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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：
9150686
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/9150686
关键词：
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 Components Gene Expression Regulation Genes Genome Genomics Growth Guide RNA Health Human Human Microbiome Immune system Immunization Invaded Investigation Lead Mass Spectrum Analysis Mediating Methods Microbe Nucleic Acid Binding Nucleic Acids Organism Outcome Pathogenesis Physiological Plasmids Play Prevalence Probability Problem Solving Process Production Proteins Proteomics Pseudomonas aeruginosa RNA Recruitment Activity Research Role System Techniques Testing Transcription Process Viral Virus Virus Diseases Work base gene function genome editing gut microbiome helicase in vivo inhibitor/antagonist member microorganism mutant novel novel therapeutics nuclease pathogen pathogenic bacteria programs protein complex research study 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.