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

虽然CRISPR-Cas系统早在十多年前就被确定为细菌和古生菌的防御系统，但我们仍然不知道该免疫系统的所有分子机制。目前已知的CRISPR-Cas系统有33种不同的变体，所有这些变体都在保护细胞免受入侵者的侵袭，但它们通过不同的蛋白质组和不同的分子过程来实现这一点。虽然我们知道一些变异体的许多细节，比如大肠杆菌的I-E系统和化脓性链球菌的II-A系统，但我们现在对I-B和I-D等其他变异体的了解相对较少。此外，尽管只有一半的细菌含有CRISPR-Cas系统，而几乎所有的古生菌都编码CRISPR-Cas系统，但人们对细菌系统的了解比对古生菌系统的了解更多。因此，我们将在本项目中对Haloferax cancanii和Halorubrum lacus深部的古生菌CRISPR-Cas系统I-B和I-D进行研究。我们的初步结果揭示了Haloferax I-B系统的干扰反应的细节：效应复合体的组成、PAM序列和功能crRNA的特征。此外，我们还进行了第一次实验，以刻画拉氏沼虾的I-D系统，并研究其适应性。在这个项目的框架内，我们想要详细地分析梭状芽孢杆菌的I-D系统：其效应复合体的组成和有效干扰反应的先决条件(PAM序列，活性crRNA的特征)。除了I-D系统外，H.lacusprofundi还包含一个I-B系统，我们想要研究这两个系统之间的潜在交叉反应。此外，我们希望为I-B系统鉴定抗CRISPR蛋白。还将在Haloferax以及Halorubrum调查适应步骤。对于Haloferax，我们观察到了自定位期间的适应，我们将详细研究这种类型的适应。利用我们发现的感染卤虫的病毒，我们将研究病毒感染卤虫过程中的适应。此外，我们还将研究CRISPR相关的CARF蛋白。在Haloferax中有两个CARF蛋白，功能未知，我们将对其进行研究。我们还想开发一个CRISPRa工具来激活基因表达。

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.