Construction of a Gene Knockdown System Based on Catalytically Inactive ("Dead") Cas9 (dCas9) in Staphylococcus aureus
Construction of a Gene Knockdown System Based on Catalytically Inactive ("Dead") Cas9 (dCas9) in Staphylococcus aureus
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基于金黄色葡萄球菌催化失活（“死”）Cas9（dCas9）的基因敲低系统的构建
DOI：
10.1128/aem.00291-17
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发表时间：
2017
影响因子：
4.4
通讯作者：
Sun Baolin
中科院分区：
文献类型：
--
作者：
Zhao Changlong;Shu Xueqin;Sun Baolin
目前还没有像真核生物中的RNA干扰那样有效的基因敲除金黄色葡萄球菌的方法。以往发展的反义RNA技术主要应用于正向遗传筛选，但由于缺乏合理的反义RNA设计策略，在特异性基因敲除方面的应用相当有限。在这里，我们报告了一个有效的和特定的系统，基因敲低在S。基于来自化脓链球菌的II型成簇规则间隔短回文重复序列（CRISPR）系统，在金黄色葡萄球菌（aureus）中表达。我们可以通过共表达dCas9（一种RNA引导的DNA结合蛋白）和与靶基因互补的小引导RNA来实现基因沉默。利用该系统，我们成功地沉默了不同S.金黄色葡萄球菌该系统对必需基因和非必需基因均表现出高效的敲除，并且其作用是可诱导的和可逆的。此外，该系统可以同时抑制多个基因的表达，并沉默整个操纵子或部分操纵子。因此，这种RNA引导的DNA靶向系统提供了一种简单，快速，经济的方法，选择性基因敲除在S。重要性金黄色葡萄球菌（Staphylococcus aureus）是一种重要的人类和动物病原体，可引起多种传染病。分子遗传学研究。金黄色葡萄球菌的研究为了解其毒力、发病机制和耐药性提供了途径，从而发现了治疗葡萄球菌感染的新疗法。然而，为S.金黄色葡萄球菌通常涉及低效率或费力的程序。在这里，我们报告了一个RNA引导的基因敲减系统在S。结果表明，该方法在不同的金黄色葡萄球菌菌株中选择性基因沉默具有高效、简便的特点。金黄色。这种简单、快速、经济的方法有望成为研究S.特别是对必需基因的研究，从而促进对该病原体及其与宿主相互作用的了解。
There has been an absence of an efficient method of gene knockdown in the important human pathogen Staphylococcus aureus like RNA interference in eukaryotes. The previously developed antisense RNA technology is mainly applied for forward genetic screening but is rather limited in specific gene knockdown because of the lack of rational antisense RNA design strategies. Here we report an efficient and specific system for gene knockdown in S. aureus based on the type II clustered regularly interspaced short palindromic repeat (CRISPR) system from Streptococcus pyogenes. We can achieve gene silencing with the coexpression of dCas9, an RNA-guided DNA binding protein, and a small guide RNA complementary to the target gene. With this system, we have successfully silenced diverse sets of genes varying in size and expression level in different S. aureus strains. This system exhibited high-efficiency knockdown of both essential and nonessential genes, and its effect is inducible and reversible. In addition, the system can repress the expression of multiple genes simultaneously and silence an entire operon or part of it. This RNA-guided DNA targeting system thus provides a simple, rapid, and affordable method for selective gene knockdown in S. aureus.IMPORTANCEStaphylococcus aureus is an important human and animal pathogen that can cause a diversity of infectious diseases. Molecular genetic study of S. aureus has provided an avenue for the understanding of its virulence, pathogenesis, and drug resistance, leading to the discovery of new therapies for the treatment of staphylococcal infections. However, methodologies developed for genetic manipulation of S. aureus usually involve either low efficiency or laborious procedures. Here we report an RNA-guided system for gene knockdown in S. aureus and show its high efficiency and simplicity for selective gene silencing in different strains of S. aureus. This simple, rapid, and affordable system may serve as a promising tool for functional gene study in S. aureus, especially for the study of essential genes, thus facilitating the understanding of this pathogen and its interaction with its hosts.