1 Introduction

The global population is forecast to reach 8.2 billion by 2030 and 9.3 billion by 2050 [1, 2]. This anticipated growth underscores the urgent need for a substantial escalation in food production. By 2050, global food demand is expected to rise by 56%, heightening the risk of hunger [3, 4]. Additionally, climate change poses a significant threat to food security, as shifts in temperature and precipitation patterns affect land suitability and crop yields [5]. Higher temperatures can directly influence pest reproduction, survival, spread, and population dynamics, while climate-stressed plants are more prone to disease [6, 7]. Ensuring plant health is crucial for economic stability, given agriculture's vital role in the economy [8, 9]. Plant diseases and pests have been shown to cause substantial economic losses, with direct yield reductions due to pathogens, pests, and weeds accounting for 20% to 40% of global agricultural productivity losses [10, 11]. Specifically, pathogens and pests can reduce yields of major crops like maize, potato, rice, soybean, and wheat by up to 40% [12]. Effective crop protection against plant diseases is essential to meet the increasing demand for both food quality and quantity [13, 14].

One of the aims in modern plant breeding is to produce disease-resistant crops, thereby enhancing yield stability and crop protection [15, 16]. In contrast to traditional breeding methods, plant genome editing offers the possibility of the development of novel plant traits [17]. Plant genome editing allows for precise chromosome restructuring, encompassing small insertions, deletions, substitutions, and complex rearrangements like inversions, duplications, and translocations [18]. Furthermore, plant genome editing allows for faster and more predictable plant breeding that is widely applicable for various species and thus plays a critical role in advancing food security and supporting the bioeconomy [19]. As one of the most significant advancements in modern genome editing, the Clustered Regularly Interspaced Short Palindromic Repeats and its associated proteins Cas (CRISPR/Cas9) technique heralds a new era in plant breeding [20, 21]. CRISPR/Cas9 demonstrates superior applicability compared to other endonuclease-based gene editing techniques like zinc-finger nuclease (ZFN) or transcription activator-like effector nuclease (TALEN) [22]. By inducing DNA double-strand breaks (DSBs), CRISPR/Cas9 with its site-specific nuclease activity triggers DNA repair mechanisms within cells, offering a wide range of genetic alterations depending on repair pathways and the availability of repair templates [23]. Another avenue through which crop protection can be achieved is by genetically engineering the culprits of crop damages, such as insects. More specifically, modern genetic engineering approaches on insects could disturb their biological functions and thus save the otherwise-damaged crops. As will be further elaborated in this article, this could be achieved by preventing mating attempts and introducing bodily defects in insects, among others [24, 25].

The advent of the CRISPR/Cas9 technology opened new possibilities for efficient and effective plant genome editing. Since the first published application of the CRISPR/Cas9 in plant genome editing in 2013 [26,27,28], a large body of scientific publications have shown its versatility for multiple purposes, including conferring resistance against pathogens, insects, and weeds. In this review, we explore the utilization of the CRISPR/Cas9 system as a genome editing strategy to safeguard crops against pathogens, insects, and weeds, addressing the challenges of food security. Additionally, we investigate alternative CRISPR/Cas9-related technologies employed to develop future crop protection measures.

2 The CRISPR/Cas9 system in modern genome editing

CRISPR/Cas9 stands as a pivotal technology in contemporary genome editing techniques and has drastically revolutionized the field of genetic manipulation [19, 29]. Approximately 84% of archaea and 45% bacteria are equipped with CRISPR/Cas systems, showcasing their natural prevalence [30]. The CRISPR array consists of small repetitive sequences that are interspersed with unique spacers which originate from viral nucleic acid fragments, serving as molecular memory to combat future infections [31]. Paired with Cas9, an endonuclease from Streptococcus pyrogenes, the CRISPR system acts as a precise genetic engineering tool [32].

First discovered in the DNA of Eschericia coli by Ishino et al., the function of the CRISPR system was not immediately clear [33, 34]. It was only later that its function became clearer, with studies such as that of Mojica et al. that hypothesised its function in the bacterial immune system [35]. Twenty years from its discovery, the first experimental demonstration of the immunological role of CRISPR was conducted by Barrangou et al. through their work with Streptococcus thermophilus [29]. Along with the development of CRISPR, the discovery and development of CRISPR-associated (Cas) enzymes was also taking place. The most studied Cas enzymes are those who possess the capability to cut nucleic acid, of which Cas9 is a prominent exemplar. The first association between Cas9 and CRISPR was made by Bolotin et al. in a study on S. thermophilus [36]. Copious amount of research has been then concerted towards studying and exploiting CRISPR and Cas9, which eventually culminated in the works that led Jennifer Doudna and Emmanuelle Charpentier to win the 2020’s Nobel Prize in Chemistry. Within the CRISPR/Cas9 system, Cas9 employs a specific guide RNA (gRNA) duplex consisting of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA), as illustrated by Fig. 1A. crRNA is 18–20 nt in length and plays a crucial role in targeting a certain DNA sequence by pairing with it. tracrRNA, on the other hand, is a longer piece of RNA, 50–150 nt in size and is an integral part of the Cas9 DNA cutting mechanism [37, 38]. For modern genetic engineering purposes, the duplex is combined into a single molecule termed the single guide RNA (sgRNA) [39], as shown by Fig. 1B. More specifically, sgRNA is created by attaching a linker to the 3’ end of crRNA and 5’ end of tracrRNA. sgRNA retains two crucial features: a 20-nucleotide sequence at the 5′ end that specifies the DNA target, and a double-stranded structure at the 3′ end that binds to Cas9 [39].

Fig. 1
figure 1

The basics of the CRISPR/Cas9 technology in genome editing. This technology exploits the naturally occurring genomic properties found in archaea and bacteria to precisely edit the genome of an organism. In the native system A the Cas9 enzyme is guided by a guide RNA (gRNA) duplex consisting of trans-activating CRISPR RNA (tracrRNA) and CRISPR RNA (crRNA). In the modern CRISPR/Cas technology B a synthetic guide RNA used in genome editing combines tracrRNA and crRNA into single guide RNA (sgRNA). In both cases, Cas9 introduces double strand breaks (DSBs), which can be repaired through one of two mechanisms (C): non-homologous end joining (NHEJ) and homology-directed repair (HDR)

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In both the native and synthetic CRISPR/Cas9 systems, a protospacer adjacent motif (PAM) acts as a marker for the target sites. PAMs are a prerequisite for precise nucleic acid cleavage, since they “instruct” Cas9 to cleave a DNA strand at a precise location [40]. The length of a PAM can vary between two to six nucleotides, although prior research has shown that most well-conserved PAMs have three or for nucleotides, such as NGG, NAG, CTT, and TTTV (V is A, C, or G) [41]. The seed region is a short DNA sequence consisting of 10–12 base pairs adjacent to the PAM. It has a crucial role in determining Cas9 specificity, and it makes this region more essential than other regions in the SgRNA [39]. The interplay between these components enables Cas9 to introduce double strand breaks (DSBs) in the DNA precisely [42]. DSBs then triggers their reparations, which can be achieved via one of two main mechanisms (Fig. 1C): non-homologous end joining (NHEJ) or homology-directed repair (HDR); both of which can be exploited to introduce genetic alterations [42, 43]. NHEJ typically results in random insertions or deletions, often causing mutations due to frameshifts and effectively knocking out the targeted gene [23]; whereas HDR allows for more precise gene insertions, known as ‘knock-ins’ [44]. In the absence of a homologous template, DSBs trigger the NHEJ mechanism [45], which is particularly efficient when overhangs are generated [46]; otherwise, when a homologous template is present, the HDR mechanism can be triggered. In the HDR system, the Cas9-gRNA complex plays a crucial role in this process by guiding the delivery of gene-carrying fragments flanked by homologous sequences to the DSB site [47].

Both NHEJ and HDR offer versatility and precision in plant genome editing for crop protection. CRISPR/Cas9-mediated gene knockouts have demonstrated efficacy in conferring resistance to diseases and insect pests [48,49,50]. However, harnessing HDR for DNA knock-in remains a challenging endeavour due to the limitations in supplying sufficient repair templates, such as donor DNA [51]. Furthermore, in plants, several delivery methods exist, including Agrobacterium-mediated delivery, bombardment-mediated delivery, and PEG-mediated delivery. Of these methods, Agrobacterium-mediated delivery is the most established one [52]. Several future potential delivery methods include pollen magnetofection-mediated delivery and nanoparticle-mediated delivery, which do not need tissue culture [53]. Despite the challenges, as will be discussed in this article, the genome editing potential of CRISPR/Cas9 enables unprecedented precision and versatility in plant genome editing, which can be materialised into the conferment of beneficial traits into plants, such as disease resistance and pest resistance.

3 CRISPR/Cas9 for crop protection to control pathogens

Plant diseases pose significant challenges to agriculture, impacting various stages from cultivation to post-harvest, with the principal pathogen categories encompassing viruses, bacteria, fungi, nematodes, and parasitic plants. These biotic constraints pose a threat to food security [13]. The extent of yield loss induced by viral infections varies widely, ranging from 0 to 100%, influenced by factors such as the virus source, vector, and environmental conditions [54]. Fungal diseases represent a crucial yield-limiting factor, with potential yield reductions ranging from 15 to 20% in cereals and, in severe cases, escalating up to 60% [55]. In parallel to fungal- and viral-induced losses, damages from bacterial infection can lead to significant quality decreases and yield losses, as exemplified by the documented 20.27% losses of maize yields due to bacterial stalk rot and 50% yield loss in rice yields from bacterial leaf streak disease [56, 57].

The CRISPR/Cas9 technology is able to introduce artificial mutations that alter the sequence of the target gene to resemble a disease-resistant type, provided the nucleotide variations do not adversely affect plant viability or productivity [58]. Not only does this approach have the potential to mitigate yield losses but it also contributes to the assurance of food security by enhancing the productivity and sustainability of agricultural systems. As such, leveraging the CRISPR/Cas9 technique for crop protection represents a pivotal step towards addressing the multifaceted challenges posed by plant diseases in agriculture.

3.1 Harnessing CRISPR/Cas9 for precision virus resistance in crops

The CRISPR/Cas9 technology can also be employed to introduce mutations in alleles associated with virus resistance, such as eukaryotic initiation factor 4E (eIF4E). RNA viruses rely on eIF genes like eIF4E to complete their life cycle [59]. The eIF protein plays a crucial role in initiating protein translation by recognizing and interacting with mRNA cap structures and ribosomes. Mutations in these host factors can impede viral proliferation and host infection [60]. Notably, the eIF4E mutation has been demonstrated to confer virus resistance in various plants, including tomato, Arabidopsis, cucumber, and Chinese cabbage [61,62,63,64].

The CRISPR/Cas9 technology can also be employed for direct targeting of the viral genome, offering a promising approach to generate virus resistance [58]. For instance, in tomato plants, CRISPR/Cas9 has been utilized to target the genome of the tomato yellow leaf curl virus (TYLCV), resulting in interference with viral replication. Notably, plants expressing sgRNA sequences targeting TYLCV's coat protein sequence exhibited enhanced resistance to the virus [65]. The viral coat protein plays a critical role in encapsulating the viral genome, facilitating virus movement, and recognizing vectors [66]. Similarly, tobacco plants employ a similar mechanism to resist infection by the cotton leaf curl mutant virus (CLCuMuV) [67]. By precisely targeting viral genomic sequences essential for infection and replication, CRISPR/Cas9 technology offers a potent strategy to confer durable resistance against viral pathogens in crops. In addition, the CRISPR/Cas9 technology has been utilised to edit the AC2 and AC3 genes of the African cassava mosaic virus (ACMV) genome. The AC2 gene encodes the multifunctional TrAP protein, which plays crucial roles in gene activation, viral pathogenicity, and suppression of gene silencing, while the AC3 gene encodes the REn protein, involved in increased viral replication [68]. The engineered plant possessed an ACMV-resistant trait [69]. Jogam et al. observed the induction of CRISPR/Cas9 mutations of the Tobamovirus multiplication 1 (TOM1) gene successfully confers tobacco resistance to the Tobacco mosaic virus (TMV) [70].

Furthermore, the CRISPR/Cas9 technology has been shown to have conferred resistance to banana streak virus (BSV) in bananas by targeting the endogenous virus sequences (ORF1, ORF2, and ORF3), thereby disrupting proper transcription and translation of functional viral proteins [71]. By exploiting the inactive Cas9 nuclease domain (dCas9), the CRISPR/Cas9 technology offers a promising strategy for combating viral infections in plants. In the case of cotton leaf curl virus (CLCuV) in tobacco, researchers have demonstrated the effectiveness of utilising dCas9 to suppress virus replication [72]. This approach involves targeting the promoter region of the AC1 gene, crucial for virus replication, with site-specific DNA-binding proteins to inhibit the function of the Rep protein, essential for viral replication [73]. CRISPR/dCas9 can serve as a DNA-binding protein to effectively block the promoter region of target genes, thereby impeding virus replication and reducing viral infection rates [72]. By harnessing dCas9/sgRNA complexes, along with transcription effector complexes, researchers induced transcriptional interference at the promoter regions of downstream target genes. This interference disrupts RNA polymerase binding or elongation, further inhibiting virus replication and minimizing the spread of viral infection within plant tissues [74]. Overall, these findings highlight the potential of CRISPR/Cas9-mediated transcriptional interference as a powerful tool for developing viral-resistant crops and enhancing plant defence mechanisms against viral pathogens.

3.2 CRISPR/Cas9 targeted gene editing for fungal disease resistance

CRISPR/Cas9 technology offers a promising approach to developing fungal-resistant plants by targeting genes associated with susceptibility to fungal diseases. Several past studies have showcased the potential of CRISPR/Cas9 in engineering fungal-resistant crops by targeting key genes involved in plant-fungal interactions. For instance, knockout of the mildew-resistance locus (MLO) gene using CRISPR/Cas9 has led to resistance against powdery mildew in various crops such as grapevine, tomato, bread wheat, cucumber, and soybean [49, 75,76,77,78]. The MLO locus encodes proteins that render plants susceptible to powdery mildew fungi, and loss-of-function mutations result in enhanced resistance to this disease [79].

Similarly, resistance to downy mildew disease can be achieved by knocking out the homoserine kinase (HSK) gene, which plays a crucial role in conditioning susceptibility to downy mildew in sweet basil [80]. Additionally, knocking out pathogenesis-related proteins (PR) has been shown to increase susceptibility to downy mildew in grapevine, accompanied by reduced accumulation of reactive oxygen species around the stomata [81]. These findings underscore the potential of CRISPR/Cas9 in engineering fungal-resistant crops by targeting key genes involved in plant-fungal interactions. The CRISPR/Cas9 technology has also demonstrated efficacy in conferring resistance to rice blast disease by targeting specific genes involved in the plant's defence mechanisms. For instance, mutations induced in the ethylene responsive factors (ERF) gene using CRISPR have been shown to enhance rice blast resistance [82]. ERF serves as a negative regulator of blast resistance in rice, and the induced frameshift mutations in this gene contribute to increased resistance to rice blast pathogens.

Knocking out susceptible genes in plants has also been proven in the cacao plant (Theobroma cacao) by repressing the expression of the TcNPR3 which had been previously shown to repress pathogen defence responses [83]. Fister et al. has successfully express CRISPR/Cas9 machinery targeting TcNPR3 in cacao plants and enhance their resistance against Phytophthora tropicalis, a bane in cacao farming [84]. Furthermore, disruption of the subunit of the exocyst complex (SEC3A) using CRISPR/Cas9 has also been linked to enhanced resistance against rice blast disease [85]. This disruption leads to an increase in salicylic acid synthesis, a key signalling molecule involved in plant defence responses against pathogens. The elevated levels of salicylic acid contribute to bolstering the plant's immune response, thereby improving its ability to withstand rice blast infections.

3.3 Enhancing plant resistance to bacterial infections using CRISPR/Cas9

Development of plants with resistance against bacterial infections has also been done using the CRISPR/Cas9 technology. For instance, modifying the promoter of the lateral organ border (LOB) gene can increase resistance to citrus canker in grapefruit and oranges [86, 87]. The primary transcription activator-like (TAL) effector of Xanthomonas citri subsp. citri (Xcc), known as PthA4, binds specifically to the effector binding element, EBEPthA4, in the LOB promoter. This binding activates gene expression, accelerating the growth of citrus canker. Mutations in EBEPthA4 can reduce or even eliminate the PthA4-inducible activity of the LOB promoter, thereby enhancing resistance [88].

Similarly, the TAL effector of Xanthomonas oryzae pv. oryzae, known as PthXo1, binds to the effector binding element, EBEPthXo1, in the promoter region of Os8N3, a member of the SWEET family of sugar transporters. This interaction promotes bacterial blight disease in tomatoes. By using CRISPR/Cas9 to disrupt the SWEET gene, plants can achieve increased tolerance to bacterial blight [89]. Another example of knockouts of susceptible genes has been demonstrated in a study by Pompili et al., in which the authors knocked out the gene encoding the MdDIPM4 protein in apple plants (Malus domesticus) [90]. The MdDIPM4 protein plays a crucial role in the infection mechanism of Erwinia amylovora which causes fire blight infection in apple plants. This technique highlights the potential of CRISPR/Cas9 to target specific genes and regulatory elements, thereby providing an effective strategy to enhance plant resistance against bacterial pathogens. The ability to precisely edit genes associated with susceptibility allows for the development of crops that are better equipped to withstand bacterial infections, contributing to improved agricultural productivity and sustainability.

Furthermore, CRISPR/Cas9 can enhance resistance to bacterial speck in tomatoes by editing the jasmonate zim domain (JAZ) repressor proteins [91]. The JAZ protein is essential for coronatine to stimulate stomatal opening, which facilitates bacterial leaf colonization. Editing the JAZ gene using CRISPR/Cas9 prevents stomatal reopening, thereby hindering bacterial colonization [92]. Furthermore, CRISPR/Cas9 can target the downy mildew resistance (DMR) gene to control banana xanthomonas wilt disease (BXW). The DMR gene is a susceptibility gene that is upregulated during pathogen infection, and its modification can provide broad-spectrum resistance to bacterial pathogens [93]. Kim et al. reported the mutation of the U-box type E3 ubiquitin ligase (PUB) gene showed resistance to bacterial leaf blight in rice [94]. The Pi21 gene and the effector-binding element of the OsSULTR3;6 gene mutagenesis exhibited resistance to bacterial leaf streak in rice [95]. These approaches highlight the versatility of CRISPR/Cas9 in developing disease-resistant crops by targeting genes involved in susceptibility and enhancing the plant's natural defense mechanisms. By precisely editing specific genes, CRISPR/Cas9 can significantly improve plant resilience against various bacterial infections, contributing to sustainable agricultural practices and increased crop yields.

3.4 CRISPR/Cas9 for crop protection against insect pests

Two-thirds of insects are phytophagous, meaning they feed on living plant components, making them a significant threat to agriculture. Insects represent the most diverse group of arthropods, highlighting the breadth of potential pests [96]. Their impact is profound, causing annual yield losses of 18–20% of the world's crop production, amounting to over $470 billion in estimated losses [97]. Moreover, insects play a crucial role as vectors for transmitting various plant diseases, including viruses, phytoplasmas, and bacteria, further exacerbating agricultural losses [7]. With the exacerbating effects of climate change, there is a looming threat of increased insect pest outbreaks and their global spread. In new habitats, invasive pest species often exhibit rapid proliferation, leading to heightened concerns about insect-borne plant diseases [98]. Consequently, there's a pressing need for genome editing to counteract insect pests. Insect genome editing presents two viable approaches for genetic modification to enhance resistance to insect pests [99].

Genome editing techniques offer promising avenues for controlling insect pests by targeting crucial developmental genes (Fig. 2). CRISPR/Cas9-mediated knockout of vitellogenin, a precursor for egg yolk essential for insect egg maturation and embryonic development, has resulted in incomplete embryo development in Plutella xylostella [100]. Similarly, knockout of the abdominal-A homeotic gene induces severe abdominal morphological defects in Plutella xylostella and Spodoptera frugiperda [101, 102]. The abdominal-A (abd-A) gene, a member of the homeotic (Hox) gene family, plays a pivotal role in segmental identity during embryogenesis by encoding transcription factors that modulate segment development through interactions with downstream target genes [103]. Mutations in abdominal-A lead to various deleterious phenotypes, including deformed segments, abnormal prolegs, abnormal gonads, and even embryonic lethality, with abnormal gonads potentially contributing to male and female infertility [101].

Fig. 2
figure 2

The CRISPR/Cas9 systems approach for developing resistant crops to control diseases, insect pests, and weeds

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Additionally, knockout of pheromone binding proteins (PBPs) in Spodoptera litura disrupts female sex pheromone perception, thereby preventing mating attempts [24]. Similarly, knockout of the intersex (ix) gene in Bombyx mori results in infertility and irregular external genitalia, accompanied by developmental defects in imaginal discs, affecting wings, antennae, and legs [25]. These findings underscore the potential of CRISPR/Cas9 technology to precisely target key genes involved in insect development, offering a promising strategy for controlling insect pests in agriculture.

On the side of the plants, insect-resistant plants can be achieved by amplifying the generation of salicylic acid to repel insects [104]. In rice, the cytochrome P450 gene CYP71A1 encodes tryptamine 5-hydroxylase, which catalyses the conversion of tryptamine to serotonin [105]. By using CRISPR/Cas9 to inactivate the CYP71A1 gene, serotonin production can be halted, resulting in higher levels of salicylic acid. This genetic modification makes the engineered plant more resistant against insect pests such as planthoppers and stem borers [50]. In addition, salicylic acid also acts as an immune signal, and its increase often coincides with elevated expression of antimicrobial pathogenesis-related (PR) genes, leading to enhanced disease resistance [106]. By targeting specific genes and pathways, scientists can develop crops with improved resistance to pests, contributing to sustainable agricultural practices and reducing the reliance on chemical pesticides. This strategy not only helps in protecting crops from damage but also supports environmental conservation efforts by minimizing pesticide use.

Gene drives are utilized by introducing an effector gene that causes mortality or sex bias that leads to a suppressed population in each generation. Besides, this strategy can also be used to modify the population by spreading a genetic variant that removes a harmful trait but sustains the population of the target [107,108,109]. Several selfish genetic elements have been adopted for gene drive systems, including meiotic drives such as transposons and maternal effect dominant embryonic arrest (Medea), which is a region of nuclear DNA found only in some flour beetles [110, 111]. Moreover, synthetic systems such as artificial Medea systems, engineered under dominance or homing endonuclease genes (HEGs) have been proposed to drive genes into a population [112]. Asad et al. developed a single CRISPR/Cas9-mediated gene-drive construct for Plutella xylostella, a highly destructive lepidopteran pest of cruciferous crops [113]. The gene drive constructed contains a Cas9 gene, a gRNA sequence, and a marker gene (EGFP), with their promoters to the target sites. This study resulted in gene-drive efficiency due to HDR being 6.67–12.59% and resistant-allele formation due to NHEJ being 80.93–86.77%.

3.5 CRISPR/Cas9 for crop protection to control weeds

Weeds are among the most significant biotic limitations on agricultural output, posing great potential yield losses to crops alongside viruses and pests [11]. Weeds compete with crops for essential resources such as space, sunlight, water, and nutrients, significantly reducing crop productivity. Furthermore, they serve as hosts for various insects and viruses that can harm crop plants, exacerbating the damage. Beyond agricultural impacts, weeds also devastate native habitats, threatening local flora and fauna and disrupting ecosystems [114].

Addressing these challenges is critical for ensuring food security and maintaining biodiversity. One promising solution is the creation of herbicide-resistant plants through genome editing. This approach allows engineered plants to withstand herbicide and thus its application can selectively eliminate unwanted weeds. The CRISPR/Cas9 technology offers precise and efficient methods for developing such herbicide-resistant crops, contributing to sustainable agricultural practices and better parasite management. In addition, through plant genome editing, it is possible to engineer crops that not only resist herbicides but also adapt to the dynamic and evolving challenges posed by weed species. The herbicide glyphosate inhibits the biosynthesis of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme in plants, disrupting the shikimate pathway and leading to plant death upon exposure to glyphosate [115]. Glyphosate interferes with the production of essential amino acids by binding to the EPSPS enzyme, which is crucial for plant growth and survival. To combat this, genetic engineering can be employed to create glyphosate-resistant plants.

A more conventional approach is transferring the EPSPS gene from Agrobacterium strain CP4 to crops such as potatoes. The bacterial EPSPS enzyme from Agrobacterium has a small modification that prevents glyphosate binding, thereby allowing the plant to continue producing amino acids even in the presence of glyphosate [116]. This modification ensures that while the plant's native EPSPS is inhibited by glyphosate, the bacterial EPSPS remains functional, conferring resistance to the herbicide. A more modern approach is to use the CRISPR/Cas9 technology to achieve constitutive expression of the EPSPS gene and thus heightened glyphosate resistance. Hummel et al. succeeded in replacing the endogenous EPSPS promoter and the first two exons with a strong constitutive promoter, resulting in glyphosate tolerance in cassava [117]. Li et al. developed gene replacement and insertion strategies targeting general introns via the NHEJ pathway using CRISPR/Cas9 [118]. This strategy was successfully employed to introduce the targeting induced local lesions in genomes (TILLING) amino acid substitution to the rice EPSPS gene, conferring resistance to glyphosate in rice.

The base editing technique mediated by CRISPR/Cas9 in the acetolactate synthase (ALS) gene has been used to generate herbicide resistance in rice, soybean, and watermelon [119,120,121]. In plants, ALS is an essential enzyme for the production of the branched-chain amino acids valine, leucine, and isoleucine. Single point mutations at multiple conserved locations of ALS genes can inhibit the interaction between the ALS protein and herbicides, offering a significant level of herbicide resistance across various plant species [122, 123]. The CRISPR/Cas9 technique has proven viable and successful in precise gene replacement. For instance, two specific amino acid residues in the ALS gene were precisely replaced to develop herbicide-resistant rice plants with homozygous resistance [124]. This precise gene editing approach provides a robust method for developing herbicide-resistant crops, offering an efficient alternative to traditional genetic modification techniques. By targeting specific genes and inducing precise mutations, CRISPR/Cas9 facilitates the development of crops that can withstand herbicide application, ensuring higher yields and more sustainable agricultural practices. The success of CRISPR/Cas9 in editing the ALS gene underscores its potential as a powerful tool in crop improvement and herbicide resistance management. Here is a comprehensive list (Table 1) detailing the various applications of CRISPR/Cas9 technology in crop protection, specifically aimed at managing plant diseases, pests, and weeds.

Table 1 A comprehensive list of CRISPR/Cas9 applications in the field of crop protection to address plant diseases, pests, and weeds
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These methods illustrate how CRISPR/Cas9 can be used to create herbicide-resistant crops by precisely modifying specific genes or regulatory elements. This approach not only offers an alternative to traditional genetic engineering methods but also provides a more targeted and efficient way to enhance crop resilience against herbicides. Over the last eight decades, the constant use of herbicides has resulted in the widespread evolution of herbicide resistance in numerous weed species. Currently, there are 269 weed biotypes (154 dicots and 115 monocots) have been reported to be resistant to 21 of the 31 known herbicide sites of action to date [125]. Therefore, the CRISPR/Cas9 gene drive has come as a novel genetic control strategy in agricultural weed management. There are several benefits of the utilization of gene drive in weed control, such as the specific elimination of invasive species. Its precise manner of gene drive could prevent harmful effects on non-target organisms and closely related species [126]. As an alternative to herbicides, gene drive has no safety concerns of exposure to hazardous chemicals that can lead to a lack of disturbances to the soil or environment [127]. Added advantages of the use of gene drive is reduced cost for invasive plant management and would require minimal human intervention [126].

Regulations on genome editing crops vary significantly across countries, reflecting different public perception and agricultural policies. In the U.S., the regulation of genome-edited crops is primarily overseen by three agencies: the U.S. Department of Agriculture (USDA), the Environmental Protection Agency (EPA), and the Food and Drug Administration (FDA) [133]. The USDA's 2020 SECURE (Sustainable, Ecological, Consistent, Uniform, Responsible, Efficient) rule clarified that certain genome-edited crops, such as those with small deletions or edits that could be achieved by conventional breeding, are not subject to the same regulations as traditional GMOs [134]. The FDA typically requires a safety assessment if the genome-edited crop introduces a new protein or has the potential to change the crop’s nutritional content [135]. The European Union (EU) has a more stringent approach to regulating genome-edited crops [136]. Genome-edited organisms are generally regulated under the same framework as genetically modified organisms (GMOs), following the precautionary principle. Regulation is mainly governed by Directive 2001/18/EC, which requires a thorough risk assessment, labelling, and traceability for GMOs [137]. China regulates genome-edited crops through the Ministry of Agriculture and Rural Affairs (MARA) [138]. The regulatory system is evolving as China invests heavily in biotechnology research. Canada regulates genome-edited crops based on the "novelty" of the trait rather than the technique used to develop it [139]. The Canadian Food Inspection Agency (CFIA) and Health Canada are the primary regulatory bodies [140]. CFIA oversees environmental safety, while Health Canada evaluates food safety. Japan’s regulatory approach is somewhat similar to that of Canada, focusing on the novelty of the product rather than the technique. The Ministry of Agriculture, Forestry and Fisheries (MAFF) and the Ministry of Health, Labour and Welfare (MHLW) are the primary bodies overseeing genome-edited crops [141].

4 Conclusion and future perspectives

As we look to the future, the potential of CRISPR/Cas9 technology in crop protection is both vast and promising. The increasing global population and the impacts of climate change will continue to exert pressure on agricultural systems, necessitating innovative approaches to enhance crop resilience and productivity. CRISPR/Cas9 stands at the forefront of these innovations, offering unprecedented precision and efficiency in genome editing. One of the key future perspectives is the integration of CRISPR/Cas9 with other emerging technologies, such as synthetic biology and bioinformatics, to create multi-faceted solutions for crop protection. By combining CRISPR/Cas9 with advanced data analysis and modelling techniques, researchers can better predict the outcomes of genetic modifications and optimize editing strategies for maximum effectiveness. This integrative approach will enable the development of crops that are not only resistant to diseases, pests, and weeds but also tailored to thrive in specific environmental conditions. Additionally, there is significant potential for CRISPR/Cas9 to be used in developing crops that can adapt to abiotic stresses such as drought, salinity, and extreme temperatures. Shi et al. utilized CRISPR/Cas9-enabled advanced breeding technology to create new variants of ARGOS8 [142]. In their earlier research, they demonstrated that transgenic plants with constitutive overexpression of ARGOS8 exhibit reduced sensitivity to ethylene and enhanced grain yield under drought stress conditions. Zhang et al. reported enhancing rice salinity tolerance by engineering a vector expressing Cas9 and OsRR22-gRNA, which targets the OsRR22 gene in rice [52]. Malzahn et al. compared the editing efficiencies and NHEJ repair profiles of AsCas12a, FnCas12a, and LbCas12a across four different temperatures in rice [143].

Another promising area is the use of CRISPR/Cas9 for the enhancement of plant nutritional profiles and the reduction of allergens and antinutritional factors. By precisely editing genes involved in metabolic pathways, crops can be engineered to produce higher levels of essential nutrients, vitamins, and antioxidants, contributing to improved human health and nutrition. Some prominent examples include the study by Sun et al. in which CRISPR/Cas9 was used to regulate the expression of MdMKK9 to increase anthocyanins in apple (Malus sp.) and one by Endo et al. where CRISPR/Cas9 was used to modify the OsOr gene in rice which resulted in β-carotene hyper accumulation [144, 145]. These examples are still in their nascent periods and are promising for further development. Editing genes associated with stress tolerance can create crops that maintain high yields even under adverse conditions, thus contributing to global food security. Future research should focus on identifying and validating target genes involved in abiotic stress responses and developing robust editing protocols to enhance crop resilience.

Another promising area is the use of CRISPR/Cas9 for the enhancement of plant nutritional profiles and the reduction of allergens and antinutritional factors. By precisely editing genes involved in metabolic pathways, crops can be engineered to produce higher levels of essential nutrients, vitamins, and antioxidants, contributing to improved human health and nutrition. Furthermore, as regulatory frameworks evolve, the commercialization of CRISPR/Cas9-edited crops is expected to accelerate. It is crucial for policymakers, scientists, and stakeholders to work together to establish guidelines that ensure the safety and efficacy of genome-edited crops while addressing public concerns and ethical considerations. In conclusion, the future of crop protection lies in the continued advancement and application of CRISPR/Cas9 technology. By leveraging its potential, we can develop crops that are better equipped to withstand biotic and abiotic stresses, thereby ensuring sustainable agriculture and food security for future generations. The transformative impact of genome editing in agriculture underscores the importance of sustained research, collaboration, and innovation in this rapidly evolving field.