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

点击翻译按钮获取中文摘要

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.