Bioscience Methods 2024, Vol.15, No.5, 216-225 http://bioscipublisher.com/index.php/bm 221 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-ModifiedBt 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
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