Bt Research 2024, Vol.15, No.1, 53-64 http://microbescipublisher.com/index.php/bt 59 reduced off-target effects compared to plasmid-based methods. Furthermore, optimizing the cellular environment, such as by enhancing the expression of repair proteins, can improve the efficiency of CRISPR-Cas9-mediated genome editing (Wei et al., 2020). Continuous advancements in these areas are essential for achieving high-efficiency and precise genome editing in Bt. 6 Case Studies of CRISPR-Cas9 in Bt Research 6.1 Successful gene editing examples CRISPR-Cas9 technology has been successfully applied in Bacillus thuringiensis (Bt) for gene editing, demonstrating its potential in both chromosomal and plasmid gene deletions. One notable example is the deletion of the protease genes nprA (neutral protease A) and aprA (alkaline protease A) in Bt. This was achieved by using a Lactobacillus plantarum-derived plasmid, which reduced Cas9 toxicity due to its lower copy number, thereby facilitating efficient gene editing (Soonsanga et al, 2020). Additionally, the successful editing of the plasmid vip3A gene required the use of a Bacillus-derived plasmid with longer homology sequences for the repair template, showcasing the versatility of CRISPR-Cas9 in different genetic contexts within Bt (Figure 3) (Mollashahi et al., 2023). Mollashahi et al. (2023) found that advancements in CRISPR technology have enabled precise genetic modifications through a series of innovations. Initially, CRISPR-Cas9 was utilized to create double-strand breaks in DNA, guided by synthetic single guide RNA (sgRNA) designed for specific targets. Subsequent modifications led to the development of nickase Cas9 (nCas9), which introduced single-strand cuts, allowing for targeted base editing by coupling base editor domains to the Cas9 protein. The latest iteration, prime editing, employs nCas9 to make precise cuts, with sgRNA designed to guide the reverse transcriptase enzyme to convert the sgRNA into cDNA, facilitating highly accurate genetic modifications. These advancements highlight the progressive refinement of CRISPR tools, enhancing their applicability and precision in genetic engineering. 6.2 Insights gained from functional studies Functional studies using CRISPR-Cas9 in Bt have provided significant insights into gene function and regulation. For instance, the modulation of Cas9 levels was found to be crucial for efficient gene editing. By replacing the Cas9 promoter with a sporulation-specific promoter, researchers were able to obtain a Bt ΔnprA clone, although reproducibility was an issue with this construct. These studies highlight the importance of promoter selection and plasmid copy number in optimizing CRISPR-Cas9-mediated gene editing in Bt. Furthermore, the differential impact of plasmid copy number and homology arm length on gene editing efficiency underscores the need for tailored approaches depending on the target gene and desired outcome (Li et al., 2018). 6.3 Lessons learned and best practices Several lessons have been learned from the application of CRISPR-Cas9 in Bt research, leading to the development of best practices for future studies. One key lesson is the importance of selecting appropriate plasmid vectors to minimize Cas9 toxicity and enhance editing efficiency. Using plasmids with lower copy numbers, such as those derived fromLactobacillus plantarum, can significantly reduce Cas9-induced lethality and improve the chances of obtaining desired mutants. Additionally, the length of homology arms in the repair template plays a critical role in the success of plasmid gene editing, with longer sequences being more effective. Best practices also include careful consideration of promoter selection to control Cas9 expression levels, as this can impact the efficiency and reproducibility of gene editing. Employing sporulation-specific promoters or other context-specific regulatory elements can help fine-tune Cas9 activity to achieve optimal results. Finally, the use of multiple screening steps to identify successful gene edits is recommended to ensure the accuracy and reliability of the edited strains. By adhering to these best practices and leveraging the insights gained from previous studies, researchers can continue to advance the application of CRISPR-Cas9 technology in Bt, paving the way for new discoveries and innovations in genetic research and biotechnology (Zhou et al., 2024).
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