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

在上一个资助期，我们表征了Haloferax volcanii的CRISPR-Cas I-B型系统。我们可以确定Haloferax的PAM序列，使其成为第二个确定PAM序列的古细菌。我们可以证明，与大肠杆菌的I-E系统类似，I-B型系统需要一个种子序列用于crRNA和入侵DNA的有效相互作用。杆菌此外，我们可以首次证明I-B型系统具有与核心亚基Cas 5、Cas 7和crRNA的级联样复合物。这些观测结果一起为迄今为止尚未描述的I-B型系统增加了新的数据。在下一个资助期内，我们希望完成I-B型Haloferax系统的表征。使用Haloferax的优点是提供了大量的遗传工具，使我们能够在体内检查系统。我们将详细研究Cascade复合物中蛋白质-蛋白质和蛋白质-RNA的相互作用。此外，我们还将分析适应模块，并为古细菌开发CRISPRi工具。使用合成的CRISPR基因座，我们将确定有效处理的要求。为了查明功能性crRNA的要素，我们将在体内开发不依赖于Cas 6的crRNA成熟途径。下一个资助期的第二个主要重点将是I-D型系统的研究。迄今为止，几乎没有关于该系统的报道。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体：Thermofilum pendens和Halorubrum lacusprofundi。在E. coli中的T. pendens，一种嗜热古菌。初步结果表明，该菌的基因可以在大肠杆菌中可溶性表达。杆菌重组蛋白将用于进行生物化学研究（与crRNA结合，crRNA加工）和结构研究（阐明单个蛋白质和I-D级联复合物的结构）。我们将用盐古菌H进行体内研究。深湖在这里，我们将删除单个蛋白质基因，并分析产生的缺失菌株产生crRNA和干扰活性的能力。我们还将调查来自Halorubrum栖息地的环境样本，以寻找能够感染Halorubrum的病毒。Halorubrum病毒的分离将使我们能够研究I-D系统的适应过程。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.