Generation of DNA memory by bacterial CRISPR-Cas9 systems

项目摘要 基因水平转移（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，包括了解天然抗生素的耐药性。 微生物用来限制HGT的屏障大多数细菌依靠CRISPR-Cas系统来建立适应性 对移动的遗传因子的免疫力。这些入侵者基因组中的DNA片段可以被捕获， 在CRISPR基因座上作为称为间隔区的免疫记忆储存。产生的小的反义RNA， CRISPR（crRNA）将引导Cas酶摧毁具有匹配靶位点的入侵者。在过去的十年里， 在理解CRISPR干扰酶及其在生物学中的应用方面已经取得了很大进展， 基因工程然而，微生物如何获得CRISPR记忆仍然知之甚少。 在这项提案中，我们的目标是揭示CRISPR记忆（即间隔区适应）的分子基础。我们 使用革兰氏阴性病原体脑膜炎奈瑟菌（Nme）作为模式生物，由于其临床 重要性和易处理的遗传学。目前关于间隔区适应性的知识主要来自于对以下方面的研究： I型CRISPR是E.其保守的cas 1-cas 2整合酶基因的产物可以产生功能性的 不依赖于干扰酶的记忆我们最近的初步调查 结果 表明第二类 N.脑膜炎通过一种独特的机制产生记忆。干扰基因Nmecas 9和 tracrRNA辅因子在功能间隔区的获得中发挥重要但非常规的作用。我们将 利用分子遗传学、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tracrRNA在间隔区获取中的分子作用是什么？有哪些 记忆DNA选择的规则Cas9/tracr如何与Cas 1 -2整合酶合作？和 最后，抗CRISPR蛋白会如何影响记忆过程？ 拟议的研究将阐明致病细菌，它们的CRISPR系统， HGT这项工作还有望指导技术进步，包括基于CRISPR的新型抗菌剂 杀死特定细菌病原体，以及基于Cas9-Cas 1-Cas 2的基因组标记设备， 细胞/疾病史。

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.