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

CRISPR-CAS(簇状规则间隔短回文重复序列-CRISPR相关基因) 基因座存在于几乎所有的古生菌和一半的真细菌中。它们保护原核生物免受外来生物的侵袭 遗传因素。虽然高度多样化，但所有CRISPR-CAS系统都通过三个共同步骤发挥作用： 1)适配，即获取短的外源DNA序列(间隔区)进入CRISPR阵列；2) 成熟的保护性CRISPR RNAs(CrRNAs)的产生，以及3)当CaS核酸酶时的干扰 在crRNA的引导下，它们会破坏含有互补靶标的核酸。关于干扰的研究 CRISPR反应的一部分使基因组编辑领域发生了革命性的变化。较少研究的适应 部分限制了全球水平基因转移，并可用于创建基于DNA的记录 抗生素耐药基因传播的装置和控制。初始CRISPR免疫性内置于 不分青红皂白地获取细胞内短DNA分子的过程--前步长--AS 将间隔物插入CRISPR阵列。这种情况很少发生，可能会导致自杀性的自我干扰。一个 在I型CRISPR-CAS系统中运行的称为“启动”的显著机制：间隔区的获取 与天真相比，来自与先前存在的间隔区匹配的序列的DNA被戏剧性地刺激 从没有这种序列的DNA中获取。启动适应对宿主非常有益：它 迅速导致从遗传寄生虫中获得额外的干扰熟练间隔区 并确保没有选择自定位间隔物。两者之间的机械性关系 启动过程中的干扰和适应尚不完全清楚。这项提案的目的是剖析 启动过程中干扰和适应的相互关系及识别细胞过程 在幼稚和启动的适应过程中为适应机制提供营养。我们将使用FragSeq-an 创新的高通量方法，识别细胞内短DNA片段，这是 在上一个供资期间开发--以确定前起搏器和其他起搏器的结构 不同CRISPR-CA在幼稚和启动适应过程中产生的体内适应中间产物 系统分类和类型，并鉴定对前起搏器生成和 间隔器获取。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 所需的间隔区含量/免疫图谱，以及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生存能力。

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.