Bt Research 2024, Vol.15, No.1, 53-64 http://microbescipublisher.com/index.php/bt 53 Research Insight Open Access CRISPR-Cas9 Technology in Bt Genome Editing and Functional Studies Xiuhua Liu, Jie Zhang Biotechnology Research Center of Cuixi Academy of Biotechology, Zhuji, 311800, Zhejiang, China Corresponding author: jie.zhang@cuixi.org Bt Research, 2024, Vol.15, No.2 doi: 10.5376/bt.2024.15.0006 Received: 08 Jan., 2024 Accepted: 20 Feb., 2024 Published: 15 Mar., 2024 Copyright © 2024 Liu and Zhang, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Liu X.H., and Zhang J., 2024, CRISPR-Cas9 technology in Bt genome editing and functional studies, Bt Research, 15(2): 53-64 (doi: 10.5376/bt.2024.15.0006) Abstract CRISPR-Cas9 technology, with its unparalleled precision and efficiency, has revolutionized the field of genome editing. In Bacillus thuringiensis (Bt), widely used as a biopesticide, genome editing holds great promise for significantly enhancing its efficacy and functional understanding. This study provides an overview of the mechanisms and advancements of CRISPR-Cas9, comparing it with other genome editing technologies. It delves into its applications in Bt, including gene knockout, knock-in strategies, multiplex genome editing, and targeted mutagenesis. Additionally, it explores the functional studies of Bt genes, covering gene function identification, overexpression, gene silencing, and toxin gene analysis. This study discusses methodological advancements and challenges such as delivery methods, off-target effects, and optimization. Case studies showcase successful gene editing examples, insights gained, and best practices. It also examines ethical and regulatory issues as well as public perception. Finally, it discusses future directions, emerging trends, novel applications, and the potential impact on industrial and agricultural biotechnology. Continuous research and development in CRISPR-Cas9 technology are crucial to fully realizing its potential in Bt genome editing. Keywords CRISPR-Cas9; Bacillus thuringiensis; Genome editing; Gene knockout; Biopesticides 1 Introduction CRISPR-Cas9 technology has revolutionized the field of genome editing since its discovery as a bacterial adaptive immune system (Wang et al., 2016). The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, coupled with the Cas9 (CRISPR-associated protein 9) endonuclease, allows for precise and targeted modifications of DNA sequences in a wide range of organisms (Hsu et al., 2014). This system operates by utilizing a guide RNA (gRNA) to direct the Cas9 protein to specific genomic loci, where it introduces double-strand breaks. These breaks can then be repaired through non-homologous end joining (NHEJ) or homology-directed repair (HDR), enabling the insertion, deletion, or modification of genetic material (Ran et al., 2013). The simplicity, efficiency, and versatility of CRISPR-Cas9 have made it an indispensable tool in genetic research, with applications spanning from basic biology to therapeutic interventions (Hossain, 2021). Bacillus thuringiensis (Bt) is a gram-positive bacterium renowned for its ability to produce insecticidal toxins, which are widely used in agriculture to control pest populations (Li et al., 2021). The genetic manipulation of Bt is crucial for enhancing its insecticidal properties, understanding its pathogenic mechanisms, and developing new biotechnological applications (Chen et al., 2017). Traditional methods of genetic modification in Bt have been labor-intensive and time-consuming. However, the advent of CRISPR-Cas9 technology has provided a powerful and efficient means to edit the Bt genome with high precision (Kirchner and Schneider, 2015). This has opened new avenues for the functional study of Bt genes, the development of novel Bt strains with improved insecticidal activity, and the exploration of Bt's potential in other biotechnological fields (Demirci et al., 2018). This study is to provide a comprehensive overview of the current state of CRISPR-Cas9 technology as it pertains to genome editing and functional studies in Bacillus thuringiensis. This review aims to summarize the advancements in CRISPR-Cas9 technology and its applications in genome editing and highlight the specific challenges and solutions associated with using CRISPR-Cas9 in Bt, discuss the potential biotechnological applications of CRISPR-Cas9-mediated genome editing in Bt and Identify future research directions and technological improvements needed to enhance the efficiency and precision of CRISPR-Cas9 in Bt. By
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