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 来建立适应性免疫。可以捕获这些入侵者基因组中的 DNA 并将其存储为免疫学。由 CRISPR (crRNA) 产生的小型反义 RNA 将被称为间隔区。引导Cas酶以匹配的靶标消灭入侵者在过去的十年中取得了很大进展。旨在了解 CRISPR 干扰酶及其在基因工程中的应用。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。使用革兰氏阴性病原体脑膜炎奈瑟菌（Nme）作为模式生物，因为其临床表现目前关于间隔适应的知识主要来自于对间隔适应的研究。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的我们最近的初步研究表明，记忆不受干扰酶的影响。发现建议采用II型脑膜炎奈瑟氏球菌的 CRISPR 通过一种独特的机制产生记忆：干扰酶 NmeCas9 和其 tracrRNA 和 crRNA 伙伴在功能间隔区的获得中发挥着关键但非传统的作用。我们使用分子遗传学、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？ Cas9/tracr 如何与 Cas1-2 整合酶合作？最后，抗 CRISPR 蛋白将如何影响记忆过程？该管理设备补充请求将允许访问 ddPCR 仪器，从而实现高核酸的通量和灵敏定量这对于破译 CRISPR 免疫至关重要。超越 DNA 转化，在丝状噬菌体感染的背景下，研究支持的影响。因此，所提出的研究将大大增强家长的资助。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。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Exploiting activation and inactivation mechanisms in type I-C CRISPR-Cas3 for genome-editing applications.

利用 I-C 型 CRISPR-Cas3 的激活和失活机制进行基因组编辑应用。

DOI：
10.1016/j.molcel.2023.12.034
发表时间：
2024-01-01
期刊：
Molecular cell
影响因子：
16
作者：
Chunyi Hu;Mason T. Myers;Xufei Zhou;Zhonggang Hou;Macy L. Lozen;Ki Hyun Nam;Yan Zhang;Ailong Ke
通讯作者：
Ailong Ke

Cas11 enables genome engineering in human cells with compact CRISPR-Cas3 systems.

Cas11 能够通过紧凑的 CRISPR-Cas3 系统在人类细胞中进行基因组工程。