背景:由于CRISPR-Cas9和衍生技术,对微生物的遗传研究急剧增加。然而,由于Cas9的毒性,CRISPR-Cas9系统仍然难以在许多野生型芽孢杆菌菌株中利用。此外,毒性较低的系统,如胞嘧啶碱基编辑器,产生不需要的脱靶突变,可能干扰野生型菌株的遗传研究。因此,野生型芽孢杆菌菌株的遗传研究和基因组工程需要一个方便的替代系统。由于野生型芽孢杆菌菌株的转化效率较差,新系统应基于广泛宿主范围的质粒递送系统。
结果: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)的质粒通过反式提供的Rep蛋白在供体芽孢杆菌菌株中复制,而不是在芽孢杆菌受体中复制。通过修饰的整合和接合元件将质粒转移到受体,这是一个宽宿主范围的质粒递送系统。通过转移的质粒和基因组之间的同源重组在受体中产生遗传突变。通过添加合成基因电路来改进该系统,以通过受体菌株中的双交叉重组来有效筛选所需的突变。改进的系统在测试的野生型芽孢杆菌属菌株中显示出大约100%的靶基因突变效率。
结论:本研究开发的芽孢杆菌整合质粒系统在野生型芽孢杆菌菌株中与合成基因回路结合时,可以高效地产生目标突变。该系统没有毒性和不需要的脱靶突变,因为它通过传统的双交叉重组产生所需的突变。因此,我们的系统可能成为Cas9敏感野生型芽孢杆菌菌株的遗传研究和基因组编辑的有力工具.
BACKGROUND: 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.
RESULTS: 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.
CONCLUSIONS: 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.