Bt Research 2024, Vol.15, No.1, 53-64 http://microbescipublisher.com/index.php/bt 54 synthesizing the latest research findings, this study seeks to underscore the transformative impact of CRISPR-Cas9 on Bt genome editing and its broader implications for biotechnology and agriculture. 2 Overview of CRISPR-Cas9 Technology 2.1 Mechanism of CRISPR-Cas9 The CRISPR-Cas9 system, derived from the adaptive immune system of bacteria, has revolutionized genome editing by providing a precise and efficient method for modifying DNA sequences. The core components of this system include the Cas9 protein, an RNA-guided DNA endonuclease, and a guide RNA (gRNA) that directs Cas9 to specific genomic loci. The gRNA contains a 20-nucleotide sequence complementary to the target DNA, enabling Cas9 to introduce double-strand breaks (DSBs) at the desired location (Ran et al., 2013). Upon binding to the target DNA, the Cas9 protein induces DSBs, which can be repaired by the cell's endogenous repair mechanisms, primarily non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ often results in insertions or deletions (indels) that can disrupt gene function, while HDR can be used to introduce precise genetic modifications using a donor DNA template (Zhang et al., 2014). To enhance specificity and reduce off-target effects, a double-nicking strategy using a Cas9 nickase mutant with paired gRNAs has been developed (Gupta et al., 2019). 2.2 Advances in CRISPR-Cas9 applications CRISPR-Cas9 technology has seen rapid advancements and diverse applications across various fields. In biomedical research, it has been employed for gene knockouts, regulation of gene expression, and creation of disease models (Wang et al., 2016). The versatility of CRISPR-Cas9 extends to its use in live-cell imaging and epigenetic modifications, where a nuclease-deactivated form of Cas9 (dCas9) is used to target specific genomic regions without inducing DSBs (Sander and Joung, 2014). In agriculture, CRISPR-Cas9 has been instrumental in crop improvement by enabling precise genetic modifications to enhance traits such as disease resistance, drought tolerance, and nutritional content (Bortesi et al. 2015). The development of CRISPR ribonucleoproteins (RNPs) has further improved the efficiency and specificity of genome editing in plants, addressing some limitations associated with plasmid-based systems8. 2.3 Comparison with other genome editing techniques CRISPR-Cas9 stands out among genome editing technologies due to its simplicity, efficiency, and versatility. Compared to earlier methods such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR-Cas9 is easier to design and implement, as it requires only the synthesis of a gRNA complementary to the target sequence (Arora and Narula, 2017). This ease of use has democratized genome editing, making it accessible to a broader range of researchers. While ZFNs and TALENs also induce DSBs at specific genomic loci, they rely on protein-DNA interactions, which are more complex to engineer and less flexible than the RNA-DNA interactions used by CRISPR-Cas9. Additionally, CRISPR-Cas9 can be multiplexed to target multiple genes simultaneously, a feature that is more challenging to achieve with ZFNs and TALENs. Despite its advantages, CRISPR-Cas9 is not without challenges. Off-target effects, where unintended genomic sites are edited, remain a concern. However, ongoing improvements in gRNA design, delivery methods, and the development of high-fidelity Cas9 variants are addressing these issues, enhancing the precision and reliability of CRISPR-Cas9-mediated genome editing (Wang et al., 2017). In summary, CRISPR-Cas9 technology has transformed the landscape of genome editing, offering unprecedented precision and flexibility. Its continued development and application hold great promise for advancing our understanding of genetics and improving human health and agriculture. 3 Applications of CRISPR-Cas9 in Bt Genome Editing 3.1 Gene knockout and knock-in strategies CRISPR-Cas9 technology has revolutionized the field of genome editing by enabling precise modifications such as gene knockouts and knock-ins. This system utilizes small guide RNAs (sgRNAs) to direct the Cas9 nuclease to specific genomic loci, where it creates double-strand breaks. These breaks can be repaired in an error-prone manner to generate knockouts or through homologous recombination with donor DNA to create knock-ins
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