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: 10227166
• 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/10227166
• 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。 必须在多个方面进行与抗生素抗性的斗争，包括对自然的理解 微生物用来限制HGT的障碍。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。这些入侵者基因组中的DNA碎片可以捕获，并且 在CRISPR基因座中存储为被称为垫片的免疫记忆。小型反义rNA由 CRISPR（CRRNA）将引导CAS酶使用匹配的目标部位摧毁入侵者。在过去的十年中， 在了解CRISPR干扰酶及其在 基因工程。但是，微生物如何获得其CRISPR记忆仍然非常了解。 在此提案中，我们旨在发现CRISPR记忆的分子基础（即间隔者适应）。我们 由于其临床 重要性和可处理的遗传学。当前关于间隔适应的知识主要来自于研究 大肠杆菌本地的I型CRISPR；其保守的Cas1-Cas2整合酶基因的产品可以创建功能 记忆独立于干扰酶。我们最近的初步 发现 建议II型 N. meningitidis的CRISPR通过独特的机制创造记忆。干扰基因NMECAS9和 tracrrna共同因素，在功能间隔者的采集中起重要但非规定的作用。我们将 使用分子遗传，基因组和生化方法来解决基本问题，包括： CAS9和CRISPR编码的旋风在间隔者采集中的分子作用是什么？什么是 控制记忆DNA选择的规则？ CAS9/TRACR如何与CAS1-2积分酶合作？和 最后，抗Crispr蛋白将如何影响记忆过程？ 拟议的研究将阐明致病细菌，其CRISPR系统和 HGT。这项工作还有望指导技术进步，包括基于CRISPR的小说抗菌素 杀死特定的细菌病原体和基于CAS9-CAS1-CAS2的基因组标签设备，有助于记录 细胞/疾病史。