权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Generation of DNA memory by bacterial CRISPR-Cas9 systems

通过细菌 CRISPR-Cas9 系统生成 DNA 记忆

基本信息

批准号：
10792662
负责人：
Yan Zhang
金额：
$ 18.43万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2025-07-31
项目状态：
未结题

项目摘要

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 CRISPR-Cas to establish adaptive immunity against invasive elements. DNA 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. 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 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 enzyme, NmeCas9 and its tracrRNA and crRNA partners, play key but non-conventional roles in the acquisition of functional spacers. We 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? This administrative equipment supplement request will allow access to ddPCR instrument, which enables high throughput and sensitive quantification of nucleic acids. This is essential to decipher CRISPR immunization beyond DNA transformation, in the context of filamentous phage infection. The impact of research supported by the parent grant will therefore be greatly enhanced. Collectively, the proposed research will illuminate the interplay between pathogenic bacteria, their CRISPR systems, and different horizontal gene transfer routes.

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。大多数细菌依赖CRISPR-Cas来建立适应性免疫对抗入侵者来自这些入侵者基因组的DNA可以被捕获并储存在免疫系统中。在CRISPR基因座上的称为间隔区的记忆。由CRISPR产生的小的反义RNA（crRNA）将引导Cas酶摧毁具有匹配目标的入侵者。在过去的十年里，在理解CRISPR干扰酶及其在基因工程中的应用方面取得了进展。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。干扰酶NmeCas 9和其tracrRNA和crRNA伴侣在功能间隔区的获得中发挥关键但非常规的作用。我们使用分子遗传学，基因组学和生物化学方法来解决基本问题，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？这一行政设备补充申请将允许访问ddPCR仪器，通量和核酸的灵敏定量。这对于破译CRISPR免疫至关重要在丝状噬菌体感染的背景下，研究支持的影响家长的支持力度将大大提高。总的来说，拟议的研究将阐明病原菌、其CRISPR系统和不同水平基因转移途径之间的相互作用。