A Simplified Method for CRISPR-Cas9 Engineering of Bacillus subtilis.
A Simplified Method for CRISPR-Cas9 Engineering of Bacillus subtilis.
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一种简化的枯草芽孢杆菌CRISPR-Cas9工程化方法
DOI：
10.1128/spectrum.00754-21
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发表时间：
2021-10-31
影响因子：
3.7
通讯作者：
Helmann JD
中科院分区：
文献类型：
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作者：
Sachla AJ;Alfonso AJ;Helmann JD
来自化脓性链球菌的成簇规则间隔短回文重复序列（CRISPR）-Cas9系统已被广泛用作细菌菌株构建的工具。传统的CRISPR-Cas9编辑策略需要设计和分子克隆适当的指导RNA（gRNA）以靶向基因组切割和修复模板以引入所需的位点特异性基因组修饰。在这里，我们提出了一种简化的方法，利用现有的近4,000株枯草芽孢杆菌菌株（BKE收集），其中单个基因被整合的红霉素（EMT）抗性盒取代。具有靶向ESTs的gRNA的单个质粒（pAJS 23）允许在任何非必需基因和许多必需基因附近的位点切割基因组。该质粒可以被工程化以包括修复模板，或者修复模板可以作为PCR产物或基因组DNA与质粒共转化。我们证明了该系统的效用产生基因置换，位点特异性突变，基因间区域的修饰，并引入基因报告融合。总之，这种策略绕过了对gRNA设计的需要，并且允许突变和遗传构建的容易转移，而不需要中间克隆步骤。重要性枯草芽孢杆菌是一种特征良好的革兰氏阳性模式生物，也是生物技术的流行平台。尽管已经为B开发了许多不同的基于CRISPR的基因组编辑策略。对于枯草芽孢杆菌，它们通常涉及针对每个应用的特定指导RNA（gRNA）和修复模板的设计和克隆。通过用抗BKE gRNA靶向耐药基因盒，基因组编辑可以针对现有BKE菌株集合中的近4，000种基因破坏物中的任何一种。修复模板可以被工程化为PCR产物，或者特定的等位基因和构建体可以被转化为染色体DNA，从而绕过质粒构建的需要。所描述的方法是快速的，并有利于广泛的基因组操作。
The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system from Streptococcus pyogenes has been widely deployed as a tool for bacterial strain construction. Conventional CRISPR-Cas9 editing strategies require design and molecular cloning of an appropriate guide RNA (gRNA) to target genome cleavage and a repair template for introduction of the desired site-specific genome modification. Here, we present a streamlined method that leverages the existing collection of nearly 4,000 Bacillus subtilis strains (the BKE collection) with individual genes replaced by an integrated erythromycin (erm) resistance cassette. A single plasmid (pAJS23) with a gRNA targeted to erm allows cleavage of the genome at any nonessential gene and at sites nearby to many essential genes. This plasmid can be engineered to include a repair template, or the repair template can be cotransformed with the plasmid as either a PCR product or genomic DNA. We demonstrate the utility of this system for generating gene replacements, site-specific mutations, modification of intergenic regions, and introduction of gene-reporter fusions. In sum, this strategy bypasses the need for gRNA design and allows the facile transfer of mutations and genetic constructions with no requirement for intermediate cloning steps. IMPORTANCE Bacillus subtilis is a well-characterized Gram-positive model organism and a popular platform for biotechnology. Although many different CRISPR-based genome editing strategies have been developed for B. subtilis, they generally involve the design and cloning of a specific guide RNA (gRNA) and repair template for each application. By targeting the erm resistance cassette with an anti-erm gRNA, genome editing can be directed to any of nearly 4,000 gene disruptants within the existing BKE collection of strains. Repair templates can be engineered as PCR products, or specific alleles and constructions can be transformed as chromosomal DNA, thereby bypassing the need for plasmid construction. The described method is rapid and facilitates a wide range of genome manipulations.