Spacer acquisition during the type III-A CRISPR-Cas immune response

III-A 型 CRISPR-Cas 免疫反应期间间隔区的获取

• 批准号: 10638980
• 负责人: Luciano A Marraffini
• 金额: $ 43.55万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10638980
• 关键词: Address; Archaea; Bacteria; Bacteriophages; Base Pairing; Basic Science; Binding; Biotechnology; Cells; Cellular Immunity; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; DNA Sequence; Deoxyribonucleases; Development; Disease; Event; Generations; Genes; Genetic Diseases; Genetic Transcription; Genome; Genus staphylococcus; Growth; Guide RNA; Human Genetics; Immune response; Immune system; Immunity; Infection; Integration Host Factors; Invaded; Investigation; Knowledge; Libraries; Lytic Phase; Mediating; Memory; Micrococcal Nuclease; Molecular; Mutagenesis; Nucleic Acids; Periodicity; Population; Process; Production; RNA; RNA Phages; Ribonucleases; Second Messenger Systems; Single-Stranded DNA; Staphylococcus aureus; System; Testing; Transcript; Translational Research; Viral; Viral Genes; Viral Genome; Virus; Virus Replication; adaptive immunity; adenylate; bacterial community; enzyme activity; experimental study; gene therapy; human pathogen; mutant; nuclease; prevent; response; sample fixation; success

Project Summary CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against their viruses (phages). The hallmark of the CRISPR-Cas immune response is the acquisition of a “memory” of infection in the form of a short DNA sequence from the invading phage genome. This sequence, known as “spacer”, is integrated into the CRISPR locus of the host and then transcribed and processed into a CRISPR RNA (crRNA) guide. CRISPR-associated (Cas) effectors use the crRNA to recognize the nucleic acids of the invading phage through base-pair complementarity and trigger different defense strategies. For Type III CRISPR systems, commonly present in the human pathogen Staphylococcus aureus, target recognition leads to the dormancy of the infected cell, an event that prevents viral replication and propagation. Here, we propose to investigate a central, yet unanswered, question about this mechanism: if there are spacers that trigger a growth arrest in the host, how are they maintained in the bacterial community after they are acquired? Our central hypothesis is that the degradation of the phage DNA eventually eliminates the viral genome from the host, enabling growth and the fixation of the spacer in the population. To investigate this, we will explore several aspects of the Csm6-mediated response required for the defense mediated by type III CRISPR spacers that match late- expressed viral genes. First, we will define whether dormant cells eventually die or are able to exit this state, survive infection and continue growing. Second, we will determine whether spacers that match late-expressed phage genes can provide a selective advantage to the cell that harbors them, even when they trigger host dormancy. Third, we will determine if these spacers are actually acquired during infection. In all these experiments we will test our central hypothesis by using mutant staphylococci lacking in the expression of several nucleases to determine if they are required for the fixation of dormancy-triggering spacers. Finally, we will use a transposon library of mutants to investigate, in an unbiased manner, the impact of host genes that could be involved in the exit from dormancy. Our proposed experiments, aimed at understanding how spacers from dormancy-inducing CRISPR systems are fixed in the host population, will fill in a fundamental knowledge gap in our understanding of the hallmark feature of CRISPR immunity: the generation of a memory of infection. In addition, by directly addressing a fundamental mechanism of phage defense of staphylococci, our proposal can facilitate the success of phage therapies for the treatment of staphylococcal disease. In a more indirect manner, the characterization of the molecular mechanisms of type III CRISPR systems can lead to avenues to repurpose these immune systems for gene editing, particularly for the development of gene therapies to treat genetic diseases.

项目概要 CRISPR-Cas 系统为细菌和古细菌提供针对病毒（噬菌体）的适应性免疫。 CRISPR-Cas免疫反应的标志是获得感染的“记忆” 来自入侵噬菌体基因组的一段短 DNA 序列，该序列被称为“间隔区”，被整合到其中。 宿主的 CRISPR 基因座，然后转录并加工成 CRISPR RNA (crRNA) 向导。 CRISPR 相关 (Cas) 效应器使用 crRNA 识别入侵噬菌体的核酸 通过碱基对互补性并触发不同的防御策略对于III型CRISPR系统， 通常存在于人类病原体金黄色葡萄球菌中，目标识别导致其休眠 受感染的细胞，这是一种阻止病毒复制和传播的事件。 关于该机制的一个核心但尚未得到解答的问题是：是否存在引发细胞生长停滞的间隔基？ 宿主，它们获得后如何在细菌群落中维持？我们的中心假设是 噬菌体 DNA 的降解最终将病毒基因组从宿主中消除，从而促进生长 为了研究这一点，我们将探讨该问题的几个方面。 Csm6 介导的反应是由与晚期匹配的 III 型 CRISPR 间隔区介导的防御所需的 首先，我们将定义休眠细胞是否最终死亡或能够退出这种状态， 其次，我们将确定间隔区是否与晚期表达相匹配。 噬菌体基因可以为容纳它们的细胞提供选择性优势，即使它们触发宿主 第三，我们将确定这些间隔物是否确实是在感染过程中获得的。 实验中，我们将通过使用缺乏表达的突变葡萄球菌来检验我们的中心假设 几种核酸酶以确定它们是否是固定休眠触发间隔区所必需的。 将使用突变体转座子库以公正的方式研究宿主基因的影响 可能参与休眠的退出。 我们提出的实验旨在了解休眠诱导 CRISPR 系统中的间隔物如何 固定在东道国人口中，将填补我们对该标志的理解中的基本知识空白 CRISPR免疫的特点是：通过直接寻址产生感染记忆。 葡萄球菌噬菌体防御的基本机制，我们的建议可以促进噬菌体的成功 以更间接的方式治疗葡萄球菌疾病的疗法。 III型CRISPR系统的分子机制可以为重新利用这些免疫系统提供途径 基因编辑，特别是开发治疗遗传疾病的基因疗法。