CRISPR-Cas tools for Haloarchaea

Abstract

Due to their complex and unusual biology, the development of tools for archaea is not simple and so far, no tool for the regulation of gene repression has been available. However, all of archaea have CRISPR-Cas systems, which are an excellent source for the development of new methods. Using haloarchaeon Haloferax volcanii as an example, we show that these CRISPR-Cas systems can be transformed into tools for gene repression: If the degradation of the target DNA does not take place upon deletion of the effector nuclease Cas3, the Cascade complex remains bound and blocks the access for RNA polymerase (CRISPRi). Cascade-based gene repression is an efficient and valuable tool to study essential genes in archaea. CRISPRi is highly modular and can quickly and easily be directed towards a different target gene by the exchange of the crRNA. Here we summarize the steps to repurpose the CRISPR-Cas system of H. volcanii and report on applications in our laboratory.