Bioscience Methods 2024, Vol.15, No.5, 216-225 http://bioscipublisher.com/index.php/bm 222 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 inBt 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
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