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Defining CRISPR adaptation and interference mechanisms in E. coli

定义大肠杆菌中的 CRISPR 适应和干扰机制

基本信息

批准号：
10387608
负责人：
Dipali Gurudutt Sashital
金额：
$ 2.4万
依托单位：
IOWA STATE UNIVERSITY
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：
2021-06-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10387608
关键词：
Adaptive Immune System Affect Antibiotic Resistance Archaea Automobile Driving Bacteria Bacteriophages Base Pairing Binding Binding Proteins Biochemical Biophysics CRISPR interference Clustered Regularly Interspaced Short Palindromic Repeats Communities Complex DNA Dependence Detection Development Disease Ecosystem Effectiveness Elements Escherichia coli Event Evolution Genes Genetic Genome Goals Guide RNA Health Horizontal Gene Transfer Human Human Microbiome Immune Immune response Immune system Immunity Infection Invaded Investigation Knowledge Lead Life Measures Metabolism Methods Modeling Molecular Molecular Conformation Movement Mutation Nucleic Acids Organism Plasmids Point Mutation Population Population Dynamics Production Proteins RNA Interference Race Seeds Shapes Small RNA Specificity Structure System Therapeutic Time Virus Diseases Work antimicrobial arm biophysical tools combat endonuclease in vivo microbiome mutant pathogenic virus response tool viral resistance virus host interaction

项目摘要

Project Summary Viral resistance is essential in all kingdoms of life, although diverse organisms have evolved equally diverse mechanisms for combatting infection. In bacteria and archaea, the CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system clears invading DNA during infection through a small-RNA guided interference mechanism. CRISPR immunity proceeds through two stages: adaptation, in which fragments of invasive DNA from bacteriophages or plasmids are inserted as spacers within the CRISPR locus of the host genome and subsequently serve as templates for the production of small guide CRISPR (cr)RNAs;; and interference, during which the crRNA and its effector CRISPR associated (Cas) proteins bind complementary target regions of the invading DNA, leading to its destruction by a Cas endonuclease. Our goal is to define how bacteria maximize their immune capacity to gain an advantage in the molecular arms race against their invaders. Our first goal is to understand the sequence-dependence of immune system evasion through the development of point mutations within the invading DNA. Our previous studies have revealed that spacer sequence greatly influences the effectiveness of these “escape” mutations, suggesting for the first time that some spacer sequences provide stronger immunity than others. In addition, we have discovered that during initial infection, bacteria use a two-tiered defensive system to broaden their adaptation capacity. We will evaluate the impact of this tactic on host immunity and elucidate the molecular mechanisms underlying this defense strategy. Finally, we will determine the structural basis for rapid adaptation triggered when the CRISPR machinery senses non- canonical target sequences. Our studies will have major implications on the understanding of host-virus interactions and co-evolution, an important determinant of the compositional dynamics within complex ecological systems including the human microbiome.

项目摘要病毒抵抗力在生命的所有领域都是必不可少的，尽管不同的生物体具有进化出同样多样化的对抗感染机制。在细菌和古细菌中， CRISPR（成簇规则间隔短回文重复）适应性免疫系统通过小 RNA 引导的干扰机制清除感染过程中入侵的 DNA。 CRISPR 免疫经历两个阶段：适应，其中侵入性片段来自噬菌体或质粒的 DNA 作为间隔子插入到 CRISPR 基因座内宿主基因组，随后作为生产小向导 CRISPR 的模板 (cr)RNA；；和干扰，在此期间 crRNA 及其效应子 CRISPR 相关 (Cas) 蛋白质结合入侵 DNA 的互补目标区域，导致其被破坏 Cas 核酸内切酶。我们的目标是确定细菌如何最大限度地提高其免疫能力在对抗入侵者的分子军备竞赛中具有优势。我们的首要目标是了解通过点的发展来逃避免疫系统的序列依赖性入侵 DNA 内的突变。我们之前的研究揭示了间隔序列极大地影响了这些“逃逸”突变的有效性，这是首次提出某些间隔序列比其他间隔序列提供更强的免疫力。此外，我们还有发现在初始感染期间，细菌使用两层防御系统来扩大感染范围他们的适应能力。我们将评估这种策略对宿主免疫力的影响，并阐明这种防御策略背后的分子机制。最后，我们将确定当 CRISPR 机器感知到非-时，就会触发快速适应的结构基础规范目标序列。我们的研究将对理解宿主-病毒相互作用和共同进化，组成的重要决定因素包括人类微生物组在内的复杂生态系统内的动态。