Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain. Issue 17 (15th September 2022)

• Record Type: Journal Article
• Authors: Wörle, Elisabeth; Newman, Anthony; D'Silva, Jovita; Burgio, Gaetan; Grohmann, Dina
• Is Part Of: Nucleic acids research. Volume 50:Issue 17(2022)
• Page Start: 10153
• Page End: 10168
• Publication Date: 2022-09-15
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkac767
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: Nucleases derived from the prokaryotic defense system CRISPR-Cas are frequently re-purposed for gene editing and molecular diagnostics. Hence, an in-depth understanding of the molecular mechanisms of these enzymes is of crucial importance. We focused on Cas12a from Francisella novicida (FnCas12a) and investigated the functional role of helix 1, a structural element that together with the bridge helix (BH) connects the recognition and the nuclease lobes of FnCas12a. Helix 1 is structurally connected to the lid domain that opens upon DNA target loading thereby activating the active site of FnCas12a. We probed the structural states of FnCas12a variants altered in helix 1 and/or the bridge helix using single-molecule FRET measurements and assayed the pre-crRNA processing, cis- and trans- DNA cleavage activity. We show that helix 1 and not the bridge helix is the predominant structural element that confers conformational stability of FnCas12a. Even small perturbations in helix 1 lead to a decrease in DNA cleavage activity while the structural integrity is not affected. Our data, therefore, implicate that the concerted remodeling of helix 1 and the bridge helix upon DNA binding is structurally linked to the opening of the lid and therefore involved in the allosteric activation of the active site.