Bioscience Methods 2024, Vol.15, No.5, 216-225 http://bioscipublisher.com/index.php/bm 219 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 andPxABCC3 genes to study their roles in resistance to Cry1Ac toxin (Guo et al., 2019).
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