Bt Research 2024, Vol.15, No.1, 53-64 http://microbescipublisher.com/index.php/bt 58 4.2 Overexpression and gene silencing Beyond gene knockouts, CRISPR-Cas9 can also be used for gene overexpression and silencing, providing a comprehensive toolkit for functional studies (Zhou et al., 2024). The CHOPCHOP web tool, for example, supports CRISPR activation and repression, allowing researchers to modulate gene expression levels in Bt and other organisms. This capability is crucial for studying gene function in a more dynamic context, as it enables the investigation of both loss-of-function and gain-of-function phenotypes. Moreover, the CRISPR-Cas9 system has been adapted to target RNA, expanding its applications to include the regulation of endogenous gene expression and the study of alternative transcript isoforms (Labun et al., 2019). 4.3 Functional analysis of toxin genes Bt is well-known for its production of insecticidal toxins, and CRISPR-Cas9 technology has been instrumental in dissecting the functions of these toxin genes. By introducing targeted mutations, researchers can study the effects of specific gene alterations on toxin production and activity. For example, CRISPR-Cas9 has been used to investigate the functional impact of gene modifications in immune cells, which can be analogous to studying toxin gene function in Bt. Additionally, the system's ability to induce large gene modifications, such as deletions and insertions, allows for a detailed analysis of the genetic elements involved in toxin gene regulation and expression (Hulton et al., 2020). In summary, CRISPR-Cas9 technology provides a versatile and powerful approach for functional studies of Bt genes, enabling precise gene knockouts, overexpression, and silencing, as well as detailed analyses of toxin gene functions. This technology continues to advance our understanding of Bt biology and its applications in biotechnology and agriculture. 5 Methodological Advances and Challenges 5.1 Delivery methods for CRISPR-Cas9 in Bt The delivery of CRISPR-Cas9 components into Bacillus thuringiensis (Bt) cells is a critical step for successful genome editing. Various strategies have been developed to enhance the efficiency and specificity of CRISPR-Cas9 delivery. One promising approach is the use of ribonucleoprotein (RNP) complexes, which consist of Cas9 protein and single guide RNA (sgRNA). This method offers the advantage of transient genome editing and reduced off-target effects (Zhang et al., 2021). Additionally, both viral and non-viral delivery systems have been explored. Viral vectors, such as adenoviruses and lentiviruses, are effective but come with limitations like immunogenicity and limited packaging capacity. Non-viral methods, including lipid- or polymer-based nanocarriers, have shown potential due to their lower risk of carcinogenesis and immune response. Despite these advancements, challenges remain in achieving efficient and targeted delivery in vivo, necessitating further research and development. 5.2 Off-target effects and specificity Off-target effects are a significant concern in CRISPR-Cas9 genome editing, as unintended modifications can lead to adverse outcomes. Various strategies have been developed to minimize these effects. High-fidelity Cas9 variants and paired nickases have been engineered to enhance specificity (Manghwar et al., 2020). Computational tools and experimental methods have been employed to predict and validate off-target sites, thereby improving the precision of genome editing (Guo et al., 2023). Additionally, the use of truncated sgRNAs and partial DNA replacement in guide RNAs has been shown to reduce off-target activity while maintaining on-target efficiency8 10. Despite these advancements, off-target effects remain a critical hurdle, and ongoing research aims to develop more robust methods to mitigate these risks (Gupta et al., 2019). 5.3 Efficiency and optimization The efficiency of CRISPR-Cas9 genome editing in Bt can be influenced by several factors, including the design of sgRNAs, the delivery method, and the cellular environment. Optimizing sgRNA design is crucial for enhancing target specificity and reducing off-target effects. Tools for sgRNA design and evaluation have been developed to assist researchers in selecting the most effective guides1. Additionally, the delivery method plays a significant role in the overall efficiency of genome editing. RNP delivery has been highlighted for its high efficiency and
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