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Cited 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, D’Silva, Jovita, Burgio, Gaetan, Grohmann, Dina·2022·Nucleic Acids Research
DOI 10.1093/nar/gkac767PMC PMC9508855

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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 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.

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Research works printed but not linked: 54

  1. Barrangou, 2007, CRISPR provides acquired resistance against viruses in prokaryotes, Science, 315, 1709, 10.1126/science.1138140 · DOI · Paper page
  2. Makarova, 2020, Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants, Nat. Rev. Microbiol., 18, 67, 10.1038/s41579-019-0299-x · DOI · Paper page
  3. Marraffini, 2015, CRISPR-Cas immunity in prokaryotes, Nature, 7571, 55, 10.1038/nature15386 · DOI
  4. Mohanraju, 2016, Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems, Science, 353, 556, 10.1126/science.aad5147 · DOI
  5. Shmakov, 2017, Diversity and evolution of class 2 CRISPR–Cas systems, Nat. Rev. Microbiol., 15, 169, 10.1038/nrmicro.2016.184 · DOI · Paper page
  6. Mojica, 2009, Short motif sequences determine the targets of the prokaryotic CRISPR defence system, Microbiology, 155, 733, 10.1099/mic.0.023960-0 · DOI · Paper page
  7. Brouns, 2008, Small CRISPR RNAs guide antiviral defense in prokaryotes, Science, 321, 960, 10.1126/science.1159689 · DOI · Paper page
  8. Makarova, 2015, An updated evolutionary classification of CRISPR-Cas systems, Nat. Rev. Microbiol., 13, 722, 10.1038/nrmicro3569 · DOI · Paper page
  9. Nishimasu, 2017, Structures and mechanisms of CRISPR RNA-guided effector nucleases, Curr. Opin. Struct. Biol., 43, 68, 10.1016/j.sbi.2016.11.013 · DOI
  10. Swarts, 2017, Structural basis for guide RNA processing and seed-dependent DNA targeting by CRISPR-Cas12a, Mol. Cell, 66, 221, 10.1016/j.molcel.2017.03.016 · DOI
  11. Zetsche, 2015, Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system, Cell, 163, 759, 10.1016/j.cell.2015.09.038 · DOI · Paper page
  12. Yan, 2019, Functionally diverse type v CRISPR-Cas systems, Science, 363, 88, 10.1126/science.aav7271 · DOI
  13. Specht, 2020, Massively parallel CRISPRi assays reveal concealed thermodynamic determinants of dCas12a binding, PNAS, 117, 11274, 10.1073/pnas.1918685117 · DOI
  14. Yamano, 2017, Structural basis for the canonical and non-canonical PAM recognition by CRISPR-Cpf1, Mol. Cell, 67, 633, 10.1016/j.molcel.2017.06.035 · DOI · Paper page
  15. Jinek, 2012, A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity, Science, 337, 816, 10.1126/science.1225829 · DOI · Paper page
  16. Dong, 2016, The crystal structure of cpf1 in complex with CRISPR RNA, Nature, 532, 522, 10.1038/nature17944 · DOI · Paper page
  17. Yamano, 2016, Crystal structure of cpf1 in complex with guide RNA and target DNA, Cell, 165, 949, 10.1016/j.cell.2016.04.003 · DOI
  18. Wörle, 2021, Decoupling the bridge helix of cas12a results in a reduced trimming activity, increased mismatch sensitivity and impaired conformational transitions, Nucleic Acids Res., 49, 5278, 10.1093/nar/gkab286 · DOI
  19. Stella, 2018, Conformational activation promotes CRISPR-Cas12a catalysis and resetting of the endonuclease activity, Cell, 175, 1856, 10.1016/j.cell.2018.10.045 · DOI
  20. Fonfara, 2016, The CRISPR-associated DNA-cleaving enzyme cpf1 also processes precursor CRISPR RNA, Nature, 532, 517, 10.1038/nature17945 · DOI · Paper page
  21. Stella, 2017, Structure of the cpf1 endonuclease R-loop complex after target DNA cleavage, Nature, 546, 559, 10.1038/nature22398 · DOI · Paper page
  22. Cofsky, 2020, CRISPR-Cas12a exploits R-loop asymmetry to form double strand breaks, Elife, 9, e55143, 10.7554/eLife.55143 · DOI
  23. Strohkendl, 2018, Kinetic basis for DNA target specificity of CRISPR-Cas12a, Mol. Cell, 71, 816, 10.1016/j.molcel.2018.06.043 · DOI
  24. Hille, 2018, The biology of CRISPR-Cas: backward and forward, Cell, 172, 1239, 10.1016/j.cell.2017.11.032 · DOI
  25. Jeon, 2018, Direct observation of DNA target searching and cleavage by CRISPR-Cas12a, Nat. Commun., 9, 2777, 10.1038/s41467-018-05245-x · DOI · Paper page
  26. Singh, 2018, Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR cpf1 (Cas12a), Proc. Natl. Acad. Sci. U.S.A., 115, 5444, 10.1073/pnas.1718686115 · DOI
  27. Fu, 2019, Target-dependent nickase activities of the CRISPR-Cas nucleases cpf1 and cas9, Nat. Microbiol., 4, 888, 10.1038/s41564-019-0382-0 · DOI
  28. Fuchs, 2019, Cas12a trans-cleavage can be modulated in vitro and is active on ssDNA, dsDNA, and RNA, 10.1101/600890 · DOI
  29. Murugan, 2020, CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects, J. Biol. Chem., 295, 5538, 10.1074/jbc.RA120.012933 · DOI
  30. Li, 2018, CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA, Cell Res., 28, 491, 10.1038/s41422-018-0022-x · DOI
  31. Chen, 2018, CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity, Science, 360, 436, 10.1126/science.aar6245 · DOI · Paper page
  32. Varble, 2019, Three new C’s for CRISPR: collateral, communicate, cooperate, Trends Genet., 35, 446, 10.1016/j.tig.2019.03.009 · DOI
  33. Li, 2018, CRISPR-Cas12a-assisted nucleic acid detection, Cell Discov., 4, 18, 10.1038/s41421-018-0028-z · DOI
  34. Gootenberg, 2018, Multiplexed and portable nucleic acid detection platform with cas13, cas12a, and csm6, Science, 6387, 439, 10.1126/science.aaq0179 · DOI
  35. Parameshwaran, 2021, The bridge helix of cas12a imparts selectivity in cis-DNA cleavage and regulates trans-DNA cleavage, FEBS Lett., 595, 892, 10.1002/1873-3468.14051 · DOI
  36. Swarts, 2018, Mechanistic insights into the cis- and trans-acting DNase activities of cas12a, Mol. Cell, 73, 589, 10.1016/j.molcel.2018.11.021 · DOI
  37. Chin, 2002, Addition of p-azido-L-phenylalanine to the genetic code of escherichia coli, J. Am. Chem. Soc., 124, 9026, 10.1021/ja027007w · DOI
  38. Grohmann, 2013, Making connections-strategies for single molecule fluorescence biophysics, Curr. Opin. Chem. Biol., 17, 691, 10.1016/j.cbpa.2013.05.020 · DOI
  39. Saxon, 2000, Cell surface engineering by a modified staudinger reaction, Science, 287, 2007, 10.1126/science.287.5460.2007 · DOI
  40. Ramachandran, 2021, CRISPR enzyme kinetics for molecular diagnostics, Anal. Chem., 93, 7456, 10.1021/acs.analchem.1c00525 · DOI
  41. Schrimpf, 2018, PAM: a framework for integrated analysis of imaging, single-molecule, and ensemble fluorescence data, Biophys. J., 114, 1518, 10.1016/j.bpj.2018.02.035 · DOI
  42. Nir, 2006, Shot-Noise limited single-molecule FRET histograms: comparison between theory and experiments, J. Phys. Chem. B, 110, 22103, 10.1021/jp063483n · DOI
  43. Hellenkamp, 2018, Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study, Nat. Methods, 15, 669, 10.1038/s41592-018-0085-0 · DOI · Paper page
  44. Torella, 2011, Identifying molecular dynamics in single-molecule FRET experiments with burst variance analysis, Biophys. J., 100, 1568, 10.1016/j.bpj.2011.01.066 · DOI
  45. Yamada, 2017, Crystal structure of the minimal cas9 from campylobacter jejuni reveals the molecular diversity in the CRISPR-Cas9 systems, Mol. Cell, 65, 1109, 10.1016/j.molcel.2017.02.007 · DOI
  46. Jiang, 2016, Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage, Science, 351, 867, 10.1126/science.aad8282 · DOI
  47. Anders, 2014, Structural basis of PAM-dependent target DNA recognition by the cas9 endonuclease, Nature, 513, 569, 10.1038/nature13579 · DOI · Paper page
  48. Tomov, 2012, Disentangling subpopulations in single-molecule FRET and ALEX experiments with photon distribution analysis, Biophys. J., 102, 1163, 10.1016/j.bpj.2011.11.4025 · DOI
  49. Tsukanov, 2013, Conformational dynamics of DNA hairpins at millisecond resolution obtained from analysis of single-molecule FRET histograms, J. Phys. Chem. B, 117, 16105, 10.1021/jp411280n · DOI
  50. Swarts, 2018, Cas9 versus cas12a/cpf1: Structure–function comparisons and implications for genome editing, Wiley Interdiscip. Rev. RNA, 9, e1481, 10.1002/wrna.1481 · DOI · Paper page
  51. Nishimasu, 2015, Crystal structure of staphylococcus aureus cas9, Cell, 162, 1113, 10.1016/j.cell.2015.08.007 · DOI
  52. Bratovič, 2020, Bridge helix arginines play a critical role in cas9 sensitivity to mismatches, Nat. Chem. Biol., 16, 587, 10.1038/s41589-020-0490-4 · DOI
  53. Gao, 2016, Type v CRISPR-Cas cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition, Cell Res., 26, 901, 10.1038/cr.2016.88 · DOI
  54. van Aelst, 2019, The effect of DNA topology on observed rates of R-Loop formation and DNA strand cleavage by CRISPR cas12a, Genes (Basel), 10, 169, 10.3390/genes10020169 · DOI

Metadata

Authors
Wörle, E., Newman, A., D’Silva, J., Burgio, G., & Grohmann, D.
Affiliations
  • University of Regensburg (DE)
  • Australian National University (AU)
  • Division of Genome Sciences and Cancer, The John Curtin School of Medical Research, The Australian National University , Canberra, ACT 2601 , Australia
Corresponding author
Grohmann, Dina
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2022-09-15
Volume
50
Issue
17
Pages
10153-10168
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gkac767
PMC
PMC9508855
OpenAlex
W4295893236
Cited by
32
References
4
Citations by year
2026: 11 · 2025: 8 · 2024: 8 · 2023: 4 · 2022: 1
FWCI
1.71
Type
journal article
Language
en
Open access
Licence
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
  • Deutsche Forschungsgemeinschaft (GR 3840/3-2)
  • Australian-Germany Joint Research co-operation scheme – University Australia/German Academic Exchange Service (UA-DAAD 575113331)
  • Australian Government
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, CRISPR-Cas Systems, CRISPR-Associated Proteins

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