Investigation of the prokaryotic immune systems I-B and I-D in archaea

在之前的资助期间，我们描述了火山盐藻的CRISPR-Cas型I-B系统。结果表明，该菌是第2个鉴定出PAM序列的古菌。我们可以证明I-B型系统需要一个种子序列来实现crRNA和入侵DNA的有效相互作用，类似于大肠杆菌的I-E系统。此外，我们可以首次证明I-B型系统具有具有核心亚基Cas5， Cas7和crRNA的级联状复合体。这些观测结果加在一起，为迄今为止尚未确定的I-B型系统增添了新的数据。在下一个资助期内，我们希望完成I-B型氟化氟醚系统的特性描述。使用Haloferax的优点是可以使用大量的遗传工具，使我们能够在体内检查系统。我们将详细研究Cascade复合体中蛋白质-蛋白质和蛋白质- rna的相互作用。此外，我们将分析适应模块并开发用于古细菌的CRISPRi工具。使用合成的CRISPR基因座，我们将确定有效处理的要求。为了确定功能性crRNA的本质，我们将在体内开发一种独立于Cas6的crRNA成熟途径。下一个筹资期间的第二个重点将是调查第1 - d类系统。迄今为止，几乎没有关于这个系统的报道。为了能够在体外和体内有效地分析它，我们将使用两种不同的生物：Thermofilum pendens和Halorubrum lacusprofundi。大肠杆菌中产生重组蛋白的基因将取自T. pendens，一种嗜热古菌。初步结果表明，该菌的基因可在大肠杆菌中可溶性表达。重组蛋白将用于生化研究（结合crRNA，加工crRNA）和结构研究（阐明单个蛋白和I-D级联复合物的结构）。我们将对深湖盐古菌进行体内研究。在这里，我们将删除单个蛋白质基因，并分析由此产生的删除菌株产生crrna的能力和在干扰中活跃的能力。我们还将调查来自盐藻栖息地的环境样本，寻找能够感染盐藻的病毒。halorrubrum病毒的分离将使我们能够研究I-D系统的适应过程。综合这些方法将有助于我们阐明I-D系统的分子细节，并将其与I-B系统进行比较。

In the previous funding period we characterised the CRISPR-Cas type I-B system of Haloferax volcanii. We could identify the PAM sequences for Haloferax, making it only the second archaeon for which the PAM sequences were identified. We could show that the type I-B system needs a seed sequence for the efficient interaction of crRNA and invader DNA similar to the I-E system of E. coli. Further we could show for the first time that the type I-B system has a Cascade-like complex with the core subunits Cas5, Cas7 and the crRNA. Together these observations add new data about the hitherto uncharacterised type I-B system. In the next funding period we want to complete the characterisation of the type I-B Haloferax system. The advantages of using Haloferax are the availability of a plethora of genetic tools which allows us to examine the system in vivo. We will investigate the protein-protein and protein-RNA interactions in the Cascade complex in detail. In addition we will analyse the adaptation module and develop a CRISPRi tool for archaea. Using a synthetic CRISPR locus we will determine the requirements for efficient processing. To pinpoint the essentials of a functional crRNA we will develop a crRNA maturation pathway independent of Cas6 in vivo. A second major focus in the next funding period will be the investigation of the type I-D system. Hitherto almost nothing has been reported about this system. To be able to effectively analyse it in vitro and in vivo we will use two different organisms: Thermofilum pendens and Halorubrum lacusprofundi. The genes for generation of recombinant proteins in E. coli will be taken from T. pendens, a thermophilic archaeon. Preliminary results show that genes from this organism can be solubly expressed in E. coli. The recombinant proteins will be used to do biochemical studies (binding to crRNA, processing of crRNAs) and structural studies (elucidation of the structure of single proteins and the I-D Cascade complex). We will do in vivo studies with the haloarchaeon H. lacusprofundi. Here we will delete the individual protein genes and analyse the resulting deletion strains for their ability to produce crRNAs and to be active in interference. We will also investigate environmental samples from Halorubrum habitats for viruses that are able to infect Halorubrum. The isolation of Halorubrum viruses will allow us to study the adaptation process of the I-D system. Taken together these approaches will help us to elucidate the molecular details of the I-D system and to compare it to the I-B system.