The Function of Small RNA-Based viral Defense System in E. coli - Renewal 1

CRISPR-Cas（CRISPR相关基因） 基因座存在于几乎所有古细菌和一半真细菌中。它们保护原核生物不受外来的 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)适应，即，将短的外源DNA序列（间隔区）获取到CRISPR阵列中; 2） 产生成熟的保护性CRISPR RNA（crRNA），和3）当Cas核酸酶 在crRNA的引导下，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 部分限制了全球水平基因转移，并可用于创建基于DNA的记录 设备和控制抗生素耐药基因的传播。初始CRISPR免疫力建立在 “天真”的过程，非歧视性的获取短的细胞内DNA分子- prespacers -作为 插入CRISPR阵列。它很少发生，可能导致自杀性自我干预。一 称为“引发”的显着机制在I型CRISPR-Cas系统中起作用：间隔区的获取 与原始DNA相比， 从缺乏这种序列的DNA中获得。启动适应对宿主非常有益：它 快速导致从遗传寄生虫特异性获得额外的干扰有效间隔区 并确保不选择自靶向间隔区。之间的机械关系 预充期间的干扰和适应并不完全清楚。这项提案的目的是剖析 启动期间干扰和适应之间的相互关系，并确定细胞过程 在初始和启动适应过程中为适应机制提供能量。我们将使用FragSeq - an 创新的高通量方法，鉴定短的细胞内DNA片段， 在前一个资助期内制定的-以确定预定距器和其他 在不同CRISPR-Cas中的初始和引发适应期间产生的体内适应中间体 系统的类别和类型，并确定prespacer生成所必需的非Cas细胞蛋白， 间隔区获取。通过我们的工作，对CRISPR适应性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， 通过揭示限制适应的过程，可能有助于 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.