The alternative roles of Neisseria CRISPR-Cas9 in endogenous gene regulation and bacterial physiology

 描述（申请人提供）：许多生物体采用一系列迷人的基因沉默途径-包括最著名的例子，RNA干扰（RNAi）-来对抗入侵的外来核酸。2007年，在细菌和古细菌中发现了一种新的遗传干扰途径。成簇的、规则间隔的短回文重复序列（CRISPR）基因座及其相关的cas基因提供了针对病毒和移动的元件的适应性、可遗传的、基于序列的遗传干扰。在过去的7年里，在阐明CRISPR-Cas作为防御系统的功能和机制方面取得了令人难以置信的进展。然而，CRISPR-Cas组件在防御之外的非经典功能，如内源性基因调控和细菌发病机制等领域，人们知之甚少，直到最近才开始受到重视。我们使用革兰氏阴性人类病原体脑膜炎奈瑟氏球菌（Nm）作为模型系统，因为它的临床重要性和实验易处理性。我们的初步发现促使我假设脑膜炎球菌CRISPR-Cas9系统在转录后基因调控和细菌生理学中具有替代作用。本提案的目的是检验这一假设， 解决一些基本问题，包括：NmCas 9如何识别和切割RNA靶标？CRISPR-Cas9调控哪些内源性转录物？最后，Cas9是如何参与脑膜炎球菌的生理学和毒力的？在该奖项的K99阶段，我将深入分析NmCas 9在体外不寻常的RNA识别和切割活性。此外，我将鉴定与NmCas 9相关或受NmCas 9调控的内源性脑膜炎球菌RNA。此外，我还将研究NmCas 9如何参与粘附人类上皮细胞，并在脑膜炎球菌生理学和致病性的各个方面寻找其他Cas9依赖性表型。随着生物化学技能的发展和NmCas 9的调控目标的定义，我将进一步剖析和重建在R 00阶段内源性转录的NmCas 9为基础的调控。此外，在K99阶段鉴定的新的NmCas 9依赖性毒力缺陷将在R 00阶段进一步表征。综上所述，我的建议将有助于理解CRISPR-Cas系统在基因调控和细菌生理学中的替代作用。重要的是，这项提案的发现对于广泛的受众具有重要意义，他们有兴趣在抗菌治疗、人类脑膜炎球菌疾病干预以及基于Cas9的新型RNA靶向工具的开发等实际领域利用CRISPR-Cas9系统。

 DESCRIPTION (provided by applicant): Many organisms employ a fascinating array of gene silencing pathways - including the best- known example, RNA interference (RNAi) - to confront invasive foreign nucleic acids. In 2007, a novel genetic interference pathway was discovered in bacteria and archaea. The clustered, regularly interspaced, short palindromic repeat (CRISPR) loci and their associated cas genes provide adaptive, inheritable, sequence-based genetic interference against viruses and mobile elements. Over the past 7 years, incredible progresses have been made in elucidating the functions and mechanisms of CRISPR-Cas as a defense system. However, the non-canonical functions of CRISPR-Cas components beyond defense, in areas like endogenous gene regulation and bacteria pathogenesis, were poorly understood and have only recently begun to be appreciated. We are using the gram-negative human pathogen Neisseria meningitidis (Nm) as a model system, because of its clinical importance and experimental tractability. Our preliminary findings prompted me to hypothesize that the meningococcal CRISPR-Cas9 system has alternative roles in post-transcriptional gene regulation and in bacterial physiology. The goal of this proposal is to test this hypothesis and to address some fundamental questions including: How does NmCas9 recognize and cleave RNA targets? What endogenous transcripts are regulated by CRISPR-Cas9? And, finally, how is Cas9 involved in meningococcal physiology and virulence? In the K99 phase of this award, I will perform in-depth analyses of the unusual RNA recognition and cleavage activities of NmCas9 in vitro. In addition, I will identify endogenous meningococcal RNAs that associate with, or are regulated by NmCas9. Moreover, I will also examine how is NmCas9 involved in adherence to human epithelial cells, and search for additional Cas9-dependent phenotypes in various aspects of meningococcal physiology and pathogenicity. With the biochemical skills developed and NmCas9's regulatory targets defined, I will further dissect and reconstitute NmCas9-based regulation of endogenous transcripts during the R00 phase. In addition, new NmCas9-dependent virulence defects identified in the K99 phase will be further characterized during the R00 phase. Taken together, my proposal will shed light on the understandings of alternative roles of CRISPR-Cas systems in gene regulation and bacterial physiology. Importantly, findings from this proposal will be of importance to a wide audience with interests in exploiting CRISPR-Cas9 systems in practical areas like antimicrobial treatments, human meningococcal disease interventions, as well as the developments of novel Cas9-based RNA targeting tools.