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

摘要 CRISPR-CAS基因座为细菌提供针对噬菌体和质粒的获得性免疫。通过 CRISPR-Cas系统记忆和破坏外来遗传元素也限制了 水平基因转移--抗生素耐药基因传播的常见途径 和毒素。为了平衡防御和基因交换，CRISPR-CAS系统可能会经历 时空调节；然而，对CRISPR-CAS基因座的方式知之甚少 与细菌宿主或不断变化的环境相互作用。 我的实验室研究的是第二类CRISPR-CAS系统，它编码了基因编辑工具Cas9。 CRISPR-Cas9系统经常在化脓性链球菌等人类病原体中发现，而我们 重点了解CRISPR-CAS9活动如何与CRISPR-CAS9活动交织在一起并经常被定义 通过它们的细菌宿主细胞的生物学特性。我们最近发现化脓性链球菌Cas9 执行新的自动调节功能。非规范指南RNA将Cas9从 核酸酶转化为转录抑制因子，使自身的启动子沉默。这一发现很有帮助 解释CRISPR-Cas9系统如何防止针对细菌染色体的自身免疫， 但目前尚不清楚如何根据需要诱导CRISPR-Cas的表达 在噬菌体感染期间或之前。 在这个提案中，我们探索了CRISPR-CAS生物学的两个新方向。首先，我们调查 Cas9的非规范调节作用。具体地说，我们描述了条件和 允许Cas9瞬时解除其抑制并诱导CRISPR-CA的机制 表达，我们想知道Cas9是否进化来调控其他细菌基因 CRISPR-CAS基因座。这些研究将建立一个新的基础，以了解 在共生菌和病原菌的生理学方面。此外，我们对 非规范引导RNA将为可控CAS9的发展带来新的战略 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 首次现场观察CRISPR-Cas免疫事件。这个项目将照亮一个 CRISPR-CAS生物学中尚未解答的基本问题：为什么新记忆成功 每一百万个受感染的细胞中只有一个？这些答案将为我们提供新的线索 在这种情况下，细菌种群内的异质性使其能够生存。此外，这些见解 和工具对于噬菌体疗法的发展将是有价值的，这在 抗击抗生素耐药病原体日益增长的威胁。

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.