Streamlined and efficient genome editing in Cupriavidus necator H16 using an optimised SIBR-Cas system.

Cupriavidus necator H16 is a promising microbial platform strain for CO valorisation. While C. necator is amenable to genome editing, existing tools are often inefficient or rely on lengthy protocols, hindering its rapid transition to industrial applications.

Type III-B CRISPR-Cas cascade of proteolytic cleavages.

The generation of cyclic oligoadenylates and subsequent allosteric activation of proteins that carry sensory domains is a distinctive feature of type III CRISPR-Cas systems. In this work, we characterize a set of associated genes of a type III-B system from that contains two caspase-like proteases, SAVED-CHAT and PCaspase (prokaryotic caspase), co-opted from a cyclic oligonucleotide-based antiphage signaling system (CBASS). Cyclic tri-adenosine monophosphate (AMP)-induced oligomerization of SAVED-CHAT activates proteolytic activity of the CHAT domains, which specifically cleave and activate PCaspase.

Characterization of the AcrIIC1 anti‒CRISPR protein for Cas9‒based genome engineering in E. coli.

Anti-CRISPR proteins (Acrs) block the activity of CRISPR-associated (Cas) proteins, either by inhibiting DNA interference or by preventing crRNA loading and complex formation. Although the main use of Acrs in genome engineering applications is to lower the cleavage activity of Cas proteins, they can also be instrumental for various other CRISPR-based applications. Here, we explore the genome editing potential of the thermoactive type II-C Cas9 variants from Geobacillus thermodenitrificans T12 (ThermoCas9) and Geobacillus stearothermophilus (GeoCas9) in Escherichia coli.