Abiotic stresses significantly reduce agricultural productivity and crop yields, but CRISPR/Cas technology provides precise tools to enhance grain crop tolerance by targeting specific stress response genes ( Table 2 ).

Staple grain crops such as rice, wheat, and maize are particularly vulnerable to water scarcity, which poses a major challenge to food security. CRISPR/Cas technology allows precise genetic modifications to improve drought tolerance by targeting genes that regulate water use efficiency and osmotic balance (Shelake et al., 2022). A notable breakthrough in this area is the modification of the ZmHDT103 gene, a key component of the abscisic acid (ABA) signaling pathway, which has been shown to improve drought tolerance in maize by enhancing the plant’s ability to withstand water scarcity without compromising growth and yield under non-stress conditions (Wang et al., 2024f). Another promising application of CRISPR technology in combating drought stress involves engineering the TaRPK1 gene in wheat to enhance water absorption (Abdul Rahim et al., 2024). By targeting genes that influence root growth patterns and depth, CRISPR/Cas has been used to develop plants with deeper root systems capable of accessing water from further soil layers. Another innovative approach involves using CRISPR/Cas to manipulate Sal1 genes to improve the production of osmoprotectants, such as proline, which has been shown to enhance drought resistance in wheat by allowing plants to withstand dry periods more effectively through precise modulation of gene expression (Mohr et al., 2022).

Elevated temperatures can impair plant growth, reduce photosynthetic efficiency, and ultimately decrease crop yields, leading to a focus on using CRISPR/Cas technology to modify heat shock factors (HSFs) and heat shock proteins (HSPs) to enhance heat tolerance (Younas et al., 2024). These proteins protect cells from heat damage by refolding denatured proteins and preventing the aggregation of unfolded proteins. For instance, enhancing HsfA1b expression in wheat improves heat tolerance, maintaining growth and yields under high temperatures (Tian et al., 2020). Researchers are also editing genes involved in the synthesis of protective osmolytes and antioxidants to mitigate oxidative stress caused by high temperatures (Yadav et al., 2023). Furthermore, integrating CRISPR/Cas with other biotechnological tools enhances the robustness of crops against heat stress. For instance, gene drives engineered through CRISPR technology can spread heat tolerance traits rapidly through plant populations, potentially transforming the resilience of an entire crop in a few generations (Mohan et al., 2022).

Salt stress, an increasing agricultural concern particularly in regions affected by soil salinization due to improper irrigation practices and rising sea levels, is being addressed by CRISPR/Cas technology through the enhancement of salt tolerance by targeting genes that regulate ion homeostasis and osmotic balance (Hualpa-Ramirez et al., 2024). One strategy involves modifying transporters involved in sodium uptake and compartmentalization. For example, editing genes like knocking out AKT1, WRKYs reduces sodium accumulation, thereby improving salt tolerance and maintain better growth and yield in soybean and barley (Price, 2022; Feng et al., 2023). Additionally, by upregulating the DREB2A transcription factor has successfully enhanced salt tolerance in various crops, including soybeans and rice (Farhat et al., 2019; Feng et al., 2023). By fine-tuning the expression of these transcription factors, crops can activate comprehensive stress response pathways that confer enhanced tolerance to saline conditions.

CRISPR/Cas technology facilitates targeted genomic edits to confer cold tolerance by targeting the CBF (C-repeat Binding Factor) pathway, a well-documented regulatory mechanism in plants that enhances cold tolerance. CBF transcription factors activate a suite of genes that confer cold tolerance by enhancing the plant’s cellular machinery to cope with freezing stress (Perez-Garcia et al., 2023). CRISPR/Cas has been used to increase the expression of fatty acid desaturase genes, leading to changes in membrane lipid composition that better support cellular processes during cold exposure. For instance, studies have demonstrated that OsKASI-2 is required for increasing unsaturated fatty acids in rice membranes via CRISPR/Cas to improve cold tolerance (Zhang et al., 2024b). Another innovative approach involves using CRISPR/Cas for epigenetic modifications that influence gene expression related to cold stress (Jogam et al., 2022). This method offers a flexible approach to crop improvement that can be adjusted as environmental conditions change.

Heavy metals such as cadmium (Cd), arsenic (As), and lead (Pb) are toxic to plants, causing stunted growth and reduced yields, but CRISPR/Cas technology can be used to enhance plant tolerance to heavy metals by modifying genes involved in metal transport and detoxification. For instance, editing the OsLCD gene in rice reduces cadmium uptake, thereby increasing cadmium tolerance and reducing its accumulation in the edible parts of the plant (Chen et al., 2023). Similarly, targeting genes like Lsi1 and Lsi2 in rice can decrease arsenic accumulation in plant tissues, thereby improving tolerance to arsenic-contaminated soils (Xu et al., 2024). Furthermore, targeting the ZmHMA3 gene in maize has shown to increase zinc tolerance by enhancing the plant’s ability to compartmentalize and detoxify these metals (Lv et al., 2023). These advancements contribute to the development of crops capable of growing in contaminated soils and producing safer food products.

CRISPR/Cas technology offers a promising solution to the significant threat of UV radiation to crop health by enabling precise genetic modifications that enhance tolerance to DNA damage, oxidative stress, and impaired photosynthesis. Targeting the OsCOP1 gene has demonstrated potential in improving UV tolerance in rice, enhancing their resistance to UV-B radiation (Hu et al., 2024). By boosting the plant’s protective mechanisms against UV damage, CRISPR/Cas technology can help develop crops that maintain productivity and growth under high UV exposure.

Oxidative stress results from the accumulation of reactive oxygen species (ROS) under various stress conditions. CRISPR/Cas technology provides a means to enhance oxidative stress tolerance in crops by targeting genes involved in ROS detoxification and antioxidant defense mechanisms. For example, Editing the CAT (catalase) gene family, particularly OsCAT3, which is crucial for detoxifying superoxide radicals and hydrogen peroxide, can enhance rice’s ability to mitigate oxidative damage (Jiang et al., 2023). Additionally, targeting regulatory genes such as zinc finger proteins, which modulate the expression of multiple antioxidant genes, can offer a comprehensive approach to improving oxidative stress resilience in crops (Qu et al., 2024).

Biotic stresses threaten crop health and productivity, and CRISPR/Cas technology enables precise genetic modifications to enhance crop resistance.

CRISPR/Cas systems, particularly Cas13 have shown targeting and degrading the RNA genomes of RNA viruses, preventing their replication within the host plant (Sarkar et al., 2024). This approach has been effectively demonstrated in crops such as potato, where Cas13 was engineered to target and cleave the RNA of sweet potato virus disease (Zhan et al., 2023). Researchers have expanded the capabilities of CRISPR/Cas systems in viral defense by using them not only to target pathogens directly but also to modify the host plant’s genome to enhance its natural virus defense mechanisms (Uranga and Daròs, 2023). For instance, in crops like wheat and rice, CRISPR/Cas9 has been employed to knock out susceptibility genes such as TaPDIL5 or OsDjA2 and OsERF that facilitate viral infection, thus providing broad-spectrum virus resistance (Kan et al., 2022; Távora et al., 2022). Additionally, CRISPR/Cas technology was also employed to knock out ZmPDRP1 in maize, revealing that the loss of this gene significantly reduced the ability of sugarcane mosaic virus (SCMV) to replicate and spread within the plant (Xie et al., 2024). This highlights the utility of CRISPR/Cas not only for plant trait improvement but also as a powerful tool for dissecting gene functions in plant-pathogen interactions.

Enhancing bacterial resistance in crops using CRISPR/Cas technology involves targeting bacterial virulence genes and enhancing the plant’s immune response by disrupting key genes in bacterial pathogens to significantly reduce their virulence. For instance, knocking out the StNRL1 gene in potatoes using CRISPR/Cas9 increases resistance to late blight caused by Phytophthora infestans while simultaneously increasing susceptibility to early blight caused by Alternaria alternate (Norouzi et al., 2024). CRISPR/Cas technology has also been employed to modify plant immune receptors to recognize bacterial pathogens more effectively. Editing the FERONI and SlWak1 depend on FLS gene in rice and wheat, which encodes a receptor involved in pathogen recognition, has improved the plants’ ability to detect and respond to bacterial infections, thereby enhancing resistance (Huang et al., 2020; Zhang et al., 2020). This genetic modification has resulted in rice varieties with enhanced resistance to bacterial blight, leading to healthier plants and higher yields.

Fungal diseases are a major concern for crop health, and CRISPR/Cas technology offers new ways for enhancing fungal resistance in crops through precise genetic modifications. One strategy involves using CRISPR/Cas to knock out susceptibility genes that fungi exploit, such as MLO (Mildew Locus O) genes in soybean and wheat, which has been shown to confer resistance to powdery mildew by making the plants less susceptible to fungal infections (Li et al., 2022a; Bui et al., 2023). Enhancing plant defense genes involved in recognizing and responding to fungal attacks also improves resistance. For instance, editing the CNL (Coiled-Coil, Nucleotide-Binding, Leucine-Rich Repeat) gene family and MeRPPL1 in cassava has led to enhanced fungal resistance (Ramulifho and Rey, 2024). Another innovative application of CRISPR/Cas involves editing the genomes of fungal pathogens themselves. This approach has been explored in various fungal species, including Fusarium and Botrytis, which are responsible for significant agricultural losses.

CRISPR/Cas technology is also being utilized to enhance pest resistance in crops by knocking out susceptibility genes and enhancing plant defense mechanisms. For example, editing the ABC transporter gene in soybean has been shown to confer resistance to bollworms by disrupting the insect’s ability to digest plant tissues (Amezian et al., 2024).Another strategy is to enhance the expression of plant defense genes involved in producing secondary metabolites that deter insect feeding. For instance, increasing the expression of genes involved in the biosynthesis of phenolic compounds has been shown to reduce insect herbivory in crops like maize and soybean (Razzaq et al., 2023; Kumari et al., 2024). CRISPR/Cas technology has also been used to modify genes encoding insecticidal proteins, such as Cry proteins, VIP proteins improving pest resistance in crops (Dubovskiy et al., 2024). These genetic modifications result in plants that produce higher levels of natural insecticidal compounds, providing an effective defense against pests.

Nematodes, such as root-knot nematodes (Meloidogyne spp.), cause root damage and reduce nutrient and water uptake. CRISPR/Cas technology can enhance nematode resistance by targeting genes that facilitate nematode infection and reproduction. For instance, editing a susceptibility gene OsHPP04 in rice has conferred resistance to root-knot nematodes (Huang et al., 2023). Similarly, modifying the GmSNAP02 and an α-SNAP gene in soybean has enhanced resistance to cyst nematodes (Usovsky et al., 2023, Usovsky et al., 2023). Through disrupting the molecular pathways that nematodes exploit, CRISPR/Cas can develop crops with robust nematode resistance, reducing yield losses and the need for chemical nematicides.

Parasitic plants, such as Striga and Orobanche, attach to the roots of host plants and extract water and nutrients, significantly reducing crop yields. CRISPR/Cas technology can enhance resistance to parasitic plants by targeting genes involved in host-parasite interactions. For instance, editing the LGS1 gene in sorghum has conferred resistance to Striga by disrupting the production of strigolactones, which are essential for Striga seed germination and attachment (Makaza et al., 2023). By modifying specific signaling pathways and defense mechanisms, CRISPR/Cas can develop crops less susceptible to parasitic plants, thereby improving yield and sustainability, while highlighting the technology’s versatility in managing various biotic stresses to ensure better crop health and productivity.

CRISPR/Cas technology offers new opportunities to enhance crop yield by directly targeting genes that regulate plant growth and development. For example, editing the OsAPL involved in nutrient transport has been shown to increase yield in rice (Zhang et al., 2024a). Enhancing photosynthetic efficiency by targeting genes involved in chlorophyll synthesis and light capture, such as the OsSXK1 gene in rice, has improved photosynthetic rates and increased grain yield (Zheng et al., 2021). Additionally, editing genes involved in nutrient uptake and utilization, such as the ARE genes in barley or wheat, enhances nitrogen use efficiency and leads to higher yields under low nitrogen conditions (Karunarathne et al., 2022). Recent studies have demonstrated the potential of CRISPR/Cas technology in enhancing yield-related traits in various crops. Editing the DEP1 gene in rice has led to the development of semi-dwarf varieties with improved lodging resistance and higher grain yield (Zhang et al., 2023a).

CRISPR/Cas9 technology has dramatically advanced agricultural biotechnology by enabling precise genome editing to improve various crop quality attributes, including safety, taste, texture, shelf life, and industrial applicability. In cassava, CRISPR/Cas9 has been used to edit the CYP79D1 gene, significantly reducing cyanogenic glycosides, which lowers the risk of cyanide toxicity, enhancing the safety of this staple crop without affecting its agronomic performance (Juma et al., 2022). In rice, the technology has been employed to enhance aromatic qualities by editing the OsBADH2 gene, leading to increased production of 2-acetyl-1-pyrroline (2-AP), a compound that imparts a desirable fragrance, thereby catering to consumer preferences (Tian et al., 2023). In potatoes, CRISPR/Cas9 has modified the gbss gene responsible for granule-bound starch synthase, resulting in amylose-free starch that provides a smoother texture, which is highly valued in both culinary applications and industrial processes (Toinga-Villafuerte et al., 2022). The technology has also been pivotal in extending the shelf life of various crops by targeting genes involved in the ripening process, such as those regulating ethylene production, allowing for slower ripening, reduced post-harvest losses, and improved economic viability. Furthermore, in barley, CRISPR/Cas9 has been used to enhance grain hardness by editing the Hina gene, producing grains with a higher hardness index that are better suited for industrial applications, though this has also resulted in reduced grain width and thousand-grain weight (Jiang et al., 2022). Additionally, in potatoes, targeting the FtsZ1 gene has led to the development of lines with larger starch granules, significantly increasing the final viscosity of starch paste, making these potatoes more suitable for specific industrial processes, all achieved without compromising the plant’s overall phenotype or nutritional quality (Pfotenhauer et al., 2023). These diverse applications of CRISPR/Cas9 underscore its transformative potential in crop improvement, enabling tailored modifications that meet consumer demands, enhance safety, and address specific industrial needs while ensuring the sustainability and economic viability of agricultural practices.

Addressing nutritional deficiencies through crop biofortification is a key goal in agricultural biotechnology, and CRISPR/Cas technology plays a pivotal role in achieving this. For instance, CRISPR/Cas has been employed to increase pro-vitamin A content in rice, a vital intervention in combating vitamin A deficiency in populations that rely heavily on rice as a staple food (Maiti and Banik, 2023). Biofortification aims to increase the content of essential nutrients in crops, thereby improving their nutritional value. The development of “Golden Rice,” which contains higher levels of beta-carotene, was accomplished by modifying genes involved in pro-vitamin A biosynthesis (Dong et al., 2020; Datta et al., 2021). RISPR/Cas technology has also been applied to enhance the mineral content of crops. In rice and wheat, genes such as OsNAS have been edited to increase iron and zinc levels, addressing micronutrient deficiencies that often lead to anemia and impaired immune function (Dueñas et al., 2021). Similarly, in maize, targeting the PSY1, Crtl, and LCYB genes has boosted the biosynthesis of pro-vitamin A, resulting in the creation of “Golden Maize” (Sobrino-Mengual et al., 2024).The technology is also instrumental in improving the amino acid content of crops. In cassava, CRISPR/Cas has been used to enhance the levels of essential amino acids and vitamins, significantly improving the nutritional quality of this staple crop (Otun et al., 2023). In maize, editing genes involved in lysine biosynthesis has increased lysine content, addressing a common deficiency in cereal grains (Hasan, 2024). Improving the protein quality of crops is another significant area of focus for CRISPR/Cas technology. For example, targeted mutagenesis of the OsAAP6 and OsAAP10 genes in rice can reduce grain protein content, thereby improving the eating and cooking quality of the crop (Wang et al., 2020). CRISPR/Cas technology has been used to reduce antinutritional factors such as phytic acid in soybean by targeting the GmIPK1 gene, enhancing the bioavailability of iron and zinc and thereby improving the overall nutritional quality of the soybean (Song et al., 2022).

CRISPR/Cas technology has significantly advanced rice improvement by enabling precise genome modifications, enhancing traits such as yield, stress resistance, and nutritional value. This technology has been instrumental in improving resistance to both biotic and abiotic stresses in rice. For example, the clade III subfamily of OsSWEETs, including OsSWEET11a and OsSWEET14, plays a crucial role in susceptibility to bacterial blight by mediating sucrose efflux for bacterial proliferation. These genes are targeted by specific transcription activator-like effectors (TALEs) from Xoo (Wang et al., 2024g). This approach demonstrates the potential of CRISPR/Cas9 to address other pathogen-related challenges in rice. Similarly, the OsCS511 gene has been edited to improve cold tolerance in rice, which is critical for expanding rice cultivation to cooler climates (Park et al., 2024). It highlights the adaptability of CRISPR/Cas technology in enabling rice to grow in suboptimal conditions, thereby expanding its cultivation area. Furthermore, base editing has been used to increase zinc uptake and plant yield by editing the promoter region of the OsNAS2 gene, which plays a crucial role in zinc translocation and accumulation (Ludwig et al., 2024). This precise editing has led to a significant increase in zinc concentration in rice grains, addressing both yield improvement and nutritional enhancement in a single genetic modification. Additionally, the knockout of the OsDSG1 and OsbHLH024 transcription factor has been shown to significantly enhance salt stress resistance, allowing rice to thrive in saline soils (Alam et al., 2022; Ly et al., 2024). This advancement is particularly important in regions where soil salinity limits agricultural productivity. Furthermore, CRISPR/Cas9-mediated mutagenesis of the susceptibility gene OsHPP04 has conferred enhanced resistance to rice root-knot nematode, a significant pest that causes major yield losses in rice crops, demonstrating CRISPR/Cas9’s potential in sustainable rice production (Huang et al., 2023).

Despite these successes, challenges such as off-target effects, potentially lead to undesirable mutations. For instance, when editing the OsWRKY71 gene to enhance resistance against brown plant hopper, careful analysis was necessary to ensure that no off-target effects compromised the plant’s health (Li et al., 2023b). This case underscores the importance of precision in gene editing to avoid unintended consequences that could negate the benefits of the modifications. Another challenge is the relatively low efficiency of HDR in rice, which is required for precise gene insertions or complex modifications. This limitation has been addressed in part by developing more precise methods like prime editing, which does not rely on HDR. Prime editing has been successfully applied to the OsMLH1 gene to enhance the efficiency of the editing process without disturbing rice fertility, offering a promising approach for future genetic improvements (Liu et al., 2024a).

Recent advancements in CRISPR technology, particularly base editing and prime editing, have opened new avenues for improving specific traits in rice. Base editing has been successfully applied to the OsALS gene to confer herbicide resistance, allowing rice to resistant to herbicides such as imazamox, which is crucial for effective weed management (Zafar et al., 2023). This modification allows for more targeted and sustainable weed control strategies, reducing the need for broad-spectrum herbicides that can harm the environment. Additionally, prime editing has been applied to the Xa13 and Xa25 genes, creating rice varieties with enhanced resistance to bacterial blight by targeting and disrupting these susceptibility genes (Zhu et al., 2024). These innovations, including the application of base editing and prime editing to improve traits like herbicide resistance and bacterial blight resistance, are crucial for developing resilient rice varieties suited to climate change and global food security challenges.

The application of CRISPR/Cas systems, including advanced techniques like prime editing and base editing, has revolutionized the ability to enhance maize’s tolerance to various abiotic and biotic stresses. Prime editing, particularly the optimized ePE5max system, has been instrumental in generating heritable mutations in maize, enabling resistance to herbicides by targeting key enzymes such as EPSPS, ALS, and ACCase. This has significantly improved the resilience of maize to herbicide stress. Base editing has also shown potential in this area, with the introduction of specific nucleotide substitutions, such as the triple amino acid substitution in the ZmEPSPS gene, leading to heightened glyphosate tolerance (Kaul et al., 2024). Additionally, the editing of ZmSWEET1b, a sugar transporter crucial for assimilate allocation, has demonstrated improved salt stress response, showcasing the effectiveness of these gene-editing techniques in enhancing maize’s abiotic stress tolerance (Wu et al., 2023).

Further advancements in maize stress tolerance have been achieved through the targeted editing of specific genes associated with drought, salinity, and disease resistance. For example, the gene editing of ZmGA20ox3 has not only improved drought tolerance but also optimized plant architecture, while the co-expression of ZmVPP1 with ZmNAC111 has conferred robust drought resistance by enhancing the plant’s stress response mechanisms (Liu et al., 2023d; Liu et al., 2024b). Similarly, genes like ZmHAK17, which encodes a Na⁺-selective transporter, and the HKT1 family sodium transporter have been pivotal in improving maize’s tolerance to salinity by regulating sodium influx and promoting seed germination under salt conditions (Zhang et al., 2023d; Wang et al., 2024a). Moreover, the genome editing of the ZmNANMT gene has conferred multiple disease resistance without agronomic penalties, addressing major diseases like southern leaf blight, northern leaf blight, and Fusarium stalk rot (Li et al., 2023c). These targeted genetic modifications underline the versatility of CRISPR/Cas systems in addressing both biotic and abiotic stresses in maize.

However, despite these promising developments, the application of CRISPR/Cas systems in maize faces several challenges, including off-target effects, regulatory hurdles, and efficiency in gene editing techniques. For instance, while the DNA-free genome editing of the ZmPLA1 gene via ribonucleoprotein complexes has been successful in increasing haploid induction rates in tropical maize, optimizing these methods further remains crucial to enhancing efficiency and reducing unintended mutations (Rangari et al., 2023). Similarly, the generation of maize lines with enhanced cold stress tolerance through the overexpression of ZmG6PDH1 in glucose-6-phosphate dehydrogenase family, and the regulation of heat stress tolerance through the HSF20-HSF4-cellulose synthase A2 module, illustrate the complexity of stress responses in maize that require precise and efficient gene editing tools (Li et al., 2023a, Li et al., 2024b). Addressing these challenges through ongoing research and technological advancements will be key to fully harnessing the potential of CRISPR/Cas systems in improving maize resilience and productivity under various environmental stresses.

Off-target effects in CRISPR/Cas systems present a significant challenge in genome editing, where unintended cuts by the Cas enzyme at non-target sites can lead to adverse consequences. In crops, such unintended mutations can affect traits, reducing yield or introducing unwanted characteristics. Therefore, minimizing off-target effects is critical to ensuring the safety and efficacy of CRISPR-based technologies, making precision in genome editing a top priority. To address these challenges, considerable progress has been made in developing high-fidelity Cas variants and optimizing gRNA design. High-fidelity Cas9 variants, such as eSpCas9, SpCas9-HF1 and FrCas9, have been engineered to enhance precision by reducing nonspecific DNA interactions (Guo et al., 2023; He et al., 2024). These variants demonstrate lower off-target activity while maintaining robust on-target efficacy, making them suitable for applications. Additionally, studies have shown that Cas12b, with its strict PAM requirements and low tolerance for mismatches, has minimal sgRNA-dependent off-target effects, showing great promise in rice genome editing (Gurel et al., 2023). This system’s ability to precisely target specific genomic sites with minimal off-target activity underscores the importance of optimizing both the Cas enzyme and gRNA design to enhance the specificity of CRISPR/Cas systems. Future research should focus on enhancing accuracy, minimizing off-target effects, and developing novel variants or engineered Cas proteins that can better handle the complexities of multiplex editing, especially in crops with polyploid genomes. This will not only improve precision in genome editing but also reduce unintended consequences, making CRISPR technology safer and more reliable for agricultural applications.

Delivering CRISPR components into plant cells, particularly in recalcitrant staple crops like wheat, maize, and certain rice varieties, presents significant challenges due to their complex genetic makeup and poor tissue culture responses. Traditional methods, such as Agrobacterium-mediated transformation and biolistic particle delivery, are often ineffective in these crops. Agrobacterium-mediated transformation is limited by its host range and often results in random transgene integration, leading to gene silencing and unpredictable expression patterns. Similarly, while biolistic delivery bypasses some limitations of Agrobacterium, it frequently causes multiple, unstable transgene insertions and physical damage to plant tissues, reducing transformation efficiency. Nanoparticle-mediated delivery, a more recent approach, shows promise but faces challenges related to the precise release and stability of CRISPR components within plant cells, as well as potential cytotoxicity concerns (Antony Ceasar and Ignacimuthu, 2023).

To overcome these challenges, research is increasingly focused on developing novel delivery methods that enhance the efficiency and specificity of CRISPR delivery in recalcitrant crops (Kocsisova and Coneva, 2023). Viral vectors offer a promising avenue by utilizing natural plant infection mechanisms to deliver CRISPR components, though their limited cargo capacity remains a significant hurdle (Liu et al., 2023c). Enhancements in nanoparticle systems, such as surface functionalization with specific ligands and the development of biodegradable particles, could improve the specificity and safety of CRISPR delivery. Protoplast transfection, combined with optimized regeneration protocols, offers a direct method for introducing CRISPR components, though its application is currently limited to species with efficient protoplast regeneration systems (Yang et al., 2024). Grafting techniques, which use transgenic rootstocks to deliver CRISPR components to wild-type scions, represent another innovative approach for achieving transgene-free genome editing in recalcitrant crops (Yang et al., 2023b). To achieve sustainable improvements, future research should not only focus on developing novel delivery methods but also on ensuring the long-term stability and heritability of CRISPR-induced modifications across multiple generations. Understanding mechanisms such as chromosomal rearrangements and DNA repair fidelity will be crucial to maintaining the integrity of these genetic edits. Longitudinal studies that track the persistence and effects of these modifications over time are essential to confirm their stability and effectiveness, ensuring that the benefits of CRISPR technology are retained through successive crop generations.

CRISPR technology offers significant potential in crop improvement, but it also presents challenges in ethics, regulation, and socioeconomic impact. Ethically, the precision and speed of CRISPR modifications, such as those used in rice and wheat to enhance yield and nutritional content, raise concerns about the moral acceptability of altering plant genomes. These concerns are especially pronounced in regions like Europe, where public sentiment is cautious about genetic modifications. The ethical debate includes potential unintended consequences, the creation of “unnatural” organisms, and the long-term ecological impacts (Marone et al., 2023). Additionally, the regulatory landscape for CRISPR-edited crops varies significantly across countries. In European Union classifies CRISPR-edited crops as genetically modified organisms (GMOs), subjecting them to rigorous regulations that have hindered the commercialization of crops like CRISPR-edited wheat. Countries like Argentina and Brazil have adopted more flexible regulatory frameworks, focusing on the final product rather than the process, allowing for the development and commercialization of crops like CRISPR-edited sugarcane with less regulatory burden. Meanwhile, China’s significant investment in CRISPR research, particularly in crops like rice, positions it as a major player, although commercialization remains tightly regulated (Ghouri et al., 2023; Kumawat et al., 2024). Socioeconomic challenges further complicate the integration of CRISPR technology into global agriculture. The contentious patent landscape, dominated by the U.S. and European nations, restricts access to CRISPR technology, particularly for smaller entities or developing countries. This is evident in the development of CRISPR-edited crops like tomatoes with enhanced GABA levels, where patent issues could limit access in less affluent regions (Akhtar et al., 2023). Additionally, the concentration of CRISPR technology within a few large corporations could exacerbate inequalities in the agricultural sector, particularly for staple crops like maize and wheat, crucial for food security in developing regions (Molinari et al., 2024). Countries like India and several African nations face the challenge of ensuring that the benefits of CRISPR technology, such as drought-resistant maize or disease-resistant cassava, are accessible to all farmers, not just large agribusinesses (Munawar et al., 2024). As CRISPR technology advances, ensuring that regulatory frameworks keep pace with these developments is essential. Future research must focus on addressing safety concerns, including off-target effects and the long-term ecological impacts of CRISPR-modified crops. This requires the development of comprehensive risk assessment models and strong collaboration between scientists, regulators, and policymakers. By aligning technological advancements with robust regulatory measures, we can ensure the safe and responsible integration of CRISPR into global agricultural practices, ultimately fostering public trust and acceptance.

The intersection of CRISPR technology with emerging fields such as nanotechnology, synthetic biology, and machine learning (ML) offers a transformative potential to advance genome editing in staple crops like rice, maize, wheat, and potato. Nanotechnology, in particular, addresses one of the critical challenges in CRISPR-based genome editing: the efficient and precise delivery of CRISPR components into plant cells. Nanomaterials like carbon nanotubes and mesoporous silica nanoparticles can bypass the plant cell wall, enabling targeted and controlled delivery of CRISPR components, which increases transformation efficiency and reduces off-target effects (Khanna et al., 2023). Additionally, nanoparticle-mediated delivery systems are species-independent, democratizing CRISPR technology across diverse crops (Naik et al., 2022). Future research should focus on optimizing these nanotechnologies for larger CRISPR complexes, organelle-specific editing, and direct transformation of germline cells, potentially bypassing tissue culture. This synergy holds great potential for advancing sustainable agriculture, improving crop resilience, nutritional content, and reducing chemical input dependency.

The intersection of CRISPR and Synthetic Biology offers a promising path to enhance genome editing efficiency and precision in staple crops. CRISPR/Cas systems enable targeted modifications, but challenges such as off-target effects, variable efficiency, and polyploid genome complexity persist. Synthetic Biology addresses these issues by providing tools to design and control genetic circuits, thereby reducing off-target effects and improving adaptability across different species (Yang and Reyna-Llorens, 2023). Key advancements include regulatory circuits that fine-tune CRISPR activity and feedback loops that adjust editing in real-time, enhancing precision (Wang and Demirer, 2023). Integrating CRISPR with synthetic metabolic pathways could yield crops that are higher-yielding and more resilient to environmental stressors, crucial for addressing global food security challenges posed by climate change. Future research should focus on developing synthetic promoters for efficient editing in polyploid crops and combining CRISPR with metabolic engineering to produce bioactive compounds, advancing sustainable agriculture in both traditional and controlled environments like vertical farming.

Machine learning (ML) adds another layer of innovation by exponentially enhancing CRISPR’s potential for precise genome modifications. However, when combined with the predictive capabilities of ML, the potential of CRISPR can be vastly expanded. One of the key challenges in CRISPR genome editing is ensuring specificity and efficiency in targeting the correct genomic sites while minimizing off-target effects. ML models can address this challenge by analyzing large datasets from CRISPR experiments to predict the most effective guide RNA sequences, thereby enhancing the precision of the Cas9 enzyme and reducing unintended consequences (Chen et al., 2024). Additionally, ML can predict the phenotypic outcomes of specific gene edits, which is particularly complex due to the multifactorial nature of traits like yield and stress tolerance (Das et al., 2023). In crops like rice and maize, where multiple genes interact to influence traits such as drought resistance, ML can identify the most impactful gene edits by considering various genetic and environmental factors. Future research should focus on developing sophisticated ML algorithms capable of handling the complexity of polygenic traits and creating extensive datasets to train these models. Integrating CRISPR-ML into precision agriculture systems could provide tailored recommendations, optimizing crop performance in specific field conditions and ultimately contributing to more sustainable and efficient agricultural practices.

While the CRISPR/Cas9 system has predominantly been used for gene knockouts, the development of CRISPR/Cas-based knock-in strategies has significantly expanded its potential, enabling precise gene integration and enhancing crop traits with high accuracy. This system allows for the insertion of desired genes into specific genomic locations, facilitating complex genetic modifications such as the introduction of large DNA sequences or multiple genes, which are crucial for stacking beneficial traits like disease resistance, stress tolerance, and improved nutritional content. However, the knock-in approach relies primarily on HDR, which is less efficient in plants compared to NHEJ. To address this limitation, researchers are developing strategies to enhance HDR efficiency, including the use of HDR enhancers, dual-gRNA systems, and advanced delivery methods like nanoparticle-mediated delivery and viral vectors, making the knock-in process more feasible for large-scale agricultural applications.

The application of the CRISPR/Cas knock-in system in specific crops has demonstrated its versatility and promise in sustainable agriculture. For instance, in rice, this system has been used to confer glyphosate resistance by precisely editing the acetolactate synthase (ALS) gene and to upregulate genes involved in key metabolic pathways, significantly enhancing the crop’s nutritional content and stress tolerance (Sony et al., 2023). In wheat, researchers are focusing on improving HDR efficiency using fusion proteins like Cas9-VirD2 to enhance traits such as disease resistance and grain quality (Schreiber et al., 2024). Similarly, in maize, the knock-in of regulatory elements to more precisely control gene expression has been explored to improve yield, nutrient use efficiency, and stress tolerance (Kaul et al., 2024). These examples underscore the knock-in system’s crucial role in developing next-generation crops that are more resilient, productive, and adaptable to changing environmental conditions.

Despite its potential, the CRISPR/Cas knock-in system faces challenges, including low HDR efficiency and off-target effects. To overcome these, researchers are exploring various strategies, such as enhancing HDR pathways, developing modified CRISPR/Cas variants, and utilizing alternative genome editing tools like CRISPR nickase and prime editing, which reduce the risk of off-target mutations. Improving delivery systems, such as nanoparticle-based methods, is also critical for increasing the efficiency and precision of gene insertion. Future directions for this technology include further engineering of Cas proteins for enhanced specificity, optimizing guide RNA design to minimize off-target effects, and integrating the knock-in system with emerging technologies like base editing and prime editing. These advancements are essential for refining the CRISPR/Cas knock-in system and maximizing its impact on agricultural innovation.