Bacillus spp. are a group of Gram-positive bacteria that have shown significant potential for development in recent years. It is capable of utilizing low-cost substrates to produce various high-value-added compounds, making it widely applicable in fields such as feed, pharmaceuticals, and food. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system-mediated genetic modification is recognized as one of the most efficient technologies. The application of this technology for the genetic improvement of Bacillus spp. greatly enhanced the production performance of these strains. In this review, we summarize the various CRISPR-Cas systems that have been applied to Bacillus spp., with a particular focus on systematically outlining the strategies for implementing CRISPR-Cas-mediated genetic modification in these bacteria. Notably, homologous recombination is the most widely used strategy, while base editing is emerging as a novel and precise approach. Additionally, we discuss the importance of expression regulation strategies in establishing Bacillus spp. as a cell factory. Finally, we propose potential solutions to current technical challenges, providing insights for the development of high-performance genetically modified Bacillus spp. production strains.

中文翻译：

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CRISPR-Cas 系统介导的芽孢杆菌基因改造研究现状与未来

芽孢杆菌属是一组革兰氏阳性菌，近年来显示出巨大的发展潜力。它能够利用低成本的基材生产各种高附加值的化合物，使其广泛应用于饲料、制药和食品等领域。成簇的规则间隔短回文重复序列 （CRISPR）-CRISPR 相关 （Cas） 系统介导的基因修饰被认为是最有效的技术之一。该技术应用于芽孢杆菌属的遗传改良，大大提高了这些菌株的生产性能。在这篇综述中，我们总结了应用于芽孢杆菌属的各种 CRISPR-Cas 系统，特别侧重于系统地概述了在这些细菌中实施 CRISPR-Cas 介导的基因修饰的策略。值得注意的是，同源重组是使用最广泛的策略，而碱基编辑正在成为一种新颖而精确的方法。此外，我们讨论了表达调控策略在将芽孢杆菌属建立为细胞工厂中的重要性。最后，我们提出了针对当前技术挑战的潜在解决方案，为高性能转基因芽孢杆菌生产菌株的开发提供了见解。