Generation of DNA memory by bacterial CRISPR-Cas9 systems

项目摘要 原核水平基因转移(HGT)强调了抗生素耐药性和致病性状的传播。 对抗抗生素耐药性的斗争必须在多条战线上进行，包括对自然 微生物用来限制HGT的屏障。大多数细菌依靠CRISPR-Cas系统建立适应 对移动遗传因素的免疫力。这些入侵者基因组中的DNA片段可以被捕获并 以免疫记忆的形式存储在CRISPR基因座上，称为间隔区。小的，反义RNA产生的 CRISPR(CrRNAs)将引导Cas酶摧毁具有匹配靶点的入侵者。在过去的十年里， 在了解CRISPR干扰酶及其应用方面取得了很大进展 基因工程。然而，微生物如何获得CRISPR记忆仍然知之甚少。 在这项提议中，我们旨在揭示CRISPR记忆(即间隔区适应)的分子基础。我们 以革兰氏阴性杆菌脑膜炎奈瑟氏菌(NME)作为模式生物，由于其临床特点。 重要性和易驯服的遗传学。目前关于间隔区适应的知识大多来自于对 大肠杆菌固有的I型CRISPR；其保守的Cas1-Cas2整合酶基因的产物可以产生功能性的 不受干扰酶影响的记忆。我们最近的初步调查 发现 表明第二类病毒 脑膜炎奈瑟氏菌的CRISPR通过一种不同的机制创造记忆。干扰基因Nmecas9和 TrrRNA辅助因子，在获得功能间隔区方面发挥着重要但非常规的作用。我们会 使用分子遗传学、基因组学和生物化学方法解决基本问题，包括： Cas9和CRISPR编码的trrRNA在间隔区获得中的分子作用是什么？什么是 管理记忆DNA选择的规则？Cas9/TRAIR如何与Cas1-2整合酶协同工作？和 最后，抗CRISPR蛋白将如何影响记忆过程？ 拟议的研究将阐明病原菌、它们的CRISPR系统和 HGT。这项工作也有望指导技术进步，包括基于CRISPR的新型抗菌剂 杀死特定细菌病原体的，以及基于Cas9-Cas1-Cas2的有助于记录 细胞/病史。

Project Summary Prokaryotic horizontal gene transfer (HGT) underlines the spread of antibiotic resistance and pathogenic traits. The battle against antibiotic resistance must be fought on multiple fronts, including the understanding of natural barriers that microbes use to restrict HGT. Most bacteria rely on the CRISPR-Cas system to establish adaptive immunity against mobile genetic elements. DNA pieces from these invaders' genome can be captured and stored as immunological memories termed spacers, at the CRISPR loci. Small, antisense RNAs produced from CRISPR (crRNAs) will guide Cas enzymes to destroy invaders with a matching target site. In the past decade, much progress has been made in understanding the CRISPR interference enzymes and their applications in genetic engineering. However, how microbes acquire their CRISPR memories remains very poorly understood. In this proposal, we aim to uncover the molecular basis for CRISPR memorization (i.e. spacer adaptation). We use the gram-negative pathogen Neisseria meningitidis (Nme) as a model organism, due to of its clinical importance and tractable genetics. Current knowledge about spacer adaptation mostly comes from studies of the type I CRISPR native to E. coli; products of its conserved cas1-cas2 integrase genes can create functional memories independently of the interference enzymes. Our recent preliminary findings suggest that the type II CRISPR of N. meningitidis creates memory by a distinct mechanism. The interference genes, Nmecas9 and tracrRNA co-factor, play important but non-conventional roles in the acquisition of functional spacers. We will use molecular genetic, genomic and biochemical approaches to address fundamental questions, including: What are the molecular roles of Cas9 and the CRISPR-encoded tracrRNA in spacer acquisition? What are the rules governing memory DNA selection? How does Cas9/tracr cooperate with the Cas1-2 integrase? And finally, how would the anti-CRISPR proteins affect the memorization process? The proposed research will illuminate the interplay between pathogenic bacteria, their CRISPR systems, and HGT. This work also promises to guide technology advances, including CRISPR-based novel antimicrobials that kill specific bacterial pathogens, and Cas9-Cas1-Cas2 based genome-tagging devices that help record cellular/disease history.