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

尽管CRISPR-Cas系统在十多年前就被确定为细菌和古细菌的防御系统，但我们仍然不知道关于这种免疫系统的分子机制的所有细节。目前已知的CRISPR-Cas系统有33种不同的变体，它们都在保护细胞免受入侵，但它们使用不同的蛋白质和不同的分子过程。虽然我们知道一些变体的许多细节，比如E.大肠杆菌和化脓性链球菌的II-A型系统，目前对I-B和I-D型系统的研究相对较少。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要多于古细菌系统，而几乎所有的古细菌都编码CRISPR-Cas系统。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crRNA的特征。此外，我们还首次在H. lacusprofundi和研究适应。在本项目的框架下，我们想对H. lacusprofundi详细：其效应复合物的组成和有效干扰反应的前体（PAM序列，活性crRNA的特征）。H.除了I-D系统外，lacusprofundi还包含I-B系统，我们希望研究两个系统之间的潜在交叉反应性。此外，我们还希望鉴定I-B系统的抗CRISPR蛋白。适应步骤也将在Haloferax以及Halorubrum中进行研究。对于Haloferax，我们在自我靶向过程中观察到了适应，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在病毒感染过程中的适应性。此外，我们将研究CRISPR相关的CARF蛋白。在Haloferax中有两种CARF蛋白，没有已知的功能和我们将要研究的那些。我们还想开发一种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.