Bt Research 2024, Vol.15, No.1, 53-64 http://microbescipublisher.com/index.php/bt 55 (Tavakoli et al., 2021). For instance, in mice, CRISPR-Cas9 has been employed to rapidly generate both knockout and knock-in models by microinjecting Cas9, sgRNA, and donor DNA into zygotes (Hall et al., 2018). Additionally, the system has been optimized to enhance targeting efficiency and specificity, which is crucial for successful genome editing experiments (Campenhout et al., 2019). 3.2 Multiplex genome editing One of the significant advancements of CRISPR-Cas9 technology is its ability to perform multiplex genome editing, where multiple genes can be targeted simultaneously. This is particularly useful for studying complex genetic interactions and pathways. For example, in Dictyostelium, the CRISPR-Cas9 system has been used to generate mutants in five different PI3Kgenes simultaneously, achieving high mutagenesis frequencies. The use of an all-in-one vector containing Cas9 and multiple sgRNAs facilitates this process (Figure 1), allowing for efficient and transient expression without integrating drug resistance cassettes into the genome (Sekine et al., 2018). This multiplex capability has also been demonstrated in primary cell cultures, where CRISPR-Cas9 ribonucleoprotein complexes have been used to achieve high-efficiency gene knockouts and targeted deletions (Hoellerbauer et al., 2020). Sekine et al. (2018) found that using an endogenous tRNA system to express sgRNAs can lead to efficient sgRNA expression, as demonstrated by RT-PCR analysis. The study also predicted the secondary structure of tRNA-sgRNA and identified the tRNA cleavage site necessary for releasing sgRNA. Sequence analysis of the tRNA-sgRNA junction through cRT-PCR revealed consistent sequences across multiple clones, with specific recognition sites for inserting oligonucleotides. Additionally, the sub-nuclear localization of dCas9-GFP was examined under different concentrations of hygromycin B, showing varying fluorescence distributions. This indicates the uniformity and efficiency of dCas9-GFP expression when maintained with specific antibiotic concentrations. The findings suggest that tRNA systems can be effectively utilized for sgRNA expression and dCas9 targeting within the nucleus. 3.3 Targeted mutagenesis CRISPR-Cas9-mediated targeted mutagenesis offers a powerful tool for functional genomics and crop improvement. This technology allows for precise modifications at specific genomic sites, enabling the study of gene function and the development of crops with enhanced traits. For instance, targeted mutagenesis has been employed to improve crop yields under biotic and abiotic stresses by creating gene knockouts and modifications. The high efficiency and accuracy of CRISPR-Cas9 make it a valuable tool for generating targeted mutations, which can lead to the development of crops with greater resilience to environmental stressors (Abdelrahman et al., 2018). Additionally, the system's ability to perform high-throughput gene screening and live-cell labeling further expands its applications in functional genomics (Gupta et al., 2019). 4 Functional Studies of Bt Genes 4.1 Identifying gene function through knockouts CRISPR-Cas9 technology has revolutionized the ability to perform gene knockouts, allowing researchers to precisely disrupt specific genes to study their functions. This method has been widely used in various organisms, including Bt (Bacillus thuringiensis), to understand the roles of individual genes. For instance, the CRISPR-Cas9 system has been employed to generate gene knockouts in primary T cells, achieving near-complete loss of target gene expression without the need for selection, thus simplifying the process of gene function discovery (Figure 2) (Seki and Rutz, 2018). Additionally, the use of CRISPR-Cas9 in patient-derived xenografts (PDXs) has enabled the analysis of genetic dependencies by targeted gene disruption, demonstrating the system's utility in functional genomics (Hulton et al., 2020). Seki and Rutz (2018) found that using an endogenous tRNA system for expressing sgRNAs significantly enhances sgRNA production, as shown by RT-PCR results. They also predicted the secondary structure of the tRNA-sgRNA complex and identified a crucial tRNA cleavage site required for sgRNA release. Sequence analysis confirmed the accuracy of the tRNA-sgRNA junction, indicating consistent integration across multiple
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