Generation of DNA memory by bacterial CRISPR-Cas9 systems

通过细菌 CRISPR-Cas9 系统生成 DNA 记忆

• 批准号: 10454868
• 负责人: Yan Zhang
• 金额: $ 37.8万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/10454868
• 关键词: Address; Affect; Animal Model; Antibiotic Resistance; Antisense RNA; Bacteria; Biochemical; Biology; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Devices; Disease; Enzymes; Escherichia coli; Generations; Genes; Genetic; Genetic Diseases; Genetic Engineering; Genome; Genomics; Health; Horizontal Gene Transfer; Human; Human Genetics; Immune system; Immunologic Memory; Integrase; Knowledge; Memory; Microbe; Mobile Genetic Elements; Molecular; Molecular Genetics; Neisseria meningitidis; Pathogenicity; Play; Process; Proteins; Recording of previous events; Research; Research Design; Role; Site; System; Technology; Work; adaptive immunity; antimicrobial; base; genetic element; novel; pathogen; pathogenic bacteria; tool; trait

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.

项目概要 原核水平基因转移（HGT）强调了抗生素抗性和致病性状的传播。 对抗抗生素耐药性的斗争必须在多个战线上进行，包括了解天然抗生素 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 对移动遗传元件的免疫力。这些入侵者基因组的 DNA 片段可以被捕获并 存储在 CRISPR 位点上的称为间隔区的免疫记忆中。产生的小反义 RNA CRISPR (crRNA) 将引导 Cas 酶消灭具有匹配目标位点的入侵者。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。 在本提案中，我们的目标是揭示 CRISPR 记忆（即间隔适应）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，因为其临床表现 重要性和易处理的遗传学。目前关于间隔适应的知识主要来自以下研究 大肠杆菌天然的 I 型 CRISPR；其保守的 cas1-cas2 整合酶基因的产物可以产生功能性 记忆独立于干扰酶。我们最近的初步 发现 建议采用II型 脑膜炎奈瑟氏球菌的 CRISPR 通过独特的机制创造记忆。干扰基因 Nmecas9 和 tracrRNA 辅因子在功能间隔区的获得中发挥着重要但非常规的作用。我们将 使用分子遗传学、基因组和生化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Cas1-2整合酶配合？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、其 CRISPR 系统和细菌之间的相互作用。 HGT。这项工作还有望引导技术进步，包括基于 CRISPR 的新型抗菌药物 杀死特定细菌病原体，以及基于 Cas9-Cas1-Cas2 的基因组标记设备，帮助记录 细胞/疾病史。