Discovering New Roles for CRISPR-Cas in Bacterial Pathogenesis

 描述（由申请人提供）：CRISPR-Cas（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，可防御微生物免受外来入侵者（如病毒）的侵害。这种免疫系统将来自入侵元件的DNA整合到CRISPR基因座中，基本上产生了过去病毒感染的免疫记录。CRISPR基因座的后续转录和加工产生小CRISPR RNA（crRNA），其被掺入基于序列互补性介导核酸破坏的蛋白质复合物中。crRNA与其核酸靶标之间的不完全互补性可消除切割，但仍在体内募集蛋白质复合物。这种招聘可能会导致替代 CRISPR-Cas的功能，如基因调控，但这些非典型的CRISPR-Cas功能的流行还没有被详细研究。事实上，许多病原体和重要的人类微生物组成分都拥有没有已知作用的CRISPR-Cas系统和没有预测靶点的CRISPR阵列，这就提出了一个关于它们的生物学功能是什么的问题。该项目的目的是鉴定可能调节CRISPR-Cas活性的内源性细菌蛋白，并发现和表征新的CRISPR-Cas功能。这项研究是我的研究生工作的延伸，在那里我确定了CRISPR-Cas相互作用蛋白的第一个例子，这是一组直接与不同CRISPR-Cas组分相互作用的不同抑制剂。这表明，更多的内源性相互作用者仍有待确定。总之，在不存在切割和存在CRISPR-Cas相互作用蛋白的情况下核酸结合的可能性表明可能存在可以调节或重定向CRISPR-Cas功能的整类细菌蛋白。为了扩大我们对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相互作用物并表征其生理相关性。我还将进行生物信息学分析，以确定在病原生物中发挥作用的CRISPR-Cas系统，并筛选它们的活性。考虑到由单个CRISPR-Cas系统产生的crRNA的序列多样性，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，并且将开发功能测定来测试这些可能性。最近对CRISPR-Cas系统进行工程改造，以在真核细胞中提供基因组编辑和调控工具（其中这些系统并不天然存在），完美地证明了RNA引导系统固有的可能性。细菌天然具有类似功能（即CRISPR-Cas介导的转录因子募集）的可能性尚未研究。评估这些系统在许多不同生物体中的作用不仅将增强我们对CRISPR-Cas系统的理解，还将增强我们对细菌病原体如何保护其基因组和调节重要细胞过程的理解。

 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.