Discovering New Roles for CRISPR-Cas in Bacterial Pathogenesis

发现 CRISPR-Cas 在细菌发病机制中的新作用

基本信息

批准号：
9349378
负责人：
Joseph Bondy-Denomy
金额：
$ 39.63万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-25 至 2020-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9349378
关键词：
Adaptive Immune System Affinity Chromatography Animal Model BCAR1 gene Bacteria Bacterial Proteins Bacteriophages Biochemical Bioinformatics Biological Assay Biological Models Biological Process Cell physiology Cells Cleaved cell Clustered Regularly Interspaced Short Palindromic Repeats Complement Complex Coupled DNA Defense Mechanisms Disease Elements Engineering Escherichia coli Eukaryotic Cell Evolution Gene Expression Regulation Genes Genetic Transcription Genome Genomics Growth Guide RNA Human Human Microbiome Immune system Immunization Invaded Investigation Lead Mass Spectrum Analysis Mediating Methods Microbe Microbiology Nucleic Acid Binding Nucleic Acids Organism Outcome Pathogenesis Pathogenicity Physiological Plasmids Play Prevalence Probability Problem Solving Process Production Proteins Proteomics Pseudomonas aeruginosa RNA Recruitment Activity Research Role System Techniques Testing Viral Virus Virus Diseases Vitronectin Work base experimental study genome editing gut microbiome helicase in vivo inhibitor/antagonist member microorganism mutant novel novel therapeutics nuclease pathogen pathogenic bacteria programs protein complex protein function public health relevance ribosome profiling tool transcription factor transcriptomics

项目摘要

DESCRIPTION (provided by applicant): The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes) system is a prokaryotic adaptive immune system that defends microbes from foreign invaders such as viruses. This immune system incorporates DNA from invading elements into the CRISPR locus, essentially generating an immunization record of past viral infection. The subsequent transcription and processing of the CRISPR locus generates small CRISPR RNAs (crRNAs) that are incorporated into a protein complex that mediates the destruction of nucleic acids based on sequence complementarity. Imperfect complementarity between the crRNA and its nucleic acid target can abrogate cleavage, but still recruit the protein complex in vivo. This recruitment can result in alternative functions such as gene regulation, but the prevalence of these non-canonical CRISPR-Cas functions has not been investigated in detail. In fact, many pathogens and important human microbiome constituents possess CRISPR-Cas systems with no known role and CRISPR arrays with no predicted targets, raising a question as to what their biological functions are. The objective of this project is to identify endogenous bacterial proteins that may modulate CRISPR-Cas activity and discover and characterize novel CRISPR-Cas functions. This research is an extension of my graduate work, where I identified the first examples of CRISPR-Cas interacting proteins, which are a diverse group of inhibitors that directly interact with different CRISPR-Cas components. This suggests that more endogenous interactors remain to be identified. Together, the potential for nucleic acid binding in the absence of cleavage and the existence of CRISPR-Cas interacting proteins, suggests that there may be entire classes of bacterial proteins that can modulate or redirect CRISPR-Cas function. To broaden our understanding of the roles for CRISPR- Cas, I will utilize proteomic techniques to identify endogenous CRISPR-Cas interactors and characterize their physiological relevance. I will also conduct bio-informatic analyses to identify CRISPR-Cas systems that appear functional in pathogenic organisms and screen them for activity. Given the sequence diversity of the crRNAs generated by a single CRISPR-Cas system, both canonical (i.e. foreign genome cleavage) and non-canonical (i.e. gene regulation) functions could be mediated concurrently and functional assays will be developed to test these possibilities. The recent engineering of CRISPR-Cas systems to provide genome editing and regulatory tools in eukaryotic cells (where these systems do not naturally exist) perfectly exemplify the possibilities that are intrinsic to an RNA-guided system. The possibility that bacteria naturally possess similar functions (i.e. CRISPR-Cas-mediated recruitment of a transcription factor) has not been investigated. Assessing the roles of these systems in many different organisms will enhance our understanding of not only CRISPR-Cas systems, but also of how bacterial pathogens defend their genomes and regulate vital cell processes.

描述（由申请人提供）： CRISPR-Cas（成簇规则间隔短回文重复序列和 CRISPR 相关基因）系统是一种原核适应性免疫系统，可保护微生物免受病毒等外来入侵者的侵害。该免疫系统将来自入侵元素的 DNA 纳入其中。 CRISPR 基因座，本质上生成过去病毒感染的免疫记录 CRISPR 基因座的后续转录和加工生成小 CRISPR RNA。 crRNA 结合到蛋白质复合物中，根据序列互补性介导核酸的破坏。crRNA 与其靶核酸之间的不完美互补性可以消除切割，但仍会在体内募集蛋白质复合物。选择但这些非经典 CRISPR-Cas 功能的普遍性尚未得到详细研究。事实上，许多病原体和重要的人类微生物组成分都拥有未知作用的 CRISPR-Cas 系统和无法预测的 CRISPR 阵列。该项目的目的是鉴定可能调节 CRISPR-Cas 活性的内源细菌蛋白，并发现和表征新的 CRISPR-Cas 功能。在我的研究生工作中，我发现了第一个 CRISPR-Cas 相互作用蛋白的例子，它们是直接与不同 CRISPR-Cas 成分相互作用的多种抑制剂，这表明更多的内源性相互作用蛋白仍有待鉴定。在没有裂解的情况下的酸结合以及 CRISPR-Cas 相互作用蛋白的存在表明可能存在整个类别的细菌蛋白可以调节或重定向 CRISPR-Cas 功能以扩大我们对 CRISPR-Cas 作用的理解，我将利用蛋白质组学技术来鉴定内源性 CRISPR-Cas 相互作用因子并表征其生理相关性。我还将进行生物信息学分析，以鉴定在病原生物体中具有功能的 CRISPR-Cas 系统，并根据其序列多样性筛选它们。由单个 CRISPR-Cas 系统生成的 crRNA，可以同时介导规范（即外源基因组切割）和非规范（即基因调控）功能，并且可以进行功能测定最近开发的 CRISPR-Cas 系统在真核细胞（这些系统并不自然存在）中提供基因组编辑和调控工具，完美地体现了 RNA 引导系统固有的可能性。天然具有相似功能（即 CRISPR-Cas 介导的转录因子的募集）尚未得到研究。评估这些系统在许多不同生物体中的作用不仅可以增强我们对 CRISPR-Cas 系统的理解，而且可以增强我们对 CRISPR-Cas 系统如何发挥作用的理解。细菌病原体保护其基因组并调节重要的细胞过程。