea (Cajanus cajan) and groundnut (Arachis hypogaea), prominent dryland legume crops in India and other arid regions, are primarily cultivated by resource-limited farmers in areas prone to drought and depleted soils. These crops serve as a crucial source of high-value protein, playing a vital nutritional role, particularly for vegetarian and economically disadvantaged populations in Asia and Eastern Africa (Ojiewo et al. 2020; Singh et al. 2022). Both crops contribute to environmental sustainability by enhancing soil productivity through nitrogen fixation (Móring et al. 2021). Nevertheless, the yield of these legumes has stagnated for numerous decades due to various biotic and abiotic stresses. To address this challenge, the integration of novel breeding technologies (NBTs), such as gene editing, aims to develop crops that are resilient to evolving environmental conditions.
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system has been modified to serve as a potent gene editing technology, enabling precise alterations at specific sites in a diverse array of plant species. This technology is employed for both validating gene function and enhancing crops by introducing desired phenotype for traits of interest (Ma et al. 2015). This mechanism induces DNA double-strand breaks (DSBs) at predetermined locations in the genome, guided by a specific RNA called guide RNA (gRNA) that binds with the Cas9 endonuclease. The inherent nonhomologous end-joining (NHEJ) pathway typically repairs these DSBs, and this process is error-prone, often leading to the insertion or deletion of a few nucleotides at the designated target site (Jinek et al. 2012). The CRISPR/Cas9 technology has been effectively harnessed for accurate gene editing in a variety of crops, encompassing model plants like Medicago (Meng et al. 2017) and Arabidopsis (Jiang et al. 2014), legumes including, soybean (Lu and Tian 2022), and cowpea (Che et al. 2021; Ji et al. 2019), cereals including rice (Biswas et al. 2023; Zhang et al. 2014), wheat (Wang et al. 2019; Zhang et al. 2021), and maize (Liu et al. 2024).
Numerous initiatives have been undertaken within legume breeding programs to fully leverage the capabilities of gene editing technologies for enhancement. The application of CRISPR/Cas9-mediated knockdown has been successfully illustrated in legumes like soybean, cowpea, chickpea, Medicago truncatula, and Lotus japonicus. However, the challenge lies in the resistance to invitro regeneration, hindering the widespread application of gene editing in legumes such as pigeonpea and groundnut (Ochatt et al. 2018; Pratap et al. 2018). Numerous investigators have endeavored to establish a proficient Agrobacterium-mediated genetic transformation system for pigeonpea (Ghosh et al. 2017; Sharma et al. 2006) and groundnut (Mehta et al. 2013; Prasad et al. 2013). However, these endeavors have been marked by a low level of transformation efficiency and reproducibility. Gene editing studies in groundnut are sparse, and the investigations primarily focus on groundnut protoplast/hairy root transformation systems (Neelakandan et al. 2022; Shu et al. 2020). Notably, there is an absence of reports on the successful application of Agrobacterium-mediated CRISPR/Cas9 gene editing in pigeonpea.
In the present study, we establish a stable and efficient CRISPR/Cas9-mediated mutagenesis system in pigeonpea and groundnut through Agrobacterium mediated transformation. We selected pigeonpea and groundnut phytoene desaturase (PDS) genes to validate gene editing methodology due to the distinctive phenotypes of the loss of function mutant (Qin et al. 2007). The alteration or knockout of the PDS gene impairs carotenoid biosynthesis, leading to albino phenotype and plant growth retardation (Tian 2015), and is widely used as a marker for gene editing in plants (Bánfalvi et al. 2020; Hooghvorst et al. 2019; Lu and Tian 2022; Naim et al. 2018; Ntui et al. 2020). One homolog of PDS gene in pigeonpea and two in groundnut are present. We designed two constructs employing a single guide RNA targeting one and two homologs of PDS, respectively in pigeonpea and groundnut. Subsequently, we conducted Agrobacterium-mediated transformations for pigeonpea and groundnut, respectively. Among the resulting transformants from both constructs, mutations at the intended loci were introduced in T0 plants for both pigeonpea and groundnut PDS genes, leading to conspicuous albino phenotypes. To our knowledge, this is the first successful Agrobacterium-mediated CRISPR/Cas9 gene editing system established in pigeonpea and groundnut, ushering in a new era of gene functionality and laying the groundwork for further advancements in crop improvement.
Identification of PDS gene in pigeonpea and groundnut and designing Guide RNAs
The coding sequence of AtPDS (AT4G14210.1) served as the query in BLAST searches against the genomic databases of pigeonpea (Cajanus cajan ICPL87119 v2 genome) and groundnut (Arachis hypogaea Tifrunner v2 genome) within the Legume Information System (LIS) (https://www.legumeinfo.org/). This search aimed to identify sequences encoding PDS genes. Utilizing the CHOPCHOP sgRNA (single guide) design online tool (https://chopchop.cbu.uib.no/) (Labun et al. 2019), single guide sequences targeting one homolog in pigeonpea (CcPDS) and two homologs of groundnut (AhPDS) PDS gene were designed separately. The selected guide sequences exhibited minimal off-target impact, as assessed by Cas-OFFinder (http://www.rgenome.net/cas-offinder/). Subsequently, the 20-nucleotide guide sequences were used as queries for nucleotide BLAST searches provided by the National Center for Biotechnology Information (NCBI) to identify similar sequences in the pigeonpea and groundnut genomes, respectively. Guides with minimal off-target effects were retained for further analysis.
Construction of CRISPR/Cas9 plasmid
The plasmids pFH52 (Plasmid #128181) (Hahn et al. 2020) and pYPQ150 (Plasmid # 69301) (Lowder et al. 2015) were procured from The Addgene Repository (http://www.addgene.org) (Kamens 2015). The promoter sequence, 2X35S, was amplified from the pFH52 vector utilizing a forward primer (FP) with KpnI and a reverse primer (RP) with BamHI restriction sites. Additionally, the plant codon-optimized Cas9 (pcoCas9) and Nos terminator were amplified from pYPQ150 with FP-BamHI and RP-XbaI restriction sites (Table S1). These two fragments were then ligated to create a pcoCas9 gene driven by 2X35S promoter through sequential cloning and mobilised into pCAMBIA2300 expression vector (Fig. S1).
A 20-nucleotide sgRNA targeting the CcPDS/AhPDS gene was cloned after the MtU6 promoter, along with a SpCas9-specific conserved scaffold sequence spanning 76 nucleotides and a Poly-T terminator sequence. The MtU6 promoter was amplified by PCR from Medicago truncatula genomic DNA using SbfI-MtU6-FP/MtU6-RP primers and confirmed by sequencing. An overlap PCR approach was employed to construct sgRNA driven by MtU6 promoter utilizing four primers (Table S1). The SbfI and HindIII restriction sites were subsequently employed to integrate the sgRNA into SbfI- and HindIII- digested pCAMBIA2300-2X35S-pcoCas9-NosT, generating the CRISPR/Cas9 binary vector for plant transformation (Fig. 1 and S1).
Genetic transformation of Pigeonpea and Groundnut
The gene editing constructs were introduced into Agrobacterium tumefaciens strain C58 through electroporation. The pCAMBIA2300 vector carries an aminoglycoside phosphotransferase (Apt) gene, providing resistance to kanamycin in bacteria, and the neomycin phosphotransferase (NptII) gene for plant kanamycin selection. A. tumefaciens colonies grown on yeast extract peptone (YEP) media (Sigma-Aldrich Chemicals Pvt. Ltd, Bangalore, India), containing 50 mg/L kanamycin (Sigma-Aldrich Chemicals Pvt. Ltd, Bangalore, India) and 25 mg/L rifampicin (Sigma-Aldrich Chemicals Pvt. Ltd, Bangalore, India), were confirmed via polymerase chain reaction (PCR) using vector-specific primers (Table S1). A single PCR positive colony was inoculated into 5 mL YEP starter broth having 50 mg/L kanamycin and 25 mg/L rifampicin and grown overnight at 28°C with shaking at 200 rpm. The overnight grown starter culture was used to inoculate a 25 mL fresh YEP containing same antibiotic concentrations and allowed to grow until the OD 600 reached 0.6–0.8. Before transformation, the bacterial pellet was collected by centrifugation at 5000 Xg for 10 min at room temperature. The cells were re-suspended in 25 mL of co-cultivation infection media (for pigeonpea) or half MS medium (liquid, for groundnut) and stored at 4°C for 2 hours.
Pigeonpea transformation: Pigeonpea transformation was done using a previous standardized protocol (Dayal et al. 2003) with slight modifications. To enhance transformation efficiency, we supplemented the Agrobacterium culture with 100 µm acetosyringone along with 0.1% silwet L-77. We adjusted the shoot induction and elongation media by introducing MES (0.59 g/L) to ensure pH stability. Additionally, we incorporated silver nitrate (2 mg/L) into the shoot elongation and root induction media to counteract senescence caused by ethylene. Transformed plants underwent screening using varying concentrations of kanamycin selection: 100 mg/L in shoot development and 150 mg/L in shoot elongation media. Elongated shoots (> 3 cm) were then transferred to MS basal medium to discontinue antibiotic selection and strengthen the shoots. Subsequently, they were sub-cultured onto root induction medium composed of MS medium supplemented with 11.54 µM indole acetic acid (IAA). Rooted plants were transplanted into pots containing a mixture of 2 parts cocopeat and 1 part perlite. After a week of acclimatization, during which they were initially covered with a plastic bag and gradually exposed to the open environment, the plants were transferred to a glasshouse (Fig. S2).
Groundnut transformation
Previously reported transformation procedure was used with slight modifications (Sharma and Bhatnagar-Mathur 2006). The previous transformation protocol demonstrated limited shoot induction in the elite cultivar ICGV 15083. To overcome this limitation, we introduced 5 µM TDZ (equivalent to 1.2 mg/L) to facilitate shoot formation, replacing the previously utilized BAP. Additionally, modifications were made to the shoot elongation medium, where 2µM GA3 was employed instead of BAP, and to the root induction medium, which now includes 2.5 µM IBA along with 5 µM NAA, aiming to enhance regeneration and transformation efficiency. Moreover, we incorporated the inclusion of 100 µM acetosyringone in both the co-cultivation medium and the Agrobacterium culture. Furthermore, we optimized the immersion of explants in Agrobacterium culture for 8–10 minutes before transfer to the co-cultivation medium to activate virulence genes, thereby augmenting the transformation efficiency. Finally, well-rooted plants were acclimatized and transferred to the glasshouse for further cultivation (Fig. S3).
Molecular characterization of edited plants
To extract total genomic DNA, leaf samples from pigeonpea and groundnut putative transformants were collected and homogenized using liquid nitrogen. Homogenized tissue was resuspended in DNA extraction buffer made of 2% hexadecyltrimethylammonium bromide (CTAB), 100 mM Tris pH 8.0, 20 mM ethylenediaminetetraacetic acid (EDTA) pH 8.0, 1.4 M sodium chloride (NaCl), 2% polyvinylpyrrolidone (PVP)-40 (Sigma-Aldrich Chemicals Pvt. Ltd, Bangalore, India), followed by centrifugation at 5000 rpm for 10 min to settle the debris. The supernatant was incubated at 65°C for 1h before being mixed with 1 volume of chloroform: isoamyl alcohol (24:1) (Sigma-Aldrich Chemicals Pvt. Ltd, Bangalore, India), then centrifuged at 13000 rpm for 10 min to separate the aqueous and organic phases. The aqueous phase was mixed with 1 volume of isopropanol (Sigma-Aldrich Chemicals Pvt. Ltd, Bangalore, India) for DNA precipitation. Following centrifugation at 13000 rpm for 10 min at 4°C, the DNA pellet was washed with 70% ethanol. The final DNA pellet was dried in a 60°C oven for 10 min and resuspended in 1XTE buffer. DNA samples were quantified by fluorometry using Qubit ™ dsDNA BR Assay Kit (Life Technologies, Carlsbad, California, USA) on Qubit™ 3 Fluorometer (Invitrogen, Life Technologies, Carlsbad, California, USA) as per the manufacturer’s instructions and assessed for purity by gel electrophoresis.
To verify transformation, PCR was carried out using Cas9 and NptII-specific primers (Table S1) and Emerald Amp® GT PCR 2X master mix (Takara Bio Inc., San Jose, CA, USA) as per the manufacturer’s instructions. To detect putative edits, target gene fragments were amplified by PCR using gene specific primers (Table S1) by PrimeSTAR GXL DNA polymerase (Takara Bio Inc., San Jose, CA, USA) and were sequenced using Sanger’s dideoxy method.. Sequences were further analyzed using Geneious R11 (http://www.geneious.com) (Kearse et al. 2012) and the ICE v2 CRISPR analysis tool (ICE v2) from Synthego (Conant et al. 2022).
Identification of PDS gene in Pigeonpea and Groundnut
The AtPDS3 protein sequence was used to identify a homologous gene in pigeonpea on chromosome 1 (cajca.ICPL87119.gnm2.ann1.Cc_00066.1; CcPDS01) and two homologous genes in groundnut on chromosome 14 (arahy.Tifrunner.gnm2.ann1.TUF7J0.1; AhPDS14) and 04 (arahy.Tifrunner.gnm2.ann1.M5MKEZ.1; AhPDS04). The protein sequences of AtPDS exhibit an 80% identity with CcPDS01, AhPDS14, and AhPDS04, indicating a conserved function in both pigeonpea and groundnut. However, AhPDS14 and AhPDS04 display a high similarity of 98% to each other. The genomic sequence of CcPDS01 spans 6978 bp, with 1713 bp encoding transcript for 570 amino acids. Similarly, the genomic sequences of groundnut PDS genes - AhPDS14 and AhPDS04 - are 6386 bp and 6159 bp in size, respectively, with transcripts coding for 583 and 580 amino acids. Both the pigeonpea and groundnut PDS genes consist of 13 exons and 12 introns.
Selection of sgRNA targets and vector construction for the CRISPR/Cas9 system
Guide RNAs (gRNAs) targeting the disruption of the PDS gene were designed based on the most conserved regions within the pigeonpea and groundnut genomes, utilizing the CHOPCHOP sgRNA design online tool. In pigeonpea, a single gRNA (5′-GCGGCCACAGAAACCCTTGA-3′) was designed to target exon 1, while in groundnut, a corresponding single gRNA (5′-TGCCTTAAACCGATTTCTTC-3′) was designed to target exon 6 of both homologous genes (Fig. 2, Table 1). Subsequently, these sequences were employed as queries for a BLAST search on NCBI to verify their specificity. All sgRNA target sequences exhibit minimal off-target effects.
Table 1. Overview of the gene editing constructs targeting PDS gene (s)
The pCAMBIA 2300 vector was modified as mentioned in the materials and method (Fig. S1). Briefly, a 2X35S promoter driving the expression of a plant codon-optimized Cas9 and a MtU6 promoter directing the expression of sgRNA (Fig. 1) were incorporated into the binary vector pCAMBIA2300, with kanamycin resistance. The sgRNAs were integrated into the T-DNA region of the binary vector pCAMBIA2300, resulting in the creation of the CRISPR/Cas9 constructs pCAMBIA2300-pcoCas9-CcPDS and pCAMBIA2300-pcoCas9-AhPDS (Fig. 1).
Agrobacterium mediated delivery of CRISPR/Cas9 system into pigeonpea and groundnut targeting PDS gene
Pigeonpea cv. ICPL 88039 was transformed using Agrobacterium utilizing leaf petiolar explants. Out of 1000 explants co-cultivated in shoot initiation media for 7–10 days, 675 exhibited regeneration. Following subculturing to shoot development media with kanamycin selection (100 mg/L) after 7–10 days, 450 explants survived. Among these survivors, 209 explants-producing shoots were subcultured to shoot elongation media supplemented with 0.5 mg/L GA3 (gibberellic acid) and a higher concentration of kanamycin selection (150 mg/L), being maintained for 10–15 days. Cas9 and NptII gene-specific primers were employed to test 163 shoots (symptomatic/albino and non-symptomatic), of which 152 (approximately 90%) tested positive for both genes (Fig. 3). Within this group, 56 shoots exhibited varying degrees of albino phenotype (Fig. 4, Table 2). Albino plants did not develop further, and asymptomatic cas9 positives were transferred to soil pots, where they were maintained under controlled glasshouse conditions.
|
Crop |
Total No. of explants |
No. of elongated shoots |
Plants established |
Transformation efficiency (Cas9 and NptII positive) |
Albino Phenotype |
No. of mutated plants and efficiency |
|---|---|---|---|---|---|---|
|
Pigeonpea |
1000 |
209 (20.9%) |
163 (16.3%) |
152 (15.2%) |
56 (5.6%) |
8 (5.26%) |
|
Groundnut |
250 |
80 (32%) |
70 (28%) |
50 (20%) |
25 (10%) |
2 (4%) |
In groundnut, Agrobacterium-mediated transformation was conducted in cv. ICGV 15083 employing cotyledonary node explants. A total of 250 explants were co-cultivated with the CRISPR/Cas9 construct, resulting in 80 explants producing multiple shoots within 30–40 days under shoot initiation medium. The explants generating multiple shoots were isolated and subcultured in a shoot elongation medium with kanamycin selection (125 mg/L) every 10–15 days. In total, 70 shoots were subjected to testing with Cas9 and NptII gene-specific primers. Among these, 50 (approximately 70%) tested positive for both Cas9 and NptII genes (Fig. 3). Within this group, 25 shoots (about 25%) exhibited with varying degree of albino phenotype (Fig. 4, Table 2). Albino shoots did not survive beyond three months post-regeneration. Some of the albino plant with green shoots and asymptomatic positives (Cas9) were transferred to soil pots and maintained under controlled glasshouse conditions.
Molecular analysis of CRISPR/Cas9 induced mutations of PDS genes
To confirm whether the observed albino phenotype resulted from PDS gene modification, we sequenced PCR amplicons of genomic DNA spanning the targeted PDS regions in Cas9-positive plants (Fig. 5A, 6A). The sequencing data revealed the presence of mutations in the targeted gene.
In pigeonpea, we selected 120 plants positive for Cas9 and NptII, along with wild-type plants for sequencing.. The sequencing results revealed an A nucleotide deletion in plants #98, 12, 13, 11, 17, and 26 (Fig. 5). This deletion occurred 65 base pairs upstream of the PAM sequence (Fig. 5B) leading to a premature stop codon resulting in an albino phenotype compared to the wild-type plants (Fig. 5C). Additionally, plant #34 exhibited a heterozygous (T/C) mutation in the third nucleotide upstream of the PAM sequence (Fig. S4). To further analyse this mutation, we cloned and sequenced it. Among the five colonies examined, four displayed a T nucleotide, while the fifth colony still exhibited the heterozygous (T/C) mutation.
We selected twenty plants positive for Cas9 and NptII, including wild-type plants, for sequencing to identify mutations in groundnut (Fig. 6). The sequencing results uncovered a mutation in the PAM sequence of plant #110 (Fig. 6B) and the insertion of a single A nucleotide in plant #125 (Fig. 6B), resulting in albino phenotype compared to the wild-type plants (Fig. 6C) due to premature stop codon. The CRISPR Edits of Synthego (ICE) inference indicated a 39% InDel percentage for the albino groundnut plant #125, with a knockout score of 39 representing the proportion of cells with the mutation (Fig. S5). In the case of plant #110, the amino acid sequence alignment (Fig. S6) with the wild type of control revealed the substitution of a Glutamine (Q) residue with Arginine (R) in the phytoene desaturase domain. As a result, the albino phenotype in this plant may be attributed to the partially functional PDS protein.
The CRISPR/Cas9-based gene editing has been developed as a site-specific, precise, and efficient technology for editing genes in plants. It holds significant potential for gene functional studies and crop improvement, allowing the desired phenotypes for traits such as enhanced nutritional quality and increased resilience to biotic and abiotic stresses (Kumar et al. 2021). To evaluate the efficiency of gene editing in pigeonpea and groundnut, we employed the CRISPR/Cas9-based gene editing system to target the phytoene desaturase (PDS) gene. PDS plays a crucial role in the biosynthesis of carotenoids, and its disruption results in an albino phenotype (Qin, et al. 2007). Thus, the PDS gene has been employed as a marker to establish a gene editing platform in numerous plant species (Bánfalvi, et al. 2020; Hooghvorst, et al. 2019; Lu and Tian 2022; Naim, et al. 2018; Ntui, et al. 2020).
Gene editing methods have been established in model legume plants such as Medicago (Wolabu et al. 2020) and lotus (Wang et al. 2019) along with other legume crops such as cowpea (Che, et al. 2021) and soybean (Bao et al. 2019; Cai et al. 2020; Sun et al. 2015). While gene editing tools have proven effective in various crops, groundnut and pigeonpea have lagged behind due to the intricate nature of their genomes and their inherent resistance to regeneration (Pratap, et al. 2018). The success of efficient gene editing is primarily contingent on the regeneration efficiency of the transformation method. Transformation studies in highly recalcitrant crop species such as pigeonpea and groundnut face significant challenges, including issues related to the generation of chimeras, genotype specificity, prolonged crop duration, and low regeneration efficiency (Karmakar et al. 2019). In our investigation, Agrobacterium-mediated transformation was employed in pigeonpea, utilizing leaf petiolar explants, and in groundnut, using cotyledonary node explants. We observed a regeneration efficiency of 21% in pigeonpea and 32% in groundnut, coupled with a transformation efficiency of 15.2% in pigeonpea and 20% in groundnut. Despite various attempts by different research groups to tackle this challenge, success in establishing efficient transformation systems for legumes remains elusive, especially in pigeonpea (Ghosh, et al. 2017) and groundnut (Mehta, et al. 2013; Prasad, et al. 2013). Our findings, along with prior research, indicate that the enhancement of regeneration and transformation efficiency is contingent on factors such as genotype, the initial explant, culture conditions, the Agrobacterium strain utilized for infection, and the selective agents incorporated into the culture medium.
In groundnut, which is an allotetraploid, genes predominantly exist in two copies (A and B). The existence of gene homeologs presents challenges in CRISPR-mediated knockouts in polyploid crops like groundnut, as a deletion in the A-sub genome may be compensated by a highly similar gene in the B-sub genome (Yuan et al. 2019). Therefore, in this study, we opted for sgRNAs targeting conserved regions of all homeologs of the AhPDS gene to maximize efficacy. Likewise, the disruption of the PDS gene through CRISPR/Cas9 was reported in soybean, utilizing a single guide RNA that targeted both homologs of the PDS gene, resulting in dwarf and albino phenotypes (Lu and Tian 2022).
The recent attempts at CRISPR/Cas9-based gene editing in groundnut are restricted to transient transformation methods, such as hairy root or protoplast-mediated transformations (Biswas et al. 2022; Shu, et al. 2020; Yuan, et al. 2019). Increasing the oleic acid content by targeting fatty acid desaturases, AhFAD2A and AhFAD2B was the first report on CRISPR/Cas9-based gene-editing in groundnut by using protoplast and hairy root transformation methods (Yuan, et al. 2019). However, the attempts at generating stably edited lines were unsuccessful in this study. Further, increasing the nodulation by editing Nod factor receptors (NFRs) using similar constructs with GFP in groundnut using the hairy root transformation system (Shu, et al. 2020). More recently, base editing to target FAD2 genes in groundnut using the hairy root transformation system has increased the oleic acid content (Neelakandan, et al. 2022). Furthermore, extended scaffold plus terminator increases the editing efficiency compared to normal sgRNA in groundnut while targeting FAD2 genes (Neelakandan et al. 2022). A multiplex approach was used in groundnut protoplasts to target an allergen gene, Arah2, using a polycistronic tRNA–gRNA (PTG) system and Cas9 endonuclease (Biswas, et al. 2022). Furthermore, to the best of our knowledge, there has been no prior study reporting gene editing in pigeonpea using CRISPR/Cas9 technology. This lack of reports can be attributed to the requirement for an efficient, stable, and effective gene editing system. The effectiveness of CRISPR/Cas9 relies on the design and selection of guide RNA (gRNA) that guides the Cas9 endonuclease to perform double-stranded DNA cleavage. In our study, the availability of the reference genome sequence of pigeonpea (Varshney et al. 2012) and groundnut (Bertioli et al. 2019; Zhuang et al. 2019) facilitates designing specific and efficient guide RNA. We proceeded to induce double-stranded breaks (DSBs) in the CcPDS and AhPDS genes within the genomes of pigeonpea and groundnut. This was accomplished by utilizing a CRISPR vector containing the pcoCas9 gene and sgRNAs driven by the MtU6 promoter, leading to the generation of albino plants. Our Agrobacterium-mediated transformation yielded albino phenotypes in pigeonpea (5.6%) and groundnut (10%) compared to the wild types. Furthermore, the sequencing results unveiled a deletion of an A nucleotide in the edited pigeonpea plants and insertion of an A nucleotide in the edited groundnut, resulting in the creation of a premature stop codon. This in turn, resulted in the inactivation of the PDS gene, leading to the albino phenotype.
Although the mutation of the PDS gene in both legume crops was successful, sequencing revealed a lower editing efficiency. This could be attributed to variations in intrinsic DNA repair mechanisms among plant species, the tetraploid genetic background of groundnut, the possibility that PCR amplicons may have carried a mixture of edited and unedited heterogenous DNA, or the use of non-endogenous promoters, which might have further diminished the efficiency of the CRISPR/Cas9 system (Poczai et al. 2013; Wolabu, et al. 2020). Various studies have demonstrated that Cas9 typically cleaves target sites at the fourth base upstream of the PAM sequence (Jinek et al. 2012). However, in our study mutation occurred at the 65 bp upstream of PAM in pigeonpea edited plants, and at the third base of the PAM sequence in groundnut edited plant #110. This divergence may be attributed to simultaneous activation of homologous recombination (HR) and non-homologous end joining (NHEJ) pathway for repairing double stranded breaks in the PDS region (Mainkar et al. 2023; Odipio et al. 2017).
Prior research has indicated that the editing efficiency of the CRISPR/Cas9 system is influenced by various factors. These include the expression level of Cas9-gRNA, the sequence of the guide RNA (gRNA), the promoters governing Cas9 and small guide RNA (sgRNA), terminators, the composition of the target sequence (spacer), T-DNA architecture, chromatin state, and the duration of culture incubation (Castel et al. 2019; Gao et al. 2018; Mikami et al. 2016). The employment of codon-optimized Cas9 and endogenous promoters for Cas9 and sgRNA expression has demonstrated an elevated mutation frequency in various crops, such as soybean (Sun, et al. 2015), rice (Wang et al. 2016), and M. truncatula (Wolabu, et al. 2020). We postulate that the use of an optimized construct could potentially result in higher mutation efficiencies.
The current study showcases that the Agrobacterium-mediated CRISPR/Cas9 system yields albino-phenotype mutants in the T0 generation of both pigeonpea and groundnut. Despite the relatively low editing efficiency observed in this study, the incorporation of a GFP tag, the exploration of regeneration-promoting genes, design of gRNA with higher efficiency, and the utilization of alternative promoters for gRNA or Cas9, may further enhance editing efficiency. This study opens avenues for exploring functions for various candidate genes in basic research and harnesses the potential of CRISPR/Cas9 gene-editing technologies for advancing agronomic traits in legume crops. To the best of our knowledge, this marks the initial success of establishing an Agrobacterium-mediated CRISPR/Cas9 gene editing system in pigeonpea and groundnut, bridging the gap from a basic genetic model to the contemporary gene functional era.
AUTHOR CONTRIBUTIONS
Conceptualization: KY and PSR; methodology: KP, HG, PRB, and KY; software: KY and PSR; data analysis: KY, and WT; writing—original draft preparation: KP, HG, and KY; writing—review and editing: KY, WT, and PSR; supervision and funding acquisition: KY. All authors have read and agreed to the published version of the manuscript.
ACKNOWLEDGMENT
This work was carried out with the aid of a grant from the Start-up Research Grant (SRG) (File No. SRG/2021/000422) from the Science and Engineering Research Board (SERB), Govt. of India to KY. The authors express their gratitude to Dr Prakash Gangashetty, the Pigeonpea breeder at ICRISAT, and Dr Janila Pasupuleti, the Groundnut breeder at ICRISAT, for generously supplying the pigeonpea and groundnut seeds required for plant transformation.
Data availability statement The dataset supporting the findings of this article are included within the article.
Conflict of Interest The authors declare no competing interest.
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No competing interests reported.
- Fig.S1.jpg
Fig. S1Schematic strategy for the construction of Cas9/sgRNA (CRISPR/Cas9) vector for genome editing of PDS gene through Agrobacterium-mediated transformation. A. 2X35S promoter amplified from pFH52 vector and cloned in pCAMBIA2300, B. pcoCas9 with an intron and nuclear localization signal amplified from pYPQ150 vector and cloned in pCAMBIA 2300, C. PDS gRNA cloned between MtU6 promoter and gRNA scaffold using overlap PCR D. The resultant MtU6-gRNA-polyT finally cloned in pCAMBIA 2300-Cas9 vector using SbfI and HindIII restriction site.
- Fig.S2.jpg
Fig. S2 Different stages of genetic transformation in pigeonpea through direct organogenesis. A. Sterilized and decoated seeds on MS media B. Germinated seeds C. Co-cultivated Leaf Explants, D. Shoot bud differentiation from petiolar cut end on shoot induction media, F-E. Elongated shoots on shoot elongation media, F. Rooting of elongated shoots on root induction media G. Hardening of rooted plants in jiffy cups, H. Acclimatized T0 plant under glasshouse.
- Fig.S3.jpg
Fig. S3 Different stages of genetic transformation in groundnut through direct organogenesis. A. Co-cultivated cotyledonary explants B-C. Swelling and greening of explants, D. Shoot bud differentiation on shoot initiation media, E-F. Elongated shoots on shoot elongation media, G. Rooting of elongated shoots on root induction media, H. Hardening of rooted plants in jiffy cups, I. Acclimatized T0 plant under glasshouse.
- Fig.S4.jpg
Fig. S4 Detection of mutation in CcPDS in Pigeonpea plants; Chromatogram sequence of wild type (WT) with pigeonpea edited Plant #34. Guide RNA regions are marked in black lines, and PAM sequences are marked in black dotted lines.
- Fig.S5.jpg
Fig. S5 Illustration of the outputs from the ICE (Inference of CRISPR Edits) software for a guide (sgRNA; TGCCTTAAACCGATTTCTTC targeting the groundnut PDS gene. Transformed groundnut samples were sequenced with Sanger sequencing and further analyzed with ICE. A) Representation of ICE outputs generated from sequence files (.ab1) received from Sanger sequencing edited Plant # 125 and WT samples. A 20 nucleotide gRNA sequence and PAM sequences denote the software output. Knockout score and InDel percentage received as output post ICE analysis are also exhibited.
- Fig.S6.jpg
Fig. S6 Amino acid sequence alignment of the WT- control and edited groundnut plant# 110; amino acid change marked with a red box. The phytoene desaturase domain (113-563 amino acids) is marked in a black rectangular box.
- TableS1.docx
