biostudies-literatureWorle EDeutsche ForschungsgemeinschaftAustralian-Germany Joint Research co-operation scheme – University Australia/German Academic Exchange ServiceAustralian Government10153-10168https://www.ebi.ac.uk/biostudies/studies/S-EPMC9508855biostudies-literatureUnknown50(17)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 clNucleic acids researchAllosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain.PMC9508855UA-DAAD 575113331GR 3840/3-2Grohmann DNewman AWorle ED'Silva JBurgio GfalseAllosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain.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 cl2022-01-01T00:00:00Z2022 Sep2026-05-28T01:09:28.737Z2025-04-19T22:49:04.136ZS-EPMC95088553610776710.1093/nar/gkac767