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Abstract：
Clustered regularly interspaced short palindromic repeats (CRISPR) are prokaryotic adaptive immune systems regularly utilized as DNA-editing tools. While Neisseria gonorrhoeae does not have an endogenous CRISPR, the commensal species Neisseria lactamica encodes a functional Type I-C CRISPR-Cas system. We have established an isopropyl β-d-1-thiogalactopyranoside added (IPTG)-inducible, CRISPR interference (CRISPRi) platform based on the N. lactamica Type I-C CRISPR missing the Cas3 nuclease to allow locus-specific transcriptional repression. As proof of principle, we targeted a non-phase-variable version of the opaD gene. We show that CRISPRi can downregulate opaD gene and protein expression, resulting in bacterial inability to stimulate neutrophil oxidative responses and to bind to an N-terminal fragment of CEACAM1. Importantly, we used CRISPRi to effectively knockdown all the transcripts of all 11 opa genes using a five-spacer CRISPR array, allowing control of the entire phase-variable opa family in strain FA1090. We also report that repression is reversible following IPTG removal. Finally, we showed that the Type I-C CRISPRi system can conditionally reduce the expression of two essential genes. This CRISPRi system will allow the interrogation of every Gc gene, essential and non-essential, to study physiology and pathogenesis and aid in antimicrobial development.IMPORTANCEClustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have proven instrumental in genetically manipulating many eukaryotic and prokaryotic organisms. Despite its usefulness, a CRISPR system had yet to be developed for use in Neisseria gonorrhoeae (Gc), a bacterium that is the main etiological agent of gonorrhea infection. Here, we developed a programmable and IPTG-inducible Type I-C CRISPR interference (CRISPRi) system derived from the commensal species Neisseria lactamica as a gene repression system in Gc. As opposed to generating genetic knockouts, the Type I-C CRISPRi system allows us to block transcription of specific genes without generating deletions in the DNA. We explored the properties of this system and found that a minimal spacer array is sufficient for gene repression while also facilitating efficient spacer reprogramming. Importantly, we also show that we can use CRISPRi to knockdown genes that are essential to Gc that cannot normally be knocked out under laboratory settings. Gc encodes ~800 essential genes, many of which have no predicted function. We predict that this Type I-C CRISPRi system can be used to help categorize gene functions and perhaps contribute to the development of novel therapeutics for gonorrhea.
摘要：
目的：成簇的规则间隔短回文重复序列（CRISPR）-Cas系统已被证明在遗传操纵许多真核和原核生物中具有重要作用。尽管它有用，尚未开发用于淋病奈瑟菌（Gc）的CRISPR系统，一种细菌，是淋病感染的主要病原体。这里,我们开发了一种可编程且IPTG可诱导的I-C型CRISPR干扰（CRISPRi）系统，该系统源自共生物种内酰胺酶奈瑟氏菌，作为Gc中的基因抑制系统。与产生基因敲除相反，I-C型CRISPRi系统使我们能够阻断特定基因的转录，而不会在DNA中产生缺失。我们探索了该系统的特性，发现最小的间隔区阵列足以抑制基因，同时还有助于有效的间隔区重编程。重要的是,我们还表明,我们可以使用CRISPRi敲除对Gc至关重要的基因,这些基因在实验室环境下通常无法被敲除.Gc编码约800个必需基因，其中许多没有预测功能。我们预测，这种I-C型CRISPRi系统可用于帮助对基因功能进行分类，并可能有助于开发淋病的新疗法。
