Bioscience Methods 2024, Vol.15, No.5, 216-225 http://bioscipublisher.com/index.php/bm 216 Research Insight Open Access CRISPR-based Gene Editing inBt for Improved Insecticidal Properties Wenfei Zhang College of Life Sciences, Hainan Normal University, Haikou, 571158, Hainan, China Corresponding author: wenfei2007@163.com Bioscience Methods, 2024, Vol.15, No.5 doi: 10.5376/bm.2024.15.0022 Received: 11 Jul., 2024 Accepted: 22 Aug., 2024 Published: 13 Sep., 2024 Copyright © 2024 Zhang, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Zhang W.F., 2024, CRISPR-based gene editing in Bt for improved insecticidal properties, Bioscience Methods, 15(5): 216-225 (doi: 10.5376/bm.2024.15.0022) Abstract CRISPR-based gene editing has emerged as a powerful tool for enhancing the insecticidal properties of Bacillus thuringiensis (Bt) by targeting specific genetic components associated with resistance in insect pests. This study explores the application of CRISPR/Cas9 technology in Bt to improve its efficacy against various insect species. Key studies demonstrate the successful knockout of ATP-binding cassette (ABC) transporter genes, such as ABCC2 and ABCC3, which are crucial for mediating resistance to Bt toxins in pests like the diamondback moth and cotton bollworm. These genetic modifications have resulted in significantly increased resistance levels, providing insights into the molecular mechanisms underlying Bt toxin resistance. Additionally, this study highlights the potential of CRISPR-mediated gene deletions in Bt strains to enhance their pesticidal protein profiles, thereby broadening their spectrum of activity against multiple insect pests. The findings underscore the importance of CRISPR technology in developing next-generation biopesticides with improved insecticidal properties and reduced likelihood of resistance development. Keywords CRISPR/Cas9; Bacillus thuringiensis; Insecticidal proteins; ABC transporter genes; Pest resistance 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
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