Non-canonical functions of Cas9 andthe cell biology of CRISPR-Cas immunity

Cas9的非典型功能和CRISPR-Cas免疫的细胞生物学

• 批准号: 10458754
• 负责人: Joshua Modell
• 金额: $ 40.94万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10458754
• 关键词: Antibiotic Resistance; Autoimmunity; Bacteria; Bacterial Chromosomes; Bacterial Genes; Bacteriophages; Biology; CRISPR/Cas technology; Cells; Cellular biology; Clustered Regularly Interspaced Short Palindromic Repeats; Development; Environment; Equilibrium; Event; Foundations; Functional disorder; Generations; Genes; Genetic; Genetic Diseases; Genetic Transcription; Guide RNA; Heterogeneity; Horizontal Gene Transfer; Immunity; Immunization; Infection; Laboratory Study; Memory; Physiology; Plasmids; Population; Regulation; Repression; Research Design; Role; Route; Streptococcus pyogenes; Stress; System; Technology; Time; Toxin; Transcription Repressor; adaptive immunity; commensal bacteria; drug resistant pathogen; genetic element; human pathogen; imaging platform; innovation; insight; novel; nuclease; pathogenic bacteria; prevent; promoter; resistance gene; spatiotemporal; tool

SUMMARY CRISPR-Cas loci provide bacteria with adaptive immunity against phages and plasmids. By remembering and destroying foreign genetic elements, CRISPR-Cas systems also restrict horizontal gene transfer, a frequent route for the dissemination of antibiotic resistance genes and toxins. To balance defense with genetic exchange, CRISPR-Cas systems likely undergo spatiotemporal regulation; however, little is known about the ways in which CRISPR-Cas loci interact with their bacterial hosts or changing environments. My laboratory studies type II CRISPR-Cas systems, which encode the gene-editing tool cas9. CRISPR-Cas9 systems are frequently found in human pathogens like S. pyogenes, and we are focused on understanding how CRISPR-Cas9 activities are intertwined with and often defined by the biology of their bacterial host cell. We recently discovered that S. pyogenes Cas9 performs a novel autoregulatory function. A non-canonical guide RNA repurposes Cas9 from a nuclease into a transcriptional repressor that silences its own promoter. This finding helps explain how CRISPR-Cas9 systems prevent autoimmunity against the bacterial chromosome, but it remains unclear how CRISPR-Cas expression can be induced as needed, for instance during or preceding a phage infection. In this proposal, we explore two new directions in CRISPR-Cas biology. First, we investigate the non-canonical regulatory roles of Cas9. Specifically, we characterize the conditions and mechanisms that allow Cas9 to transiently relieve its repression and induce CRISPR-Cas expression, and we ask whether Cas9 evolved to regulate other bacterial genes outside the CRISPR-Cas locus. These studies will establish a new foundation for understanding the role of Cas9 in the physiology of commensal and pathogenic bacteria. Furthermore, our studies on non-canonical guide RNAs will lead to new strategies for the development of controllable Cas9 technologies and therapies. In our second project, we establish an innovative imaging platform to observe live CRISPR-Cas immunization events for the first time. This project will illuminate a fundamental unanswered question in CRISPR-Cas biology: why are new memories successfully formed in only one in a million infected cells? The answers will provide new clues into the ways in which heterogeneity within bacterial populations enables survival. Furthermore, these insights and tools will be valuable for the development of phage therapies, which are offering promise in combatting the growing threat of antibiotic-resistant pathogens.

总结 CRISPR-Cas基因座为细菌提供针对质粒和质粒的适应性免疫。通过 CRISPR-Cas系统还可以记住和破坏外来遗传元件， 水平基因转移是抗生素耐药基因传播的常见途径 和毒素。为了平衡防御与基因交换，CRISPR-Cas系统可能会经历 时空调控;然而，关于CRISPR-Cas基因座 与细菌宿主相互作用或改变环境。 我的实验室研究II型CRISPR-Cas系统，该系统编码基因编辑工具cas9。 CRISPR-Cas9系统经常在人类病原体中发现，如S.化脓菌，我们是 专注于了解CRISPR-Cas9活动是如何与CRISPR-Cas9活动交织在一起的， 由细菌宿主细胞的生物学决定。我们最近发现S.链球菌Cas9 执行新的自动调节功能。一种非经典的指导RNA从一种非经典的指导RNA中重新利用Cas9。 将核酸酶转化为使其自身启动子沉默的转录阻遏物。这一发现有助于 解释CRISPR-Cas9系统如何防止针对细菌染色体的自身免疫， 但目前还不清楚CRISPR-Cas表达如何根据需要被诱导， 在噬菌体感染期间或之前。 在这项提案中，我们探索了CRISPR-Cas生物学的两个新方向。首先，我们调查 Cas9的非经典调节作用。具体来说，我们描述了条件和 允许Cas9暂时解除其抑制并诱导CRISPR-Cas的机制 表达，我们问Cas9是否进化到调节其他细菌基因以外的基因。 CRISPR-Cas基因座。这些研究将为理解 Cas9在肠道和病原菌生理学中的作用。此外，我们的研究 非典型的指导RNA将导致开发可控Cas9的新策略 技术和疗法。在我们的第二个项目中，我们建立了一个创新的成像平台 首次观察到活的CRISPR-Cas免疫事件。该项目将照亮一个 CRISPR-Cas生物学中一个基本的未解答的问题：为什么新记忆能成功地 百万分之一的感染细胞中形成的答案将提供新的线索， 其中细菌群体内的异质性使其能够存活。此外，这些见解 和工具将是有价值的噬菌体疗法的发展，这是提供承诺， 对抗日益增长的抗药性病原体的威胁。