1 Introduction

Bacillus thuringiensis (Bt) is a Gram-positive, spore-forming bacterium widely recognized for its insecticidal properties. It produces crystal (Cry) proteins during sporulation, which are toxic to a variety of insect pests upon ingestion. These Cry proteins have been effectively utilized in agriculture for pest control, reducing the reliance on chemical pesticides and thereby mitigating environmental pollution and health risks (Nair et al., 2020; Arsov et al., 2023; Sauka et al., 2023). Bt's specificity towards target pests, such as moths, beetles, and mosquitoes, makes it a valuable tool in integrated pest management programs (Nair et al., 2020; Arsov et al., 2023).

Despite the success of Bt-based insecticides, several limitations hinder their long-term efficacy. One significant challenge is the rapid evolution of resistance in target insect populations. For instance, resistance to Bt Cry1 toxins has been observed in various lepidopteran pests, which compromises the effectiveness of Bt crops and biopesticides (Guo et al., 2019). Additionally, the narrow spectrum of activity of certain Bt strains limits their utility against a broader range of pests (Reyaz et al., 2019; Sauka et al., 2023). Environmental factors, such as soil enzymatic activities, can also influence the persistence and efficacy of Bt proteins, necessitating careful evaluation of their environmental impact (Li et al., 2019; Li et al., 2022).

Genetic modification offers a promising approach to overcome the limitations of traditional Bt insecticides. Traditional methods of genetic modification in Bt have been labor-intensive and time-consuming (Liu and Zhang, 2024). By employing techniques such as CRISPR/Cas9, researchers can enhance the insecticidal properties of Bt strains, broaden their spectrum of activity, and mitigate resistance development in target pests (Guo et al., 2019). For example, the construction of chimeric Bt proteins with novel domain combinations has shown enhanced activity against multiple soybean pests, demonstrating the potential of genetic engineering in developing more effective biopesticides (Chen et al., 2021). Additionally, the identification and characterization of novel Cry proteins, such as Cry78Ba1, provide new avenues for specific and safe pest control (Cao et al., 2020).

This study provides a comprehensive overview of the advancements in CRISPR-based gene editing of Bt for improved insecticidal properties, including summarizing the current state of Bt as a biopesticide and its role in pest control, identifying the limitations of traditional Bt insecticides, exploring the potential of genetic modification techniques, particularly CRISPR/Cas9, in enhancing Bt's insecticidal properties, and evaluating the environmental and ecological implications of genetically modified Bt strains. By addressing these objectives, this study aims to highlight the potential of CRISPR-based gene editing in developing next-generation Bt biopesticides with improved efficacy and sustainability.

2 CRISPR-Cas9 Technology: A Tool for Genetic Enhancement

2.1 Introduction to CRISPR-Cas9 gene editing

CRISPR-Cas9, derived from a bacterial immune defense mechanism, has revolutionized genetic engineering by providing a precise, efficient, and versatile tool for genome editing. Initially recognized for its role in bacterial immunity against viruses, CRISPR-Cas9 has been adapted for use in a wide range of organisms, including plants and animals, to facilitate targeted genetic modifications (Demirci et al., 2018; Li et al., 2021). This technology employs a guide RNA (gRNA) to direct the Cas9 nuclease to a specific DNA sequence, where it introduces double-strand breaks (DSBs). These breaks are then repaired by the cell's natural repair mechanisms, leading to targeted mutations or insertions (Bao et al., 2019).

2.2 Mechanism of CRISPR-Cas9 in gene editing

The CRISPR-Cas9 system operates through a simple yet highly effective mechanism. The Cas9 protein, guided by a single-guide RNA (sgRNA), binds to a complementary DNA sequence and introduces a DSB at the target site. The cell's repair machinery then attempts to fix the break, typically through non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ often results in small insertions or deletions (indels) that can disrupt gene function, while HDR can be used to introduce specific genetic changes using a donor template (Bao et al., 2019). This precise targeting capability allows for the modification of specific genes, enabling the study of gene function and the development of organisms with desirable traits (Eş et al., 2019; Erdoğan et al., 2023).

2.3 Advantages of CRISPR in agricultural biotechnology

CRISPR-Cas9 offers several advantages over traditional breeding and earlier genome editing technologies such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). These advantages include higher efficiency, ease of design, lower cost, and the ability to target multiple genes simultaneously (multiplexing) (Demirci et al., 2018; Eş et al., 2019). In agriculture, CRISPR-Cas9 has been used to enhance crop yield, quality, and resistance to diseases and environmental stresses. For instance, it has been employed to develop crops with improved resistance to pests and pathogens, increased tolerance to abiotic stresses like drought and salinity, and enhanced nutritional profiles (Bisht et al., 2019; Chen et al., 2019; Zhu et al., 2020a). The technology's ability to create transgene-free plants also addresses regulatory and public acceptance issues associated with genetically modified organisms (GMOs) (Erdoğan et al., 2023).

2.4 Specific considerations for applying CRISPR to Bt

When applying CRISPR-Cas9 to Bacillus thuringiensis (Bt), several specific considerations must be taken into account. Bt is widely known for its insecticidal properties, which are primarily due to the production of crystal (Cry) proteins that target specific insect pests. Enhancing Bt's insecticidal properties through CRISPR-Cas9 involves precise modifications to the genes encoding these Cry proteins to increase their efficacy or broaden their spectrum of activity (Figure 1) (Komal et al., 2023). Additionally, CRISPR can be used to engineer Bt strains with improved stability and environmental persistence, ensuring sustained pest control (Bisht et al., 2019; Komal et al., 2023). However, challenges such as off-target effects, delivery methods, and regulatory hurdles must be carefully managed to ensure the successful application of CRISPR technology in Bt (Rao and Wang, 2021; Erdoğan et al., 2023).

Komal et al. (2023) highlights the use of genome-editing technologies such as ZFNs, TALENs, and CRISPR/Cas9 to develop insect-resistant crops, offering a novel approach to pest management. By targeting specific genes in either plants or insects, these technologies can increase plant resistance and reduce insecticide resistance in pests. For plants, gene editing can enhance their natural defense mechanisms, such as increasing salicylic acid levels or altering volatile compounds to repel pests. In insects, modifying genes responsible for detoxifying insecticides can make them more susceptible, reducing pest populations. These strategies present a promising path for sustainable agriculture by minimizing the reliance on chemical pesticides and improving crop productivity.

3 Genetic Modifications in Bt for Improved Insecticidal Properties

3.1 Target genes in Bt for enhanced toxin production

Genetic modifications in Bacillus thuringiensis (Bt) have primarily focused on enhancing the production and efficacy of its insecticidal toxins. Key target genes include those encoding ATP-binding cassette (ABC) transporters, which play a crucial role in the susceptibility of insects to Bt toxins. For instance, mutations in the ABCC2 and ABCC3 genes have been linked to high levels of resistance to Cry1Ac toxin in the diamondback moth, Plutella xylostella (Guo et al., 2019). Similarly, the ABCA2 gene in the pink bollworm, Pectinophora gossypiella, has been associated with resistance to Cry2Ab toxin (Fabrick et al., 2021). These findings underscore the importance of targeting ABC transporter genes to enhance Bt toxin production and efficacy.

3.2 Engineering Bt toxins: Cry and Vip proteins

Bt produces a variety of insecticidal proteins, including Cry and Vip proteins, which have been engineered to improve their insecticidal properties. Cry proteins, such as Cry1Ac and Cry1Fa, have been extensively studied and modified to enhance their toxicity. For example, CRISPR-mediated knockouts of the ABCC2 gene in Ostrinia furnacalis have conferred high-level resistance to Cry1Fa toxin, highlighting the potential for genetic modifications to improve toxin efficacy (Wang et al., 2020a). Additionally, Vip3Aa proteins have been engineered to increase their insecticidal activity. Mutations in domains IV and V of Vip3Aa have resulted in significantly higher toxicity against pests like Spodoptera frugiperda and Helicoverpa armigera (Yang et al., 2022). The coexistence of Cry9 and Vip3A genes in the same plasmid has also been shown to provide synergistic insecticidal toxicity, further enhancing the effectiveness of Bt strains (Wang et al., 2020b).

3.3 Strategies for enhancing Bt strains using CRISPR

CRISPR/Cas9 technology has emerged as a powerful tool for enhancing Bt strains by enabling precise genetic modifications. This technology has been used to knockout specific genes associated with resistance to Bt toxins, thereby increasing the susceptibility of pests. For example, CRISPR/Cas9-mediated knockouts of the ABCC2 and ABCC3 genes in Plutella xylostella have demonstrated the critical role of these genes in mediating resistance to Cry1Ac toxin (Guo et al., 2019). Similarly, CRISPR-mediated mutations in the ABCA2 gene in Pectinophora gossypiella have confirmed its role in resistance to Cry2Ab toxin (Fabrick et al., 2021). These studies highlight the potential of CRISPR technology to enhance Bt strains by targeting genes that confer resistance to Bt toxins.

3.4 Potential for multi-toxin gene editing

The potential for multi-toxin gene editing in Bt offers a promising strategy for improving insecticidal properties and delaying resistance development. By combining multiple toxin genes, such as Cry and Vip proteins, in a single Bt strain, it is possible to achieve synergistic effects and enhance overall toxicity. For instance, the coexistence of Cry9 and Vip3A genes in the same plasmid has been shown to provide synergistic insecticidal activity against pests like Chilo suppressalis (Wang et al., 2020b). Additionally, the use of chimeric toxins, such as Cry1AcF, which combine domains from different Cry proteins, has been explored to overcome resistance and enhance toxicity (Dutta et al., 2023). These multi-toxin strategies, facilitated by advanced gene editing techniques like CRISPR, hold great potential for developing more effective and sustainable Bt-based biopesticides.

4 Case Study: CRISPR-Based Enhancement of Bt Strains

4.1 Overview of the case study and selection criteria

This case study focuses on the application of CRISPR technology to enhance Bacillus thuringiensis (Bt) strains for improved insecticidal properties. The selection criteria for this study included the identification of key genes in insect pests that mediate resistance to Bt toxins and the subsequent use of CRISPR/Cas9 to modify these genes. The primary goal was to understand the genetic basis of resistance and to develop Bt strains with enhanced efficacy against resistant pest populations.

4.2 Applying CRISPR in the genetic modification of Bt

The methodology involved using CRISPR/Cas9 gene editing to target specific genes in insect pests that are known to confer resistance to Bt toxins. For instance, in the beet armyworm (Spodoptera exigua), CRISPR-mediated knockouts of five candidate Bt toxin receptor genes were performed to evaluate their roles in mediating toxicity of Cry1Ac, Cry1Fa, and Cry1Ca toxins. The genes targeted included SeAPN1, SeCad1, SeABCC1, SeABCC2, and SeABCC3 (Huang et al., 2020). Similarly, in the pink bollworm (Pectinophora gossypiella), CRISPR/Cas9 was used to introduce disruptive mutations in the ABCA2 gene, which was hypothesized to confer resistance to Cry2Ab toxin (Figure 2) (Fabrick et al., 2021). Additionally, in the diamondback moth (Plutella xylostella), CRISPR/Cas9 was employed to create knockout strains for the PxABCC2 and PxABCC3 genes to study their roles in resistance to Cry1Ac toxin (Guo et al., 2019).

Fabrick et al. (2021) focuses on the mutations in the PgABCA2 cDNA of Cry2Ab-resistant pink bollworm larvae that do not have mutations in the sgRNA target sites of their genomic DNA. The findings indicate that even without gDNA mutations, larvae exhibited significant cDNA changes, such as premature stop codons, deletions, and insertions affecting multiple exons. This suggests that resistance mechanisms can occur at the cDNA level, contributing to insecticide resistance. The study highlights the complexity of developing genome-editing strategies for pest management, as targeting only the gDNA might not be sufficient to counteract resistance. Understanding these cDNA-level mutations can help design more effective interventions to combat resistance in pest populations.

4.3 Improvement in insecticidal efficacy post-gene editing

The results demonstrated significant improvements in insecticidal efficacy post-gene editing. In the beet armyworm, the knockout of SeABCC2 resulted in a major increase in susceptibility to Cry1Ac and Cry1Fa toxins, indicating its crucial role in mediating toxicity (Huang et al., 2020). In the pink bollworm, the introduction of disruptive mutations in the ABCA2 gene led to the creation of a Cry2Ab-resistant strain, confirming the gene's role in resistance (Fabrick et al., 2021). For the diamondback moth, knockout strains for PxABCC2 and PxABCC3 exhibited high levels of resistance to Cry1Ac, with the double knockout strain showing over 10,320-fold resistance to Cry1Ac and 380-fold resistance to Cry1Fa, highlighting the synergistic effects of these genes in mediating resistance (Zhao et al., 2020).

4.4 Implications for agricultural pest management

The findings from this case study have significant implications for agricultural pest management. By using CRISPR/Cas9 to modify key resistance genes in insect pests, it is possible to develop Bt strains with enhanced insecticidal properties, thereby improving the efficacy of Bt crops and reducing the reliance on chemical insecticides. This approach not only helps in managing resistant pest populations but also contributes to sustainable agricultural practices by minimizing the environmental impact of pest control measures. The insights gained from these studies can guide the development of next-generation Bt crops with improved resistance management strategies, ensuring long-term efficacy and sustainability (Guo et al., 2019; Huang et al., 2020; Zhao et al., 2020; Fabrick et al., 2021).

5 Safety and Regulatory Aspects of CRISPR-Modified Bt

5.1 Biosafety concerns and risk assessment

The application of CRISPR/Cas9 technology in modifying Bacillus thuringiensis (Bt) for enhanced insecticidal properties brings forth significant biosafety concerns. One of the primary issues is the potential for off-target effects, which can lead to unintended genetic modifications that may have unforeseen ecological consequences (El-Mounadi et al., 2020; Movahedi et al., 2023). Additionally, there is a risk of horizontal gene transfer, where the modified genes could be transferred to non-target organisms, potentially disrupting local ecosystems (Movahedi et al., 2023). Strategies to mitigate these risks include the development of more precise gene-editing tools and robust detection methods to monitor and manage any unintended genetic changes (El-Mounadi et al., 2020; Movahedi et al., 2023).

5.2 Regulatory frameworks for gene-edited organisms

The regulatory landscape for CRISPR-modified organisms, including Bt, varies significantly across different countries. Some nations have established comprehensive guidelines to oversee the development and deployment of gene-edited crops, focusing on ensuring safety and efficacy (Tyagi et al., 2020; Zhang et al., 2020). For instance, the European Union has stringent regulations that classify CRISPR-modified organisms similarly to traditional GMOs, requiring extensive risk assessments and approval processes (Zhang et al., 2020). In contrast, countries like the United States have a more lenient approach, where gene-edited crops that do not contain foreign DNA may not be subject to the same rigorous regulations as GMOs (Zhang et al., 2020; Movahedi et al., 2023). These regulatory frameworks aim to balance the potential benefits of CRISPR technology with the need to protect public health and the environment.

5.3 Addressing ethical concerns related to genetic modification

The ethical implications of using CRISPR technology in Bt and other organisms are a subject of ongoing debate. Key concerns include the potential for unintended consequences, such as the creation of new pests or the disruption of existing ecosystems (Zhang et al., 2020; Erdoğan et al., 2023). There is also apprehension about the long-term impacts of releasing genetically modified organisms into the environment, particularly regarding their persistence and potential to revert to their original phenotypes (Ahmad et al., 2020). Addressing these ethical concerns requires transparent communication with the public, thorough ethical reviews, and the development of policies that ensure responsible use of CRISPR technology (Ahmad et al., 2020; Zhang et al., 2020; Erdoğan et al., 2023). Engaging with various stakeholders, including scientists, policymakers, and the public, is crucial to build trust and ensure that the benefits of CRISPR-modified Bt are realized in a socially and ethically responsible manner.

6 Challenges and Future Directions

6.1 Technical limitations of CRISPR in Bt Editing

CRISPR technology, while revolutionary, faces several technical limitations when applied to Bacillus thuringiensis (Bt) for enhancing its insecticidal properties. One significant challenge is the delivery of CRISPR components into Bt cells, which can be particularly difficult due to the bacterium's robust cell wall. Additionally, achieving high efficiency and specificity in gene editing remains a hurdle. For instance, the need for microinjection in preblastoderm embryos can be a limiting factor in certain insect species, as seen in the white-backed planthopper (Zhang et al., 2023). Moreover, the mosaicism often observed in gene-edited insects, where only a fraction of cells are edited as intended, complicates the establishment of homozygous lines (Zhu et al., 2020b).

6.2 Addressing off-target effects in gene editing

Off-target effects are a major concern in CRISPR-based gene editing, as unintended modifications can lead to undesirable traits or reduced fitness. Strategies to mitigate these effects include the use of high-fidelity Cas9 variants and thorough validation of guide RNA (gRNA) specificity. For example, the use of multiple sgRNAs targeting a single exon has been shown to improve the precision of gene knockouts in the fall armyworm (Zhu et al., 2020b). Additionally, the development of novel CRISPR systems, such as CRISPR/Cas12a, which has demonstrated high editing efficiencies and reduced off-target effects in Bombyx mori, offers promising alternatives (Dong et al., 2020).

6.3 Potential for resistance development in target insects

The rapid evolution of resistance in target insects poses a significant threat to the long-term efficacy of Bt crops. Studies have shown that mutations in specific genes, such as ABCC2 and ABCC3, can confer high levels of resistance to Bt toxins in insects like the diamondback moth and pink bollworm (Guo et al., 2019; Fabrick et al., 2021). This highlights the need for continuous monitoring and management strategies to counteract resistance. The use of CRISPR to create gene knockouts has provided valuable insights into the genetic basis of resistance, enabling the development of more effective pest management strategies (Douris et al., 2020; Huang et al., 2020).

6.4 Future prospects for Bt improvement using advanced gene editing

The future of Bt improvement lies in the integration of advanced gene editing technologies to create more robust and effective biopesticides. The potential of CRISPR/Cas9 and other genome editing tools to engineer durable resistance against insect pests has been demonstrated in various studies (Bisht et al., 2019; Tyagi et al., 2020). For instance, the use of CRISPR/Cas9 to knockout specific genes in insects has shown promise in enhancing the insecticidal properties of Bt (Guo et al., 2019; Fabrick et al., 2021). Additionally, the development of novel CRISPR systems, such as CRISPR/Cas12a, offers new opportunities for targeted genome engineering and improved pest resistance (Dong et al., 2020). As these technologies continue to evolve, they hold the potential to revolutionize agricultural pest management and ensure sustainable crop protection.

7 Concluding Remarks

The application of CRISPR-based gene editing in Bacillus thuringiensis (Bt) has shown significant promise in enhancing the insecticidal properties of Bt proteins. The development of chimeric Bt proteins such as Cry1A.2 and Cry1B.2 has expanded the spectrum of activity against key lepidopteran pests, demonstrating distinct receptor utilization and minimizing cross-resistance issues. Additionally, CRISPR/Cas9-mediated knockout studies have provided in vivo evidence of the roles of ABCC2 and ABCC3 proteins as functional receptors for Bt Cry1 toxins, offering insights into the molecular mechanisms of insect resistance. The integration of Bt genes into other bacterial strains, such as Bacillus suBtilis, has also been explored, resulting in enhanced biocontrol efficacy against both insect pests and plant pathogens. Furthermore, whole genome sequencing of various Bt strains has revealed a plethora of pesticidal protein genes, underscoring the genetic diversity and potential for biotechnological applications.

CRISPR technology has revolutionized the field of Bt insecticides by enabling precise genetic modifications that enhance the efficacy and specificity of Bt proteins. The ability to knockout specific genes, such as PxABCC2 and PxABCC3, has provided a deeper understanding of the genetic basis of resistance in target pests, facilitating the development of more effective Bt strains. This technology has also allowed for the creation of novel chimeric proteins with improved insecticidal properties, as seen with Cry1A.2 and Cry1B.2, which offer new tools for pest management. The integration of CRISPR with Bt technology not only enhances the insecticidal spectrum but also addresses the issue of resistance, thereby ensuring the long-term sustainability of Bt-based biopesticides.

The advancements in CRISPR-based gene editing of Bt have the potential to significantly impact global agricultural practices. By developing Bt strains with enhanced insecticidal properties and reduced resistance, farmers can achieve more effective pest control, leading to higher crop yields and reduced reliance on chemical pesticides. This is particularly important for major crops such as soybean and maize, where pest resistance has been a significant challenge. The use of genetically engineered Bt strains can also contribute to more sustainable agricultural practices by reducing the environmental impact of pest control measures and promoting the use of eco-friendly biopesticides. Overall, the integration of CRISPR technology with Bt insecticides holds great promise for improving pest management strategies and supporting global food security.

Acknowledgments

The author thanks the anonymous peer review for their critical comments and revising suggestion.

Conflict of Interest Disclosure

The author affirms that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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