Discovering New Roles for CRISPR-Cas in Bacterial Pathogenesis

 描述(申请人提供)：CRISPR-Cas(群集规则间隔短回文重复和CRISPR相关基因)系统是一种原核生物适应性免疫系统，用于保护微生物免受外来入侵，如病毒的侵袭。这种免疫系统将入侵分子的DNA合并到CRISPR基因座中，基本上生成了过去病毒感染的免疫记录。CRISPR基因座随后的转录和加工产生小的CRISPR RNAs(CrRNAs)，这些RNAs被结合到一个蛋白质复合体中，根据序列互补性介导核酸的破坏。CrRNA与其核酸靶标之间的不完全互补可以消除切割，但仍然在体内招募蛋白质复合体。这种招募可能会导致另一种选择 如基因调控等功能，但这些非规范的CRISPR-Cas功能的流行还没有详细的调查。事实上，许多病原体和重要的人类微生物组分都有CRISPR-Cas系统，但角色未知，CRISPR阵列没有预测的靶点，这就提出了一个问题，即它们的生物学功能是什么。本项目的目标是鉴定可能调节CRISPR-Cas活性的内源细菌蛋白，并发现和表征新的CRISPR-Cas功能。这项研究是我研究生工作的延伸，当时我确定了CRISPR-Cas相互作用蛋白的第一个例子，这是一组不同的抑制剂，直接与不同的CRISPR-Cas成分相互作用。这表明，还有更多的内生相互作用因素有待确定。综上所述，在没有切割和CRISPR-Cas相互作用蛋白存在的情况下，核酸结合的可能性表明，可能有整个类别的细菌蛋白可以调节或重定向CRISPR-Cas的功能。为了扩大我们对CRISPR-CA的作用的理解，我将利用蛋白质组学技术来鉴定内源CRISPR-CA的相互作用因子，并表征它们的生理相关性。我还将进行生物信息学分析，以确定在病原生物体中似乎具有功能的CRISPR-CAS系统，并筛选它们的活性。鉴于单一CRISPR-Cas系统产生的crRNAs的序列多样性，规范(即外源基因组切割)和非规范(即基因调控)功能都可以同时被介导，将开发功能分析来测试这些可能性。最近CRISPR-CAS系统的工程设计为真核细胞(这些系统并不自然存在)提供基因组编辑和调控工具，完美地例证了RNA引导系统固有的可能性。细菌天然具有相似功能(即CRISPR-Cas介导的转录因子招募)的可能性尚未被研究。评估这些系统在许多不同生物体中的作用将不仅增强我们对CRISPR-CAS系统的理解，而且还将增强我们对细菌病原体如何保护自己的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.