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

基本信息

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
项目类别：
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

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

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项目摘要

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（群集定期间隔间隔短的alindromic重复与危机相关基因）他们保护原核生物免受外国的侵害遗传因素。尽管高度多样化，但所有CRISPR-CAS系统通过三个共同的步骤运行： 1）适应，即，将简短的外源DNA序列（间隔者）获取为CRISPR阵列； 2）生产成熟的受保护的CRISPR RNA（CRRNA），3）干扰，当CAS核病在CRRNA的指导下破坏了包含完整靶标的核酸。干扰研究 CRISPR反应的一部分彻底改变了基因组编辑领域。研究较少的改编零件限制全球水平基因转移，可以利用以创建基于DNA的记录装置和抗生素耐药基因扩散的控制。最初的CRISPR免疫是内置的 “幼稚”的，无歧视的较短细胞内DNA分子的习得 - 预科剂 - 垫片进入CRISPR阵列。它很少发生，可能导致自杀的自我干扰。一个称为“启动”的杰出机制在I型CRISPR-CAS系统中运行：获取垫片与幼稚的从没有此类序列的DNA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特异性获取其他干扰的垫片并确保不选择自动靶向垫片。机械关系启动过程中的干扰和适应尚不完全清楚。该提议的目的是剖析启动过程中的干扰和适应性之间的相互关系并识别蜂窝过程在幼稚和底漆适应过程中，可以供应适应机制。我们将使用fragseq- 创新的高通量方法可以识别出短的细胞内DNA片段，那就是在上一个资金期间开发 - 确定预设的结构和其他在潜水员CRISPR-CAS中生成的体内适应性中间体系统类别和类型，并识别针对预知前生必不可少的非CAS细胞蛋白辅助收购。对我们工作将导致的CRISPR改编的理解将使我们以及其他以优化适应过程的效率的所需的间隔含量/免疫概况，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的DNA和自身释放的适应性来控制细菌群体的生存能力。