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

• Applicant: Professorin Dr. Anita Marchfelder
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2019 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 433108819

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.

• DFG Programme: Research Grants