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系统十多年前就已被确定为细菌和古细菌的防御系统，但我们仍然不知道该免疫系统分子机制的所有细节。目前已知 CRISPR-Cas 系统有 33 种不同的变体，它们都可以保护细胞免受入侵者的侵害，但它们使用不同的蛋白质组和不同的分子过程。虽然我们了解一些变体的许多细节，例如大肠杆菌的 I-E 型系统和化脓性链球菌的 II-A 型系统，但我们现在对 I-B 型和 I-D 型等其他变体知之甚少。此外，尽管只有一半的细菌含有 CRISPR-Cas 系统，而几乎所有古细菌都编码一个 CRISPR-Cas 系统，但对细菌系统的了解比对古细菌系统的了解更多。因此，我们将在本项目中研究 Haloferax volcanii 和 Halorubrum lacusprofundi 中的古细菌 CRISPR-Cas 系统 I-B 和 I-D。我们的初步结果揭示了 Haloferax I-B 系统干扰反应的细节：效应复合物的组成、PAM 序列和功能性 crRNA 的特征。此外，我们还进行了首次实验来表征 H. lacusprofundi 的 I-D 系统并研究适应性。此外，我们可以用病毒感染Halorubrum。在这个项目的框架中，我们想要详细分析H. lacusprofundi的I-D系统：其效应复合物的组成以及有效干扰反应的先决条件（PAM序列，活性crRNA的特征）。 H. lacusprofundi 除了 I-D 系统外还包含 I-B 系统，我们想要研究两个系统之间潜在的交叉反应性。此外，我们想要鉴定 I-B 系统的抗 CRISPR 蛋白。适应步骤也将在 Haloferax 和 Halorubrum 中进行研究。对于Haloferax，我们观察到自我瞄准过程中的适应，我们将详细研究这种类型的适应。利用我们发现的感染Halorubrum 的病毒，我们将研究Halorubrum 病毒感染过程中的适应性。此外，我们将研究与CRISPR 相关的CARF 蛋白。 Haloferax 中有两种功能未知的 CARF 蛋白，我们将研究它们。我们还想开发一种用于激活基因表达的 CRISPRa 工具。