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Craspase Orthologs Cleave a Nonconserved Site in Target Protein Csx30.

Sam P B van BeljouwAnna C HaagsmaKonstantinos KalogeropoulosMartin PabstStan J J Brouns
Published in: ACS chemical biology (2024)
The Craspase CRISPR-Cas effector consists of the RNA-guided ribonuclease gRAMP and the protease TPR-CHAT, coupling target RNA recognition to protease activation. The natural substrate of Craspase is Csx30, a protein cleaved in two fragments that subsequently activates downstream antiviral pathways. Here, we determined the protease substrate specificity of Craspase from Candidatus "Jettenia caeni" ( Jc -Craspase). We find that Jc -Craspase cleaves Jc -Csx30 in a target RNA-dependent fashion in A|S, which is different from the sites found in two other studied Craspases (L|D and M|K for Candidatus "Scalindua brodae" and Desulfonema ishimotonii , respectively). The fact that Craspase cleaves a nonconserved site across orthologs indicates the evolution of specific protein interactions between Craspase and its respective Csx30 target protein. The Craspase family thus represents a panel of proteases with different substrate specificities, which we exploited for the development of a readout for multiplexed RNA detection.
Keyphrases
  • amino acid
  • crispr cas
  • protein protein
  • dendritic cells
  • genome editing
  • structural basis
  • regulatory t cells
  • sensitive detection