Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 34141, South Korea.
Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology (UST), Daejeon, 34113, South Korea.
Microb Cell Fact. 2022 Dec 14;21(1):259. doi: 10.1186/s12934-022-01989-w.
Owing to CRISPR-Cas9 and derivative technologies, genetic studies on microorganisms have dramatically increased. However, the CRISPR-Cas9 system is still difficult to utilize in many wild-type Bacillus strains owing to Cas9 toxicity. Moreover, less toxic systems, such as cytosine base editors, generate unwanted off-target mutations that can interfere with the genetic studies of wild-type strains. Therefore, a convenient alternative system is required for genetic studies and genome engineering of wild-type Bacillus strains. Because wild-type Bacillus strains have poor transformation efficiencies, the new system should be based on broad-host-range plasmid-delivery systems.
Here, we developed a Bacillus integrative plasmid system in which plasmids without the replication initiator protein gene (rep) of Bacillus are replicated in a donor Bacillus strain by Rep proteins provided in trans but not in Bacillus recipients. The plasmids were transferred to recipients through a modified integrative and conjugative element, which is a wide host range plasmid-delivery system. Genetic mutations were generated in recipients through homologous recombination between the transferred plasmid and the genome. The system was improved by adding a synthetic gene circuit for efficient screening of the desired mutations by double crossover recombination in recipient strains. The improved system exhibited a mutation efficiency of the target gene of approximately 100% in the tested wild-type Bacillus strains.
The Bacillus integrative plasmid system developed in this study can generate target mutations with high efficiency when combined with a synthetic gene circuit in wild-type Bacillus strains. The system is free of toxicity and unwanted off-target mutations as it generates the desired mutations by traditional double crossover recombination. Therefore, our system could be a powerful tool for genetic studies and genome editing of Cas9-sensitive wild-type Bacillus strains.
由于 CRISPR-Cas9 和衍生技术的出现，微生物的遗传研究有了显著的增长。然而，由于 Cas9 的毒性，CRISPR-Cas9 系统在许多野生型芽孢杆菌菌株中仍然难以利用。此外，毒性较小的系统，如胞嘧啶碱基编辑器，会产生不必要的脱靶突变，从而干扰野生型菌株的遗传研究。因此，需要一种方便的替代系统来进行野生型芽孢杆菌菌株的遗传研究和基因组工程。由于野生型芽孢杆菌菌株的转化效率较低，新系统应该基于广泛宿主范围的质粒传递系统。
在这里，我们开发了一种芽孢杆菌整合质粒系统，其中没有芽孢杆菌复制起始蛋白基因 (rep) 的质粒通过提供的转座 Rep 蛋白在供体芽孢杆菌菌株中复制，但不在芽孢杆菌受体中复制。质粒通过一种改良的整合和共轭元件转移到受体中，该元件是一种广泛宿主范围的质粒传递系统。通过转移质粒和基因组之间的同源重组，在受体中产生遗传突变。通过添加一个合成基因电路，该系统得到了改进，通过双交叉重组在受体菌株中有效地筛选所需的突变。改进后的系统在测试的野生型芽孢杆菌菌株中，目标基因的突变效率约为 100%。
本研究开发的芽孢杆菌整合质粒系统在与合成基因电路结合使用时，可在野生型芽孢杆菌菌株中高效产生目标突变。该系统没有毒性和不必要的脱靶突变，因为它通过传统的双交叉重组产生所需的突变。因此，我们的系统可以成为 Cas9 敏感的野生型芽孢杆菌菌株遗传研究和基因组编辑的有力工具。
