Gene knockdown represents a powerful technology to investigate the in vivo role of essential genes, which cannot be studied by gene disruption. CRISPR type III systems, which are abundantly found in archaea, recognize and degrade RNA in their specific native immune response against invading nucleic acids. In this study, we harnessed these systems for comparative targeted gene knockdown of essential genes belonging to five different functional categories, i.e. cell division (cdvA), transcription (rpo8), translation (aIF5A), RNA metabolism (smAP2) and cell wall biogenesis (slaB) in hyperthermophilic crenarchaea of the order Sulfolobales. Comparison of over 100 individual transformants carrying differently srong silencing constructs revealed gene-specific silencing maxima, which induced specific phenotypes that could be studied in vivo. Stronger silencing exceeding the maximum was abrogated by setting specific mutations in the CRISPR array carried with the silencing construct. Thus, this study not only gives valuable insights into the function of essential genes and the required dosage of the respective protein needed for cell survival, but also reveals a new, probably CRISPR-specific mechanism that acts to mutate CRISPR arrays.
Original paper:
RNA Biology, DOI: 10.1080/15476286.2020.1813411
Comparative CRISPR type III-based knockdown of essential genes in hyperthermophilic Sulfolobales and the evasion of lethal gene silencing
Isabelle Anna Zink, Thomas Fouqueau, Gabriel Tarrason Risa, Finn Werner, Buzz Baum, Ulo Bläsi and Christa Schleper.
Full article: https://www.tandfonline.com/doi/full/10.1080/15476286.2020.1813411