Perspective Chapter: Major Insights into CRISPR-Cas9 in Edible Oilseeds Research

Abstract

Edible oilseeds significantly contribute to human nutrition and health. However, the production and consumption of edible oilseeds are facing several challenges, such as limited land and water resources, stress factors, and the quality of edible oils. Owing to its precision and versatility, the technology of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 has emerged as a vital tool in the field of oilseed research. CRISPR-Cas9 simplifies the process, allowing scientists to tailor oilseed crops more precisely for industrial applications, nutritional purposes, yield and quality, and stress tolerance. In particular, this technology is playing a crucial role in modifying the fatty acid composition in oilseed crops, addressing industry demands, and is eventually promoting sustainable agriculture. Interestingly, the focus on increasing fatty acid composition is significant for meeting the diverse needs of both industries and consumers. Taking into account relevant literature, this chapter overviews CRISPR-Cas9 system, discusses the major insights into recent applications and achievements of CRISPR-Cas9 in edible oilseed research, addresses the major challenges and proposing solutions for CRISPR/Cas9 editing applications in edible oilseed research, and suggests the themes, so far least explored in the current context.

1. Introduction

Edible oil is essential to human nutrition and health and is a renewable source of biofuel and industrial raw materials, such as rapeseeds, soybeans, sunflowers, and mustard. However, the production and consumption of edible oil face several challenges, including limited land and water resources, pollution, climate change, and rising demand [1, 2, 3]. Moreover, the quality of oil produced by these crops is also affected by different aspects, like genotype, environmental factors, and agricultural practices [4]. Therefore, the production of new oilseed varieties that can overcome these problems and meet the different needs of consumers and industries is required.

Traditional breeding methods have been used for decades to improve oil yields and the composition of oilseed crops but are often time-consuming, labor-intensive, and limited by genetic diversity [5]. Genetic engineering, on the other hand, offers more efficient and accurate methods for introducing desirable features into oilseed crops, such as higher oil content, altered fatty acid profiles, improved nutritional value, and improved resistance to biotic and abiotic challenges [6]. However, genetic engineering also faces challenges, such as public acceptance, regulatory approval, and potential off-target effects [7]. Recently, a novel technique, known as Clustered Regularly Interspaced Short Palindromic Repeats or CRISPR-Cas9, has surfaced as a transformative instrument for genome editing, enabling researchers to alter DNA within living things accurately. CRISPRs are specific DNA sequences discovered in bacteria, and CRISPR-associated protein 9, or “Cas9,” is an enzyme that works similarly to a pair of molecular scissors. CRISPR-Cas9 induces specific mutations, deletions, insertions, or replacements in the genome of any organism, including plants, by creating a guide RNA (gRNA) that matches a target DNA sequence [8]. In numerous biotechnological domains, including bioenergy, agriculture, and medicine, CRISPR-Cas9 has been extensively employed. Furthermore, enhancing the amount and quality of edible oil produced by oilseed crops is one of the potent uses for CRISPR-Cas9. Researchers alter the genes associated with oil production, metabolism, and regulation in oilseed plants to generate oils with desired fatty acid compositions [9]. Additionally, by introducing resistance to pests, diseases, and environmental stressors, this precision increases the resilience of oilseed crops and lowers the requirement for chemical inputs. Moreover, CRISPR-Cas9 enhances the nutritional value of the oilseeds, addressing dietary inadequacies globally [10]. Because of the accelerated breeding capabilities of the technology, crops with improved qualities and higher yields are being developed, contributing to more sustainable and efficient agricultural practices.

This chapter aims to overview CRISPR-Cas9 system, discuss the major insights into recent applications and achievements of CRISPR-Cas9 in edible oilseed research, address the major challenges and proposing solutions for CRISPR/Cas9 editing applications in edible oilseed research, and also suggest the themes, so far least explored in the current context.

2. CRISPR-Cas9 system: an overview

The CRISPR-Cas9 genome editing process consists of several important phases, as given in Figure 1. It begins with the precise construction of guide RNAs (gRNAs), the short RNA molecules tailored to target specific DNA sequences selectively. These gRNAs serve as navigation guides for the Cas9 nuclease, enabling it to cleave DNA at the targeted site [11]. Next, Cas9 protein is produced, which moderates double-strand breaks at the targeted genomic loci. The activation of the breaks triggers cellular repair mechanisms, leading to genomic modifications. Also, the Cas9 protein can be generated in vitro and then delivered to target cells, which allows the use of the editing tool in various organisms and cell types [12]. Besides, the Cas9 protein delivery, along with gRNA and donor DNA carrying the required genetic modifications, is carried out by several techniques, such as viral vectors, lipid nanoparticles, electroporation, and biolistics, and each technique is tailored to distinct experimental demands and biological settings [13]. Furthermore, in the process, the donor DNA is simultaneously introduced with a homologous DNA repair template, improving the efficacy of homology-directed repair (HDR), a critical process for precisely incorporating desired genetic alterations and ensuring correct alterations at the specified genomic locus [14]. Also, the Cas9 protein induces DNA cleavage, and the cellular repair mechanisms kick in. The most common of these, non-homologous end joining (NHEJ), frequently mediates mutations [15]. Post-editing, the success and accuracy of the changes are evaluated using mutation detection tools like PCR, Sanger sequencing, and next-generation sequencing. These tools provide detailed information about the genomic changes generated during the CRISPR-Cas9 process. This evaluation step is crucial for confirming the success of the editing process and ensuring the accuracy of the changes [16].

Figure 1.

Schematic representation of the major overview of CRISPR-Cas9 genome editing in plants.

As mentioned, the editing tool uses several delivery strategies, each with its own benefits and drawbacks and has been widely used in edible oilseed research. For example, Agrobacterium-mediated transformation is a common method in the study of edible oilseeds that utilizes Agrobacterium tumefaciens to transfer the components of CRISPR to plant cells [17]. Additionally, it has successfully manipulated fatty acids in oil palm and oilseed mustard cultivars [9].

The ease, excellent reproducibility, low copy number of incorporated transgenes, and capacity to transfer more extensive DNA fragments favor this approach. However, it has disadvantages, such as Agrobacterium resistance in some oilseed crops [9], inconsistent transformation efficiency [18], and unpredictable transgenic integration into the plant genome [19]. Furthermore, biolistics, also known as particle bombardment, is another versatile approach used in CRISPR-Cas9 genome editing. One significant application is its practical usage in oil palm gene editing, in which researchers introduced transformation vectors into oil palm embryogenic calli, successfully inserting mutations at the desired loci [20]. This approach is helpful as it may deliver numerous genes simultaneously and applies to a wide range of organisms. However, it also has some drawbacks, including the possibility of cellular injury due to the high velocity of the DNA-coated particles, random genomic integration, and the need for specialized equipment [21]. Despite these challenges, both Agrobacterium-mediated transformation and biolistics remain valuable tools in edible oilseed research, with ongoing efforts to improve their efficiency and applicability.

In addition, protoplast transformation has also been used successfully in edible oilseed studies. For instance, it was used for gene editing in rapeseed, a crucial oilseed crop, in a study. A practical methodology for isolating, regenerating, and transfecting rapeseed protoplasts was developed, and successful alteration of the glucosinolate transporter (GTR) genes was achieved, proving its significance in the genetic modification of rapeseed using CRISPR-Cas9 [22]. In another study of successful protoplast regeneration and transfection strategy for field cress, another potential oilseed crop was established [23]. Moreover, the protoplast transformation process has several advantages. It allows the direct delivery of CRISPR components into the plant cell, eliminates the need for bacterial vectors, and simultaneously introduces several genes, an essential aspect for complex trait development. However, because of a lack of possible plant transformation systems, genetic transformation of oilseed crops remains problematic, and protoplast regeneration becomes challenging in many plant species [24].

Additionally, there are several other delivery methods relevant to the CRISPR-Cas9 system. Viral delivery introduces CRISPR components into cells via viruses such as adeno-associated viral vectors. This highly efficient approach can result in insertional mutagenesis, has limited cloning capacity, and may trigger immune reactions [25]. Furthermore, non-viral techniques of CRISPR-Cas9 delivery hold significant potential, including liposomes and lipid nanoparticles. These approaches are safer than viral delivery but require substantial optimization to improve their efficacy [26]. Furthermore, physical delivery methods, such as electroporation, involve direct manipulation of cells and are mostly limited within controlled in vitro and ex vivo settings [27]. Also, delivery of Cas9 through extracellular vesicles provides a secure, transient, economical, and efficacious delivery framework in both in vitro and in vivo environments compared to other techniques [28]. Overall, this comprehensive approach underscores the power of CRISPR-Cas9 and the potential in defining the future of genetic engineering across numerous domains.

3. CRISPR-Cas9 in edible oilseed research: recent applications and achievements

The CRISPR-Cas9 editing tool has been widely used in edible oilseed research, especially in improving the oil content and its composition, enhancing resistance against biotic and abiotic challenges, and improving yield and quality. The major glimpses of the applications and achievements of CRISPR-Cas9 in oilseeds research are briefly discussed hereunder.

3.1 Oil content and composition

The CRISPR-Cas9 technology has dramatically facilitated the genetic manipulation of oilseed crops, particularly rapeseed, soybean, and mustard, by targeting key genes (Table 1). In rapeseed, this technology was used to modify the fatty acid elongase 1 gene (BnFAE1) in three germplasms, with elevated oil and erucic acid content. The CRISPR/Cas9 vectors targeted BnaA08.FAE1 and BnaC03.FAE1 genes, and A. tumefaciens transformed the plants. Later, mutants were identified via kanamycin screening, followed by Cas9 protein detection, and gene sequencing in T0 generation. The homozygous mutants were confirmed in T1 and T2, and the measurement of fatty acid content of the seeds was carried out by gas chromatography (GC). The result demonstrated reduced erucic acid content and high oleic acid content solving a major issue associated with the nutritional quality of rapeseed oil [38]. Interestingly, the mutation of BnC03.FAE1 alone reduced erucic acid content by more than 10. However, the deletion of BnA08.FAE1 or/and BnC03.FAE1 did not affect other agronomic factors, although it slightly diminished the content of oil in the seeds [29]. The study presented promising strategy for future breeding efforts focused on minimizing erucic acid levels. Additionally, CRISPR/Cas9 was used to edit BnFAE1 genes in the rapeseed cultivar CY2, resulting in knockout plants with high oleic acid content ranging from 70% to 80% [30]. For this, CRISPR sgRNA cassettes were incorporated into the pCAMBIA-1300 vector. Transformation used A. tumefaciens GV3101 via hypocotyl segment technique and mutation analysis spanned T0 to T4 generations using PCR and Sanger sequencing. Also, GC-MS assessed seed quality traits. The study demonstrated the potential of the technology to enhance the fatty acid composition of rapeseed cultivars. Furthermore, CRISPR/Cas9 was applied for knocking out four BnLPAT5 and all seven BnLPAT homologous copies of the Lysophosphatidic Acid Acyltransferase (LPAAT) gene, a critical enzyme in the Kennedy pathway [39]. Here, the rapeseed genotype Jia2016 was cultured under controlled conditions, and the CRISPR-Cas9 vectors targeted these BnLPAT2 and BnLPAT5 genes, which were later introduced into A. tumefaciens GV3101. The transformation occurred in 4–6 leaf plantlets, and the genomic DNA from plant tissues enabled mutant identification via PCR and Sanger sequencing, followed by oil extraction, fatty acid, and GC-MS analysis. The result highlighted knockout mutants with varying reductions in oil content, providing new insights into the roles of [31]. Besides, CRISPR/Cas9 also possess a remarkable multiplex genome editing capability, facilitating the efficient modification of multiple gene copies [40]. It also meticulously scrutinizes off-target locations, ensuring a high degree of precision and specificity in genome editing. This is particularly invaluable when dealing with rapeseed, a plant characterized by its intricate allotetraploid nature [41]. For instance, CRISPR/Cas9 technology targeted the fatty acid desaturase 2 (FAD2) genes to develop novel allelic variations that could positively influence fatty acid levels [42]. In the study, two gRNAs targeted two different sites in each gene and were cloned into a CaMV35S vector with the Cas9 and a hygromycin resistance gene. The vector was then transformed into rapeseed plants using Agrobacterium-mediated floral dip method, and the transgenic plants were selected on hygromycin medium. The plants were screened for mutations by PCR and Sanger sequencing, and oil quality was analyzed by gas chromatography. The result reported a significant increase in oleic acid content, reaching 80% compared to 66% in the wild type, while decreasing linoleic and linolenic acids due to the multiple mutations at two loci. Also, BnFAD2.A5 mutations had a more significant impact, and combining several BnFAD2 mutant alleles demonstrated the potential of precision gene editing for improving rapeseed oil quality [32].

Crop	Target gene(s)	Modification	Outcome	Reference
Rapeseed	BnFAE1 (BnA08.FAE1, BnC03.FAE1)	Double mutations, CRISPR/Cas9	Near elimination of erucic acid, increased oleic acid	[29]
Rapeseed	BnFAE1	CRISPR/Cas9-mediated knockout	Substantial rise in oleic acid content (70–80%)	[30]
Rapeseed	BnLPAT2 and BnLPAT5	CRISPR/Cas9-mediated knockout	Varied reductions in oil content	[31]
Rapeseed	BnFAD2	Multiple mutations induced at two sites	Rise in oleic acid content (80%)	[32]
Rapeseed	BnFAD2	Genome editing at A5 and C5 loci	Rise in oleic acid content compared to editing at a single locus	[33]
Rapeseed	BnTT8 and BnTT2	CRISPR/Cas9-mediated disruption	Creation of yellow-seeded mutants, enhanced seed oil	[34, 35]
Rapeseed	BnaLEC1	Overexpression and knockout	Positive regulation of oil content	[36]
Soybean	FAE1, FAD2, ROD1	Protoplast-based CRISPR/Cas9 gene knockout	Substantial increase in oleic acid content, reduced polyunsaturated fatty acids	[37]

Table 1.

Representative studies on the application of CRISPR/Cas9 for enhancing oil content in major edible oilseed crops.

Furthermore, a recent study demonstrated that the editing of BnFAD2 at A5 and C5 loci led to a considerable rise in oleic acid content in seeds than single locus-editing approaches. Here, rapeseed line B57-1, with high seed oil content (51.23%) and low oleic acid proportion (67.25%), underwent Agrobacterium-mediated transformation after CRISPR/Cas9 vectors targeted BnaA05.FAD2 and BnaC05.FAD2 genes. The mutants were identified via hygromycin resistance and PCR confirmation, while T1 mutant lines underwent genotyping and sequencing. Also, whole-genome sequencing analyzed the FAD2 gene sequence, while quantitative reverse transcription (qRT)-PCR assessed gene expression. Finally, gas chromatography evaluated fatty acid composition, and yield and seed oil content were compared to those of the wild type [33]. In parallel, this technology was also employed to engineer yellow-seeded mutants by disrupting the Brassica napus Transparent Testa-8 (BnTT8) and Brassica napus Transparent Testa-2 (BnTT2) genes. The TT8 homologs were cloned and characterized, and sgRNAs targeted BnTT8 and BnTT2 functional domains. Later, Agrobacterium-mediated transformation introduced constructs into the rapeseed lines J9707 and J9712, and Sanger DNA sequencing identified mutants. Moreover, phenotypic analysis evaluated effects on seed coat color, oil, protein content, fatty acid composition, and transcriptomic profiling. Overall, these experiments not only enhanced seed oil but also brought about changes in the fatty acid composition [34, 35]. In addition, researchers explored the role of Leafy Cotyledon1 (LEC1) in the oil content of rapeseed seeds. Overexpression of BnaLEC1, key transcription factors in fatty acid biosynthesis, increased oil content, while its deletion led to a decrease, underscoring the conserved role of LEC1 homologs in positively regulating seed oil content [36]. The overexpression involved cloning the coding sequence into pGreenII 62-SK vectors with 35S promoters, followed by Agrobacterium-mediated transformation that introduced constructs into plants. The mutants were subjected to comparative analysis. Meanwhile, phenotypic, molecular, and metabolic assessments explored BnaLEC1 effects on plant physiology and development. Similarly, CRISPR/Cas9 genome editing was carried out in soybean, where two FAD2 genes, GmFAD2-1A and GmFAD2-2A, were knocked down, which in turn were responsible for the production of polyunsaturated fatty acids (PUFAs) [43]. The soybean gene editing involved sgRNA design and VK005 vector construction followed by Agrobacterium-mediated transformation that introduced CRISPR/Cas9 vectors into soybean cultivar Jack. Later, mutants were identified via PCR and Sanger sequencing, and fatty acid analysis assessed seed contents. The resulting mutants showed notable rise in oleic acid concentration, up to 65.5%, while concurrently reducing the level of linoleic acid to 16%, contributing to the overall quality of this widely used edible oil [44]. In another study, oleic acid content of soybeans was amplified by employing CRISPR/Cas9-mediated gene knockout technique using protoplasts to target fatty acid desaturase2 (FAD2) and reduced oleate desaturase1 (ROD1), important for oilseed nutrition and quality [23, 45]. Here, the seeds were sown/transplanted under controlled conditions, and CRISPR/Cas9 constructs targeted TaFAD2 and TaROD1. The resulting transgenic plants were confirmed via hygromycin resistance, PCR, and sequencing, and later, the phenotypic assays were performed followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging that evaluated mutant traits and lipid profiles. The outcome demonstrated significant increase in oleic acid concentration and a concurrent decrease in polyunsaturated fatty acid (PUFA) and C22:112 [37]. The study further demonstrated the versatility of CRISPR/Cas9 in enhancing the oleic acid composition of soybean oil.

3.2 Stress tolerance

Different studies have been performed to enhance resistance against various diseases in edible oilseed crops using CRISPR/Cas9. One study focused on the development of herbicide-resistant oilseed rape, specifically employing a cytidine-deaminase-mediated base editor (CBE) [46]. The study targeted the acetolactate synthase (ALS) gene at position P197 using Cas9 nickase fused with a cytidine-deaminase enzyme, resulting in a precise C to T conversion and the acquisition of herbicide resistance. Furthermore, BnALS1 and BnALS3 were identified as ideal targets due to their essential functions for amino acid biosynthesis and conserved nature [47]. The application of the CBE system led to the generation of T₀ plants with 3.2% exhibiting editing events in BnALS1, and subsequent generations confirmed the inheritance and significant herbicide resistance of homozygous mutants. The research highlighted the potential of base editing for precision molecular breeding in polyploid crops like oilseed rape [48].

Also, the genetic editing of rapeseed involved employing the CRISPR/Cas9 system to target specific genes associated with desirable traits. For resistance to Verticillium longisporum (Vl43), the susceptibility gene AtCRT1a was edited, introducing double-strand breaks that triggered repair mechanisms, resulting in its knockout. For this, sgRNAs were designed to edit the target gene, with consideration for potential off-target effects. A vector system was constructed to deliver CRISPR/Cas components into the plants, enabling stable transformation and generation of transgenic lines. Later, functional validation confirmed the absence of AtCRT1a expression, and pathogenicity tests showed reduced sensitivity to the fungal pathogen [49]. Furthermore, herbicide tolerance was addressed through precise editing of the glyphosate-binding site in BnaC04EPSPS, resulting in rapeseed plants tolerant to glyphosate. Here, CRISPR-associated RNA endoribonuclease Csy4 cleaved sgRNAs enabling efficient gene replacement and amino acid substitutions in plant cells, achieving frequencies up to 20% with the introduction of donor templates and geminiviral replicons [50]. The sgRNAs were introduced in the plants through A. tumefaceins. Moreover, drought tolerance was also improved by editing BnaRGA genes, associated with DELLA proteins, using CRISPR/Cas9, resulting in mutations (BnaA6.rga-D) that improved resistance to drought [51]. This mutation led to the creation of the gain-of-function mutant, called BnaA6.rga-D, which led to hypersensitivity of stomatal closure to abscisic acid (ABA) treatment enhancing water conservation during drought. The resulting mutant was obtained by screening a rapeseed ethyl methanesulfonate (EMS) library, while the plasmid construction involved amplifying genes from a rapeseed cDNA library and cloning them into vectors for various assays. These genetic modifications offer a strong avenue to generate varieties of rapeseed with improved resilience to biotic and abiotic stresses, contributing to sustainable agriculture.

Additionally, the editing tool also targeted BnWRKY11 and BnWRKY70 genes in rapeseed, known for their involvement in resistance to the pathogenic fungus Sclerotinia sclerotiorum [52, 53]. Two Cas9/sgRNA constructs were designed for precise editing, resulting in 22 BnWRKY11 and eight BnWRKY70 mutants in the T0 generation. The target sequences were selected based on minimal base pairing with sgRNA scaffold or target and the constructs transformed into Escherichia coli, then Agrobacterium for plant transformation followed by mutation analysis via genomic DNA extraction, PCR, and Sanger sequencing. The S. sclerotiorum infection assays measured lesion sizes on detached leaf surfaces. The findings suggested the role of BnWRKY70 as a negative regulator impacting Sclerotinia resistance in rapeseed, showcasing the potential of CRISPR/Cas9 for developing resistant germplasm [54]. Furthermore, CRISPR/Cas9 was employed to induce targeted chromosome cleavage in two NBS-LRR gene families of soybeans, namely, Rpp1L and Rps1, known for their role in effector-triggered immunity [55]. Here, multiple sgRNA cassettes targeted gene clusters and the Cas9 and sgRNA constructs transformed the soy germplasms, A3555 and AG3931. Later, R0 transformants were analyzed for CRISPR/Cas9 activity, and the R1 generation was assessed for copy-number variations, confirming targeted mutations. The copy-number variations and sequencing revealed up to 58.8% of progenies with extensive CRISPR/Cas9-induced chromosome rearrangements, giving rise to new genetic structures with preserved open reading frames [56]. This approach demonstrated the potential to diversify plant innate immunity by creating new resistance gene paralogs, offering a promising strategy for enhancing disease resistance in crops. Another study edited the GmAITR genes, a group of transcription repressors induced by abscisic acid (ABA), in soybeans to enhance salinity tolerance [57]. In the study, six GmAITRs were simultaneously targeted, resulting in gmaitr36 double and gmaitr23456 quintuple mutations, free from Cas9. Various assays were then performed including ABA and salt tolerance, qRT-PCR gene expression, and field production analysis, and the GmAITRs were later characterized. Additionally, phylogenetic analysis was done that used amino acid sequences from soybean and related species. The qRT-PCR analysis revealed elevated GmAITRs expression in response to ABA and exposure to saline conditions. Also, the mutants exhibited increased tolerance to salinity in seedling development assays, seed germination, and importantly, in field experiments, demonstrating the efficiency of CRISPR/Cas9 in improving soybean salinity tolerance and suggesting potential agricultural significance [58].

Furthermore, the editing tool also targeted the glucosinolate transporter (GTR) genes of mustard [59]. The study created cell lines with low seed glucosinolate content (SGC) and high SGC in other plant parts for better defense against biotic threats. Using three gRNAs, four BjuGTR1 and six BjuGTR2 homologs were edited, reducing SGC from 146.09 μmoles/g to 6.21 μmoles/g. The mutated lines exhibited elevated glucosinolate content and altered distribution in foliar parts without compromising plant defense and yield indicators. Furthermore, when evaluated against Sclerotinia sclerotiorum and Spodoptera litura, the mutated lines showed defense responses on par with or superior to the wild type, highlighting the potential of this edited mustard for improved agricultural outcomes through reduced seed glucosinolate content and enhanced resistance [60].

3.3 Yield and quality

The utilization of CRISPR/Cas9 in rapeseed has reported significant enhancement in both the yield and quality by optimizing key plant traits that contribute to oilseed production. These include crucial yield-related traits of rapeseed, such as the seeds per pod, number of pods per plant, plant height, seed weight, shattering resistance, and top branch angle [61].

In an effort to mitigate yield loss due to silique shattering in rapeseed, researchers have targeted genes, such as BnaALC and BnaIND. BnaALC plays a crucial role in inhibiting the segregation layer, while BnaIND regulates the formation of lignified cells and the separation layer. Notably, successful editing of the BnaALC gene resulted in the development of mutants exhibiting enhanced shattering resistance [62]. In the study, CRISPR-Cas9 precisely edited these genes using a binary vector system and A. tumefaciens for plant transformation. The mutants were identified via PCR, Sanger and Illumina sequencing, and the shatter resistance measurements evaluated the impact of mutations on physical traits. Similarly, editing of BnaIND and its comparison with BnaALC genes revealed the higher potential of BnaIND for cracking resistance [35]. Furthermore, two subgenomes were compared, and BnaA03.IND was found to contribute more to this resistance than BnaC03.IND. Here, the rapeseed line J9707 underwent genetic transformation using CRISPR/Cas9 and specific sgRNAs and a hygromycin resistance cassette were employed. The transgenic plants were then confirmed via Agrobacterium-mediated hypocotyl transformation, and the mutants were identified through PCR, PAGE-based genotyping, and Sanger sequencing. Also, gene expression analysis was performed across various tissues, and the shattering resistance was evaluated via random impact test and light microscopy.

Besides, regulatory genes like BnJAG.A08 [63] and BnSHP1/SHP2 homologs [64] were also edited in the rapeseed variety “Zhongshuang 6” (ZS6) using three sgRNAs introduced into the plant hypocotyls using Agrobacterium-mediated transformation. The mutant lines were selected and identified using PCR, PAGE-based screening, and Sanger sequencing. The phenotypic analysis was performed, and RNA expression analysis and staining of pod transverse sections for lignin visualization were done. The mutants obtained from this approach demonstrated significantly improved pod-shattering resistance, providing valuable resources for rapeseed breeding efforts focused on enhancing yield and oil quality.

Moreover, studies have been conducted focusing on the CLAVATA (CLV) signaling pathway to understand the genetic mechanisms that regulate pod development in rapeseed and improve crop yield and quality as this pathway controls traits related to yields in various crops, including rice, tomato, and maize [41]. Specifically, the genes of this pathway, namely, CLAVATA1 (CLV1) and CLAVATA3 (CLV3), have been found to regulate the multilocular trait in Indian mustard and wild kale, respectively [65, 66]. To assess the functional significance of these CLV pathway genes in rapeseed variety J9707, the CRISPR/Cas9 tool was used for knocking out CLV3, and the receptors—CLV1 and CLV2, inducing mutations in the two copies of the BnCLV gene [67]. Here, CRISPR-P-designed sgRNAs were used, which were introduced into the plant using Agrobacterium-mediated transformation, followed by mutant identification via PCR and Sanger sequencing. The phenotypic characterization was conducted, and potential off-target effects were analyzed using CRISPR-P, Illumina sequencing, and southern blotting to assess the genomic integrity and specificity of the CRISPR/Cas9 editing system. The heritable multilocular silique phenotype was observed as a result of the dual homozygous deletion of BnaA04.CLV3 and BnaC04.CLV3. Also, hybrid phenotypes with both dual and manifold-chambered pods were also reported. Nonetheless, potential for increased yield, with increased weight of seed and leaf numbers than the natural variant, was observed. This targeted approach aimed to elucidate the specific functions of CLV3 and its receptors in rapeseed, providing insights that could contribute to enhancing yield-related traits in this important oilseed crop.

Editing has also been carried out to reduce phytic contents in rapeseeds as reduced phytic acid content in rapeseeds enhances the nutritional quality of crops, contributing to environmental sustainability and maintaining or even improving yield, making it a promising strategy for future rapeseed cultivation and utilization [68]. Given this, a CRISPR/Cas9 editing was employed to target the ITPK gene, responsible for encoding an enzyme catalyzing the second-to-last step of phytate synthesis [69]. In this study, the German spring cultivar Haydn was used for transformation. phylogenetic analysis of ITPK proteins guided CRISPR/Cas9 editing, and the CRISPR-Cas9 cassette transformed rapeseed hypocotyls. The phenotyping analysis was also carried out, and later, mutations were verified. Besides, seed phytic acid and inorganic phosphorus contents were measured. The resulting ITPK mutants showcased a 35% decrease in phytic acid levels without any discernible impact on plant performance [70].

CRISPR/Cas9 technology has also been applied to genes associated with flowering time, floral organ development, and flower coloration in rapeseed to improve yield, as these factors not only directly influence the seed-setting process but also determine the optimal sowing dates for subsequent rotation crops [71]. To achieve this, the system was directed toward the TERMINAL FLOWER 1 (TFL1) gene, which belongs to the phosphatidylethanolamine-binding protein (PEBP) family and recognized for its role in regulating flowering time [72]. The TFL1 genes were identified in the Brassica species (B. napus, B. rapa, and B. oleracea) using BLASTP algorithms examining conserved motifs and gene structures. The CRISPR/Cas9 editing using gRNAs inserted into pKSE401 vector targeted the PEBP domain, followed by transformation of the rapeseed hypocotyls. Later, mutations were verified, the T1 and T2 mutant plants were analyzed for flowering time and architecture traits, and seed phytic acid and inorganic phosphorus contents were measured. Additionally, RNA extraction and RT-PCR examined gene expression levels in different plant tissues. The results showed that the mutation in BnaC03.TFL1 resulted in earlier flowering, offering potential benefits for yield improvement. Other TFL1 copies, such as BnaA10.TFL1, BnaC03.TFL1, and BnaC09.TFL1, were observed to influence plant architecture, providing insights into multifaceted strategies for yield enhancement [73].

Furthermore, the study addressed the challenge of abnormal floral organs hindering seed yield, particularly under abiotic stress conditions. Targeting the APETALA2 (AP2) gene, classified as a class A gene within the ABCE model, that plays a crucial role in regulating floral organ development, the AP2 homologous genes of rapeseed (all four copies) were edited using CRISPR/Cas9 [74] resulting in the quadruple mutants. The target sequences within the AP2 gene were incorporated into recombinant plasmids and transformed into the rapeseed plants, which were later identified by PCR. The resulting quadruple mutants exhibited altered floral structures, including missing petals, carpels, reduced stamens, and sepals [75]. This genetic manipulation contributed to understanding the molecular basis of abnormal floral organ development, potentially providing avenues for stress-resistant rapeseed varieties with improved seed yield. Besides, to enhance rapeseed yield, CRISPR/Cas9 gene editing targeted the BnaMAX1 genes, which are associated with plant height and branch number—key factors in plant architecture [76]. This resulted in a variety of modifications in the edited plants, including homozygous, heterozygous, bi-allelic, and chimeric mutations, which were stably inherited by subsequent generations. The complete knockout of all four BnaMAX1 alleles led to the development of semi-dwarf plants with increased branching, thereby boosting the yield potential of rapeseed and providing valuable genetic resources for future breeding [77]. Here, the rapeseed variety “862” was used for transformation, and MutMap analysis identified candidate mutation sites in plants with increased branching. The complementary and CRISPR/Cas9 plasmids were transformed into the plants, and phenotypic observations evaluated genetic impact on plant height, branch number, seed number, and yield. Additionally, studies have also focused on the downregulation of BREVIPEDICELLUS (BP) homologs, specifically BnaA03.BP and BnaC03.BP, through CRISPR/Cas9 editing, resulting in more compact plants with altered branch angles [78]. Here, two sgRNAs were designed to target specific exons of BnaA03.BP and BnaC03.BP and were incorporated into the CRISPR/Cas9 system for transformation into rapeseed plants. The mutated alleles were identified by sequencing the targeted sites in the transgenic plants, and the phenotypic effects were assessed at various growth stages, including bolting and reproductive stages, by comparing them with wild-type plants.

Also, increased seed weight per rapeseed plant was also achieved by knocking out all four copies of the BnaEOD3 gene (role in seed development) [79] through CRISPR methodology using target sgRNAs. Later, the rapeseed line J9707 was transformed using Agrobacterium-mediated transformation, and the mutants were identified by PCR screening, followed by the High-Throughput Tracking of Mutations (Hi-TOM) method. This was followed by phenotypic characterization and cytological analysis and gene expression profiling. The resulting quadruple knockout effectively eliminated the redundant functions of these gene copies in seed development and the seed weight per plant in the mutants increased by an average of 13.9% compared to the wild-type rapeseed plants [80].

4. CRISPR/Cas9 editing applications in edible oilseed research: major challenges and key solutions

The application of CRISPR/Cas9 editing in edible oilseed research faces several challenges. A primary issue is the difficulty in manipulating oilseed crops like castor, sesame, and jatropha in vitro, limiting the use of this advanced technology [10]. Furthermore, the lack of genetic variability in primary germplasm for traits such as stress tolerance hinders breeding efforts, with the selection process being time-consuming [81]. Inefficient transformation systems, especially for oilseed mustard, impede progress, and the rapid generation of lines in polyploid oil crops, like rapeseed, is challenging [82, 83]. Off-target effects in rapeseed genome editing with CRISPR/Cas also pose a significant challenge, requiring strategies to minimize such effects [84].

To overcome the mentioned challenges, various strategies can be employed. Optimizing tissue culture protocols or developing new transformation methods may address the recalcitrance of oilseed crops to in vitro manipulations [85]. Efficient and reliable transformation systems for oilseed crops, using methods such as Agrobacterium-mediated transformation or biolistics, need development [86]. Research is necessary to overcome limitations related to plant organelles, transgene integration, tissue culture, and recalcitrant elite crops [87]. This includes the development of new CRISPR/Cas9 systems targeting, transgene-free genome editing methods, plant organelles, improved tissue culture efficiency, and strategies for making recalcitrant elite crops more amenable to genome editing [88]. While CRISPR/Cas9 has been revolutionary, exploring alternative systems like base editors and prime editors may address some challenges. Unlocking genome editing potential for mitochondria and chloroplasts requires discovering RNA sequences for precise editing [11]. Challenges related to plant tissue culture and transgene integration involve identifying viral vectors accommodating large inserts [89]. Advancements in DNA-free genome editing methods are transforming crop improvement, particularly in generating haploid plants for elite crop enhancement. Researchers are addressing challenges of off-target effects and regulatory constraints in rapeseed genome editing through transgene-free techniques, including nanoparticle-based delivery systems. These innovations offer non-GM rapeseed variants, bypassing stringent regulations and contributing to economic value in rapeseed production [90, 91, 92]. However, in order to fully harness the potential of CRISPR/Cas9 for edible oilseed crop improvement, regulatory considerations must ensure the safety of edited crops for consumption and the environment. Addressing these challenges will unlock its full potential in improving edible oilseed crop yield and quality, benefiting the industry and global food security [9].

5. Conclusions and future prospects

The CRISPR/Cas9 system has transformed the landscape of genome editing, providing a swift and effective approach for manipulating genes. This has significant implications for enhancing crop yield, quality, and disease resistance. Over time, the dynamic applications of CRISPR/Cas9 across various plant systems have demonstrated potential in practical studies, mitigating stress responses, and enhancing agronomic traits. However, despite its benefits, there is a continuous need for modifications to improve target effectiveness. Additionally, the regulatory landscape for genetically engineered (GE) plants necessitates precise and multidimensional rules to distinguish between GE and genetically modified (GM) crops. Also, the integration of CRISPR/Cas9 with next-generation sequencing is a critical area of emphasis, as it allows for comprehensive mutational screening. Furthermore, combining CRISPR/Cas9 with seed breeding programs could be a game-changer in achieving global food security. This combination could lead to the development of crops with enhanced features and increased yields. Besides, the ongoing advancements in CRISPR/Cas9 tools play a crucial role in shaping the future of smart crop development. However, it is important to underscore the need for careful optimization and quality checks during the gene editing process. This is to minimize off-target effects and ensure sustainable food production for the growing global population. Overall, the promise of CRISPR/Cas9 is immense, but its application must be approached with caution and precision.