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

古细菌原核免疫系统 I-B 和 I-D 的研究

• 批准号: 206942411
• 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 Units
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/206942411?language=en
• 关键词: Investigation; prokaryotic; immune; systems; archaea

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.

在上一个供资期间，我们描述了CRISPR-CAS第I-B类Haloferax火山II系统的特点。我们可以对Haloferax的PAM序列进行鉴定，使其成为第二个被鉴定PAM序列的考古子。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来实现crRNA和入侵者DNA的有效相互作用。此外，我们可以首次证明I-B型系统具有级联样复合体，其核心亚单位为Cas5、Cas7和crRNA。总而言之，这些观察结果增加了关于迄今尚未确定的I-B类型系统的新数据。在下一个供资期间，我们希望完成I-B型Haloferax系统的特征。使用Haloferax的优势是可以获得大量的遗传工具，使我们能够在体内检查系统。我们将详细研究级联复合体中蛋白质-蛋白质和蛋白质-RNA的相互作用。此外，我们将分析适应模块，并开发一个针对古生菌的CRISPRi工具。使用合成的CRISPR基因座，我们将确定有效处理的要求。为了找出具有功能的crRNA的本质，我们将在体内开发一条独立于Cas6的crRNA成熟途径。下一个供资期间的第二个主要重点将是对I-D类型系统的调查。到目前为止，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：温度丝状挂起菌和深水卤虫。在大肠杆菌中产生重组蛋白的基因将来自T.Pendens，一种嗜热古细菌。初步结果表明，这种生物的基因可以在大肠杆菌中进行可溶性表达。重组蛋白将用于生化研究(与crRNA结合，crRNA的加工)和结构研究(阐明单个蛋白质和I-D级联复合体的结构)。我们将对光环考古虫H.lacusprofundi进行活体研究。在这里，我们将删除单个蛋白基因，并分析由此产生的缺失菌株产生crRNAs的能力和在干扰中的活跃程度。我们还将调查从Halorubrum栖息地采集的环境样本中是否有能够感染Halorubrum的病毒。卤虫病毒的分离将使我们能够研究I-D系统的适应过程。综上所述，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。