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PreprintCited by 32CRISPR and Genetic EngineeringDNA and Nucleic Acid ChemistryRNA regulation and disease

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Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain

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Wörle, Elisabeth, Newman, Anthony, Burgio, Gaetan, Grohmann, Dina·2022·Nucleic Acids Research
DOI 10.1101/2022.03.15.484427PMC PMC9508855

Preprint — published as 10.1093/nar/gkac767 · Paper page

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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 BH 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 BH 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 BH upon DNA binding is structurally linked to the opening of the lid and therefore involved in the allosteric activation of the active site.

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Metadata

Authors
Wörle, E., Newman, A., Burgio, G., & Grohmann, D.
Affiliations
  • University of Regensburg (DE)
  • Australian National University (AU)
Corresponding author
Grohmann, Dina
ORCID
Venue
Nucleic Acids Research
Publisher
openRxiv
Published
2022-03-16
Volume
50
Issue
17
Pages
10153-10168
ISSN
0305-1048 (ISSN-L)
DOI
10.1101/2022.03.15.484427
PMC
PMC9508855
OpenAlex
W4295893236
Cited by
32
References
1
Citations by year
2026: 11 · 2025: 8 · 2024: 8 · 2023: 4 · 2022: 1
FWCI
1.71
Type
journal article
Language
en
Open access
Licence
cc-by-nc
Locations
  • Nucleic Acids Research (publishedVersion, cc-by-nc)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • University of Regensburg Publication Server (University of Regensburg) (acceptedVersion)
  • University of Regensburg Publication Server (University of Regensburg)
Funders
  • National Computational Infrastructure
  • Australian Government
  • Deutscher Akademischer Austauschdienst
  • Deutsche Forschungsgemeinschaft (SPP 2141, GR 3840/3-2)
  • National Cancer Institute
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
Keywords
CRISPR/Cas12a, Francisella novicida, allosteric activation, helix 1, bridge helix, lid domain, DNA binding, DNA cleavage activity, conformational stability, single-molecule FRET
MeSH terms
Gene Editing, RNA, Guide, CRISPR-Cas Systems, Allosteric Regulation, Bacterial Proteins, DNA, Endonucleases, RNA, Guide, Kinetoplastida, CRISPR-Cas Systems, CRISPR-Associated Proteins

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