The Function of Small RNA-Based viral Defense System in E. coli

大肠杆菌中基于小RNA的病毒防御系统的功能

• 批准号: 10388674
• 负责人: KONSTANTIN V SEVERINOV
• 金额: $ 7.1万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10388674
• 关键词: Antibiotic Resistance; Archaea; Bacteria; Bacterial Genome; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Sequence; Devices; Ensure; Escherichia coli; Eubacterium; Funding; Generations; Genetic; Genome; Goals; Horizontal Gene Transfer; Immunity; Infection; Lead; Memory; Molecular; Nucleic Acids; Parasites; Population; Process; Production; Prokaryotic Cells; Proteins; RNA; Small RNA; Spacer DNA; Structure; System; Viral; Virus; Work; base; genetic element; genome editing; genomic locus; in vivo; innovation; nuclease; resistance gene; response; suicidal; viral DNA

CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated genes) loci are present in almost all archaea and half of eubacteria. They protect prokaryotes from foreign genetic elements. While highly diverse, all CRISPR-Cas systems function through three common steps: 1) adaptation, i.e., acquisition of short foreign DNA sequences (spacers) into CRISPR arrays; 2) production of mature protective CRISPR RNAs (crRNAs), and 3) interference, when Cas nucleases guided by crRNAs destroy nucleic acids containing complementary targets. Studies of the interference part of CRISPR response have revolutionized the field of genomic editing. The less-studied adaptation part limits global horizontal gene transfer and can be harnessed for creation of DNA-based recording devices and control of the spread of antibiotic resistance genes. Initial CRISPR immunity is built in the course of “naïve”, non-discriminate acquisition of short intracellular DNA molecules – prespacers - as spacers into CRISPR arrays. It occurs very infrequently and can lead to suicidal self-interference. A remarkable mechanism called “priming” operates in type I CRISPR-Cas systems: acquisition of spacers from DNA with sequences matching pre-existing spacers is dramatically stimulated compared to naïve acquisition from DNA devoid of such sequences. Primed adaptation is highly beneficial to the host: it rapidly leads to specific acquisition of additional interference-proficient spacers from genetic parasites and ensures that no self-targeting spacers are selected. The mechanistic relationship between interference and adaptation during priming is not fully clear. The goal of this proposal is to dissect interrelationships between interference and adaptation during priming and to identify cellular processes that feed the adaptation machinery during naïve and primed adaptation. We will use FragSeq - an innovative high-throughput approach that identifies short intracellular DNA fragments and that was developed during the previous funding period - to determine the structure of prespacers and of other in vivo adaptation intermediates generated during naïve and primed adaptation in diverse CRISPR-Cas systems classes and types, and identify non-Cas cellular proteins essential for prespacer generation and spacer acquisition. The understanding of CRISPR adaptation that will result from our work will allow us and others to optimize the efficiency of the adaptation process, facilitating construction of strains with desired spacer content/immunity profiles and, by revealing processes that limit adaptation, may help control viability of bacterial populations by inducing adaptation from cell’s own DNA and self-interference.

CRISPR-Cas（成簇规则间隔短回文重复序列-CRISPR相关基因） 位点存在于几乎所有古细菌和一半真细菌中。它们保护原核生物免受外来物质的侵害 遗传元素。虽然高度多样化，但所有 CRISPR-Cas 系统都通过三个常见步骤发挥作用： 1) 适应，即将短的外源 DNA 序列（间隔区）采集到 CRISPR 阵列中； 2） 成熟保护性 CRISPR RNA (crRNA) 的产生，以及 3) Cas 核酸酶干扰 由 crRNA 引导破坏含有互补靶标的核酸。干扰研究 CRISPR 反应的一部分彻底改变了基因组编辑领域。研究较少的适应 部分限制全球水平基因转移，可用于创建基于 DNA 的记录 装置和控制抗生素抗性基因的传播。最初的 CRISPR 免疫是建立在 “天真的”过程，无差别地获取短的细胞内 DNA 分子 - 预间隔物 - 作为 将间隔区插入 CRISPR 阵列中。这种情况很少发生，并且可能导致自杀性的自我干扰。一个 I 型 CRISPR-Cas 系统中称为“启动”的显着机制：间隔区的获取 与naïve相比，来自与预先存在的间隔区匹配的序列的DNA受到显着刺激 从不含此类序列的 DNA 中获取。引发的适应对宿主非常有益：它 快速导致从遗传寄生虫中特异性获得额外的干扰能力强的间隔区 并确保不选择自靶向间隔物。之间的机械关系 启动期间的干扰和适应尚不完全清楚。该提案的目标是剖析 启动期间干扰和适应之间的相互关系并识别细胞过程 在幼稚适应和启动适应期间为适应机制提供动力。我们将使用 FragSeq - 一个 创新的高通量方法，可识别短的细胞内 DNA 片段 在上一个资助期间开发 - 确定预间隔器和其他的结构 不同 CRISPR-Cas 中初始适应和引发适应过程中产生的体内适应中间体 系统类别和类型，并识别预间隔子生成和生成所必需的非 Cas 细胞蛋白 间隔获取。通过我们的工作对 CRISPR 适应的理解将使我们能够 和其他优化适应过程的效率，促进菌株的构建 所需的间隔物含量/免疫特征，并通过揭示限制适应的过程，可能会有所帮助 通过诱导细胞自身 DNA 的适应和自我干扰来控制细菌种群的活力。