Investigation of the understudied CRISPR-Cas immune systems I-B and I-D in Haloarchaea

盐古菌中尚未研究的 CRISPR-Cas 免疫系统 I-B 和 I-D 的研究

• 批准号: 433108819
• 负责人: Professorin Dr. Anita Marchfelder
• 金额: --
• 依托单位: Institut für Molekularbiologie und Biotechnologie der Prokaryoten
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/433108819?language=en
• 关键词: Investigation; understudied; CRISPR; Cas; immune

Although the CRISPR-Cas system has been identified as defence system of bacteria and archaea more than ten years ago, we still do not know all details about the molecular mechanisms of this immune system. There are currently 33 different variants of the CRISPR-Cas system known, all of them are defending the cell from invaders, but they do so with different sets of proteins and different molecular processes. While we know many details about a few variants, like the type I-E system from E. coli and the type II-A system from Streptococcus pyogenes, we now comparatively little about the other variants like type I-B and I-D. In addition, more is known about the bacterial systems than about the archaeal ones although only half of the bacteria contain a CRISPR-Cas system whereas almost all archaea encode one.Therefore we will investigate in this project the archaeal CRISPR-Cas systems I-B and I-D in Haloferax volcanii and Halorubrum lacusprofundi. Our preliminary results have revealed the details about the interference reaction of the Haloferax I-B system: the composition of the effector complex, PAM sequences and characteristics for a functional crRNA. In addition, we carried out the first experiments to characterise the I-D system in H. lacusprofundi and to study adaptation. Furthermore we could infect Halorubrum with viruses.In the frame of this project we want to analyse the I-D system of H. lacusprofundi in detail: the composition of its effector complex and the pre-requisites for an effective interference reaction (PAM sequences, characteristics for an active crRNA). H. lacusprofundi contains in addition to the I-D system also a I-B system and we want to investigate potential cross reactivities between both systems. Furthermore, we want to identify anti-CRISPR proteins for the I-B system. The adaptation step will also be investigated in Haloferax as well as in Halorubrum. For Haloferax we observed adaptation during self targeting and we will study this type of adaptation in detail. Using the viruses we found to infect Halorubrum we will study adaptation during viral infection in Halorubrum.Furthermore we will investigate the CRISPR associated CARF proteins. There are two CARF proteins in Haloferax, without known function and those we will study.We also want to develop a CRISPRa tool for activation of gene expression.

尽管CRISPR-CAS系统已被确定为十多年前的细菌和古细菌的防御系统，但我们仍然不知道有关该免疫系统分子机制的所有细节。当前已知的CRISPR-CAS系统有33种不同的变体，所有这些变体都在捍卫细胞免受入侵者的辩护，但是它们以不同的蛋白质和不同的分子过程来捍卫细胞。虽然我们知道有关几种变体的许多细节，例如来自大肠杆菌的I型系统和化脓性链球菌的II-A型系统，但我们现在却几乎没有其他变体（例如I型I-B和I-D）。此外，尽管只有一半的细菌含有CRISPR-CAS系统，但对细菌系统的了解比细菌系统的了解更多，而几乎所有古细菌都包含一个细菌。我们的初步结果揭示了有关Haloferax I-B系统干扰反应的细节：效应子复合物的组成，功能性CRRNA的PAM序列和特征。此外，我们进行了第一个实验，以表征H. lacusprofundi中的I-D系统并研究适应性。此外，我们可以用病毒感染Halorubrum。在该项目的框架中，我们要详细分析H. lacusprofundi的I-D系统：其效应子复合物的组成及其有效干扰反应的先决条件（PAM序列，有效CRRNA的特征）。 H. lacusprofundi除了I-D系统外还包含I-B系统，我们还希望研究两个系统之间的潜在交叉反应性。此外，我们希望确定I-B系统的抗Crispr蛋白。适应步骤还将在Haloferax和Halorubrum中进行研究。对于Haloferax，我们观察到自我靶向过程中的适应性，我们将详细研究这种适应性。使用我们发现的病毒感染Halorubrum，我们将研究halorubrum中病毒感染期间的适应性。Furthermore我们将研究CRISPR相关的CARF蛋白。 Haloferax中有两个CARF蛋白，没有已知功能，我们还将研究这些功能。我们还想开发一种CRISPRA工具来激活基因表达。