Abstract：
While genome-wide transposon mutagenesis screens have identified numerous essential genes in the significant human pathogen Streptococcus pyogenes (group A Streptococcus or GAS), many of their functions remain elusive. This knowledge gap is attributed in part to the limited molecular toolbox for controlling GAS gene expression and the bacterium\'s poor genetic transformability. CRISPR interference (CRISPRi), using catalytically inactive GAS Cas9 (dCas9), is a powerful approach to specifically repress gene expression in both bacteria and eukaryotes, but ironically, it has never been harnessed for controlled gene expression in GAS. In this study, we present a highly transformable and fully virulent serotype M1T1 GAS strain and introduce a doxycycline-inducible CRISPRi system for efficient repression of bacterial gene expression. We demonstrate highly efficient, oligo-based single guide RNA cloning directly to GAS, enabling the construction of a gene knockdown strain in just 2 days, in contrast to the several weeks typically required. The system is shown to be titratable and functional both in vitro and in vivo using a murine model of GAS infection. Furthermore, we provide direct in vivo evidence that the expression of the conserved cell division gene ftsZ is essential for GAS virulence, highlighting its promise as a target for emerging FtsZ inhibitors. Finally, we introduce SpyBrowse (https://veeninglab.com/SpyBrowse), a comprehensive and user-friendly online resource for visually inspecting and exploring GAS genetic features. The tools and methodologies described in this work are poised to facilitate fundamental research in GAS, contribute to vaccine development, and aid in the discovery of antibiotic targets.
OBJECTIVE: While group A Streptococcus (GAS) remains a predominant cause of bacterial infections worldwide, there are limited genetic tools available to study its basic cell biology. Here, we bridge this gap by creating a highly transformable, fully virulent M1T1 GAS strain. In addition, we established a tight and titratable doxycycline-inducible system and developed CRISPR interference (CRISPRi) for controlled gene expression in GAS. We show that CRISPRi is functional in vivo in a mouse infection model. Additionally, we present SpyBrowse, an intuitive and accessible genome browser (https://veeninglab.com/SpyBrowse). Overall, this work overcomes significant technical challenges of working with GAS and, together with SpyBrowse, represents a valuable resource for researchers in the GAS field.
OBJECTIVE: While group A Streptococcus (GAS) remains a predominant cause of bacterial infections worldwide, there are limited genetic tools available to study its basic cell biology. Here, we bridge this gap by creating a highly transformable, fully virulent M1T1 GAS strain. In addition, we established a tight and titratable doxycycline-inducible system and developed CRISPR interference (CRISPRi) for controlled gene expression in GAS. We show that CRISPRi is functional in vivo in a mouse infection model. Additionally, we present SpyBrowse, an intuitive and accessible genome browser (https://veeninglab.com/SpyBrowse). Overall, this work overcomes significant technical challenges of working with GAS and, together with SpyBrowse, represents a valuable resource for researchers in the GAS field.
摘要：
虽然全基因组转座子诱变筛选已经在重要的人类病原体化脓性链球菌（A组链球菌或GAS）中鉴定出许多必需基因，他们的许多功能仍然难以捉摸。这种知识差距部分归因于用于控制GAS基因表达的有限分子工具箱和细菌的低遗传转化性。CRISPR干扰（CRISPRi），使用无催化活性的GASCas9（DCas9），是一种在细菌和真核生物中特异性抑制基因表达的强大方法，但讽刺的是,它从未被用于控制GAS中的基因表达。在这项研究中,我们提出了一种高度可转化和完全毒性的血清型M1T1GAS菌株，并引入了多西环素诱导的CRISPRi系统，以有效抑制细菌基因表达。我们证明了高效，基于寡核苷酸的单向导RNA直接克隆到GAS，能够在短短2天内构建基因敲除菌株，与通常需要的几个星期相反。使用GAS感染的鼠模型，该系统在体外和体内均可滴定和起作用。此外，我们提供了直接的体内证据，即保守的细胞分裂基因ftsZ的表达对GAS毒力至关重要，强调其作为新兴FtsZ抑制剂目标的承诺。最后,我们介绍SpyBrowse（https://veeninglab.com/SpyBrowse），一个全面和用户友好的在线资源，用于视觉检查和探索GAS遗传特征。这项工作中描述的工具和方法准备好促进GAS的基础研究，有助于疫苗开发，并帮助发现抗生素靶标。
目标：虽然A组链球菌（GAS）仍然是全球细菌感染的主要原因，可用于研究其基本细胞生物学的遗传工具有限。这里,我们通过创造一个高度可变形的，全毒力M1T1GAS菌株。此外,我们建立了一个紧密且可滴定的强力霉素诱导系统,并开发了CRISPR干扰(CRISPRi)用于GAS中受控基因表达.我们证明CRISPRi在小鼠感染模型中具有体内功能。此外,我们介绍SpyBrowse，直观和可访问的基因组浏览器（https://veeninglab.com/SpyBrowse）。总的来说,这项工作克服了与GAS合作的重大技术挑战，与SpyBrowse一起，代表了GAS领域研究人员的宝贵资源。
目标：虽然A组链球菌（GAS）仍然是全球细菌感染的主要原因，可用于研究其基本细胞生物学的遗传工具有限。这里,我们通过创造一个高度可变形的，全毒力M1T1GAS菌株。此外,我们建立了一个紧密且可滴定的强力霉素诱导系统,并开发了CRISPR干扰(CRISPRi)用于GAS中受控基因表达.我们证明CRISPRi在小鼠感染模型中具有体内功能。此外,我们介绍SpyBrowse，直观和可访问的基因组浏览器（https://veeninglab.com/SpyBrowse）。总的来说,这项工作克服了与GAS合作的重大技术挑战，与SpyBrowse一起，代表了GAS领域研究人员的宝贵资源。
