To date, a plethora of tools for molecular biology have been developed on the basis of the CRISPR-Cas
system. Almost all use the class 2 systems since here the setup is the simplest with only one protein and one
guide RNA, allowing for easy transfer to and expression in other organisms. However, the CRISPR-Cas
components harnessed for applications are derived from mesophilic bacteria and are not optimal for use in
extremophilic archaea.
Here, we describe the application of an endogenous CRISPR-Cas system as a tool for silencing gene
expression in a halophilic archaeon. Haloferax volcanii has a CRISPR-Cas system of subtype I-B, which can
be easily used to repress the transcription of endogenous genes, allowing to study the effects of their
depletion. This article gives a step-by-step introduction on how to use the implemented system for any gene
of interest in Haloferax volcanii. The concept of CRISPRi described here for Haloferax can be transferred
to any other archaeon, that is genetically tractable and has an endogenous CRISPR-Cas I systems.
Schwarz, T.S., Schreiber, S.S., Marchfelder, A. (2022). CRISPR Interference as a Tool to Repress Gene Expression in Haloferax volcanii. In: Ferreira-Cerca, S. (eds) Archaea. Methods in Molecular Biology, vol 2522. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2445-6_4
Link to chapter: https://link.springer.com/protocol/10.1007/978-1-0716-2445-6_4