Bioscience Methods 2024, Vol.15, No.5, 216-225 http://bioscipublisher.com/index.php/bm 217 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 toBt 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.
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