Department of Veterinary Medicine, University of Cambridge, Cambridge, UK.
Department of Medicine, MRC-Laboratory of Molecular Biology, Molecular Immunity Unit, University of Cambridge, Cambridge, UK.
BMC Microbiol. 2022 Oct 5;22(1):238. doi: 10.1186/s12866-022-02645-4.
Campylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuni responsible for 80% of these infections. There is an urgent need to understand fundamental C. jejuni biology for the development of new strategies to prevent and treat infections. The range of molecular tools available to regulate gene expression in C. jejuni is limited, which in turn constrains our ability to interrogate the function of essential and conditionally essential genes. We have addressed this by developing and utilising a CRISPR-based interference system known as CRISPRi in C. jejuni to control gene expression. To achieve this, a catalytically inactive ("dead") cas9 and sgRNA backbone from the Streptococcus pyogenes CRISPRi system was combined with C. jejuni-derived promoters of predetermined expression activities to develop a CRISPRi-based repression tool in C. jejuni strains M1Cam and 81-176.
The CRISPRi tool was validated through successful repression of the arylsulphatase-encoding gene astA using a range of sgRNA target sequences spanning the astA gene. The tool was also applied to target astA in an M1Cam CRISPR-Cas9 deletion strain, which showed that the presence of an endogenous CRISPR-Cas9 system did not affect the activity of the CRISPRi-based repression tool. The tool was further validated against the hippicurase-encoding gene hipO. Following this, the flagella genes flgR, flaA, flaB and both flaA and flaB were targeted for CRISPRi-based repression, which resulted in varying levels of motility reduction and flagella phenotypes as determined by phenotypical assays and transmission electron microscopy (TEM).
This is the first report of a CRISPRi-based tool in C. jejuni, which will provide a valuable resource to the Campylobacter community.
弯曲菌属是全球导致细菌性食源性疾病的主要原因，其中空肠弯曲菌导致了 80%的此类感染。为了开发预防和治疗感染的新策略，迫切需要了解空肠弯曲菌的基本生物学。目前可用于调节空肠弯曲菌基因表达的分子工具的范围有限，这反过来又限制了我们研究必需和条件必需基因功能的能力。为了解决这个问题，我们开发并利用了一种基于 CRISPR 的干扰系统，称为 CRISPRi，来控制空肠弯曲菌中的基因表达。为了实现这一目标，我们将来自化脓性链球菌 CRISPRi 系统的无催化活性（“失活”）cas9 和 sgRNA 骨架与空肠弯曲菌来源的预定表达活性启动子相结合，在 M1Cam 和 81-176 空肠弯曲菌菌株中开发了一种基于 CRISPRi 的抑制工具。
通过使用跨越 astA 基因的一系列 sgRNA 靶序列成功抑制芳基硫酸酯酶编码基因 astA，验证了 CRISPRi 工具。该工具还应用于 M1Cam CRISPR-Cas9 缺失菌株中 astA 的靶向，表明内源性 CRISPR-Cas9 系统的存在不会影响基于 CRISPRi 的抑制工具的活性。该工具进一步针对 hippuricase 编码基因 hipO 进行了验证。在此之后，flgR、flaA、flaB 以及 flaA 和 flaB 这两个 flagella 基因都被靶向用于基于 CRISPRi 的抑制，这导致了不同程度的运动能力降低和通过表型测定和透射电子显微镜（TEM）确定的鞭毛表型。
这是第一个在空肠弯曲菌中报道的基于 CRISPRi 的工具，它将为弯曲菌属社区提供一个有价值的资源。
