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Cited by 30CRISPR and Genetic EngineeringRNA regulation and diseaseBacterial Genetics and Biotechnology

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RNA targeting and cleavage by the type III-Dv CRISPR effector complex

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Schwartz, Evan A., Bravo, Jack P. K., Ahsan, Mohd, Macias, Luis A., McCafferty, Caitlyn L., Dangerfield, Tyler L., Walker, Jada N., Brodbelt, Jennifer S., Palermo, Giulia, Fineran, Peter C., Fagerlund, Robert D., Taylor, David W.·2024·Nature Communications
DOI 10.1038/s41467-024-47506-yPMC PMC11026444

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Abstract

CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction 1–5 . Target RNA cleavage at regular intervals is characteristic of type III effector complexes 6–8 . Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors 9,10 , in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2′-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.

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  1. Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007). · DOI · Paper page
  2. Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl Acad. Sci. USA 109, E2579–E2586 (2012). · DOI · Paper page
  3. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012). · DOI · Paper page
  4. Hale, C. R. et al. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139, 945–956 (2009). · DOI · Paper page
  5. Staals, R. H. J. et al. Structure and activity of the RNA-targeting Type III-B CRISPR-Cas complex of Thermus thermophilus. Mol. Cell 52, 135 (2013). · DOI
  6. Staals, R. H. J. et al. RNA targeting by the type III-A CRISPR-Cas Csm complex of Thermus thermophilus. Mol. Cell 56, 518–530 (2014). · DOI
  7. Hale, C. R., Cocozaki, A., Li, H., Terns, R. M. & Terns, M. P. Target RNA capture and cleavage by the Cmr type III-B CRISPR-Cas effector complex. Genes Dev. https://doi.org/10.1101/gad.250712.114 (2014). · DOI
  8. Makarova, K. S. et al. Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants. Nat. Rev. Microbiol. 18, 67–83 (2020). · DOI · Paper page
  9. Niewoehner, O. et al. Type III CRISPR–Cas systems produce cyclic oligoadenylate second messengers. Nature 548, 543–548 (2017). · DOI · Paper page
  10. Kazlauskiene, M., Kostiuk, G., Venclovas, Č., Tamulaitis, G. & Siksnys, V. A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems. Science 357, 605–609 (2017). · DOI · Paper page
  11. Jia, N., Jones, R., Sukenick, G. & Patel, D. J. Second messenger cA4 formation within the composite Csm1 palm pocket of type III-A CRISPR-Cas Csm complex and its release path. Mol. Cell 75, 933–943.e6 (2019). · DOI
  12. Sofos, N. et al. Structures of the Cmr-β complex reveal the regulation of the immunity mechanism of type III-B CRISPR-Cas. Mol. Cell 79, 741–757.e7 (2020). · DOI
  13. Makarova, K. S. et al. Evolutionary and functional classification of the CARF domain superfamily, key sensors in prokaryotic antivirus defense. Nucleic Acids Res. 48, 8828–8847 (2020). · DOI
  14. Mayo-Muñoz, D. et al. Type III CRISPR-Cas provides resistance against nucleus-forming jumbo phages via abortive infection. Mol. Cell 82, 4471–4486.e9 (2022). · DOI
  15. Elmore, J. R. et al. Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system. Genes Dev. 30, 447 (2016). · DOI
  16. Kazlauskiene, M., Tamulaitis, G. & Kostiuk, G. Venclovas, Č. & Siksnys, V. Spatiotemporal control of type III-A CRISPR-Cas immunity: coupling DNA degradation with the target RNA recognition. Mol. Cell 62, 295–306 (2016). · DOI · Paper page
  17. Samai, P. et al. Co-transcriptional DNA and RNA cleavage during type III CRISPR-Cas immunity. Cell 161, 1164–1174 (2015). · DOI · Paper page
  18. Tamulaitis, G. et al. Programmable RNA shredding by the type III-A CRISPR-Cas system of Streptococcus thermophilus. Mol. Cell 56, 506–517 (2014). · DOI
  19. Grüschow, S., Adamson, C. S. & White, M. F. Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector. Nucleic Acids Res. 49, 13122–13134 (2021). · DOI
  20. Steens, J. A. et al. SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation. Nat. Commun. 12, 1–12 (2021). · DOI
  21. Santiago-Frangos, A. et al. Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic. Cell Rep. Med. 2, 100319 (2021). · DOI
  22. van Beljouw, S. P. B. et al. The gRAMP CRISPR-Cas effector is an RNA endonuclease complexed with a caspase-like peptidase. Science 373, 1349–1353 (2021). · DOI
  23. Osawa, T., Inanaga, H., Sato, C. & Numata, T. Crystal structure of the CRISPR-cas RNA silencing CMR complex bound to a target analog. Mol. Cell 58, 418–430 (2015). · DOI · Paper page
  24. Goswami, H. N., Rai, J., Das, A. & Li, H. Molecular mechanism of active Cas7-11 in processing CRISPR RNA and interfering target RNA. Elife 11, e81678 (2022). · DOI · Paper page
  25. You, L. et al. Structure studies of the CRISPR-Csm complex reveal mechanism of co-transcriptional interference. Cell 176, 239–253.e16 (2019). · DOI
  26. Scholz, I., Lange, S. J., Hein, S., Hess, W. R. & Backofen, R. CRISPR-Cas systems in the cyanobacterium Synechocystis sp. PCC6803 exhibit distinct processing pathways involving at least two Cas6 and a Cmr2 protein. PLoS ONE 8, e56470 (2013). · DOI
  27. Reimann, V. et al. Structural constraints and enzymatic promiscuity in the Cas6-dependent generation of crRNAs. Nucleic Acids Res. 45, 915–925 (2017). · DOI
  28. Taylor, D. W. et al. Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning. Science 348, 581–585 (2015). · DOI
  29. Sridhara, S. et al. Structural and biochemical characterization of in vivo assembled Lactococcus lactis CRISPR-Csm complex. Commun. Biol. 5, 1–12 (2022). · DOI
  30. Kato, K. et al. Structure and engineering of the type III-E CRISPR-Cas7-11 effector complex. Cell https://doi.org/10.1016/J.CELL.2022.05.003 (2022). · DOI
  31. Hu, C. et al. Craspase is a CRISPR RNA-guided, RNA-activated protease. Science 377, 1278–1285 (2022). · DOI
  32. O’Brien, R. E. et al. Structural basis for assembly of non-canonical small subunits into type I-C Cascade. Nat. Commun. https://doi.org/10.1038/s41467-020-19785-8 (2020). · DOI
  33. McBride, T. M. et al. Diverse CRISPR-Cas complexes require independent translation of small and large subunits from a single gene. Mol. Cell 80, 971–979.e7 (2020). · DOI
  34. Schwartz, E. A. et al. Structural rearrangements allow nucleic acid discrimination by type I-D Cascade. Nat. Commun. 13, 1–11 (2022). · DOI
  35. Hayes, R. P. et al. Structural basis for promiscuous PAM recognition in type I–E Cascade from E. coli. Nature 530, 499–503 (2016). · DOI · Paper page
  36. Cannone, G., Kompaniiets, D., Graham, S., White, M. F. & Spagnolo, L. Structure of the Saccharolobus solfataricus type III-D CRISPR effector. Curr. Res. Struct. Biol. 5, 100098 (2023). · DOI
  37. Hu, C. et al. Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools. Mol. Cell 82, 2754–2768.e5 (2022). · DOI
  38. Xiao, Y. et al. Structure basis for directional R-loop formation and substrate handover mechanisms in type I CRISPR-Cas system. Cell 170, 48–60.e11 (2017). · DOI · Paper page
  39. Zhou, Y. et al. Structure of a type IV CRISPR-Cas ribonucleoprotein complex. iScience 24, 102201 (2021). · DOI
  40. Chou-Zheng, L. & Hatoum-Aslan, A. A type III—a CRISPR-Cas system employs degradosome nucleases to ensure robust immunity. Elife 8, e45393 (2019). · DOI
  41. Chou-Zheng, L. & Hatoum-Aslan, A. Critical roles for ‘housekeeping’ nucleases in type III CRISPR-Cas immunity. Elife 11, e81897 (2022). · DOI
  42. Taylor, D. W. et al. Structure of the CRISPR-Cmr complex reveal mode of RNA target positioning. Science 348, 581–586 (2015). · DOI
  43. Jia, N. et al. Type III-A CRISPR-Cas Csm complexes: assembly, periodic RNA cleavage, DNase activity regulation, and autoimmunity. Mol. Cell 73, 264–277.e5 (2019). · DOI
  44. Gorski, S. A., Vogel, J. & Doudna, J. A. RNA-based recognition and targeting: sowing the seeds of specificity. Nat. Rev. Mol. Cell Biol. 18, 215–228 (2017). · DOI
  45. Anzelon, T. A. et al. Structural basis for piRNA targeting. Nature 597, 285–289 (2021). · DOI
  46. Huo, Y., Zhao, H., Dong, Q. & Jiang, T. Cryo-EM structure and protease activity of the type III-E CRISPR-Cas effector. Nat. Microbiol. 8, 522–532 (2023). · DOI
  47. Wiegand, T. et al. Functional and phylogenetic diversity of Cas10 proteins. CRISPR J. 6, 152–314 (2023). · DOI
  48. Ding, J., Schuergers, N., Baehre, H. & Wilde, A. Enzymatic properties of CARF-domain proteins in Synechocystis sp. PCC 6803. Front. Microbiol. 13, 1046388 (2022). · DOI
  49. Bevilacqua, P. C. et al. An ontology for facilitating discussion of catalytic strategies of RNA-cleaving enzymes. ACS Chem. Biol. 14, 1068–1076 (2019). · DOI
  50. Suslov, N. B. et al. Crystal structure of the Varkud satellite ribozyme. Nat. Chem. Biol. 11, 840–846 (2015). · DOI
  51. Gaines, C. S., Piccirilli, J. A. & York, D. M. The L-platform/L-scaffold framework: a blueprint for RNA-cleaving nucleic acid enzyme design. RNA 26, 111–125 (2020). · DOI
  52. Ganguly, A. et al. Confluence of theory and experiment reveals the catalytic mechanism of the Varkud satellite ribozyme. Nat. Chem. 12, 193–201 (2020). · DOI
  53. Palermo, G. et al. Catalytic metal ions and enzymatic processing of DNA and RNA. Acc. Chem. Res. 48, 220–228 (2015). · DOI
  54. Casalino, L., Nierzwicki, Ł., Jinek, M. & Palermo, G. Catalytic mechanism of non-target DNA cleavage in CRISPR-Cas9 revealed by ab initio molecular dynamics. ACS Catal. 10, 13596–13605 (2020). · DOI
  55. Nierzwicki, Ł. et al. Principles of target DNA cleavage and the role of Mg2+ in the catalysis of CRISPR–Cas9. Nat. Catal. 5, 912–922 (2022). · DOI
  56. Seith, D. D., Bingaman, J. L., Veenis, A. J., Button, A. C. & Bevilacqua, P. C. Elucidation of catalytic strategies of small nucleolytic ribozymes from comparative analysis of active sites. ACS Catal. 8, 314–327 (2018). · DOI
  57. Jiang, W., Samai, P. & Marraffini, L. A. Degradation of phage transcripts by CRISPR-associated RNases enables type III CRISPR-Cas immunity. Cell 164, 710–721 (2016). · DOI
  58. Nussenzweig, P. M. et al. Molecular mechanisms of CRISPR-Cas immunity in bacteria. Annu. Rev. Genet. https://doi.org/10.1146/annurev-genet-022120 (2020). · DOI
  59. Mohanraju, P. et al. Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems. Science 353, aad5147 (2016). · DOI
  60. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. CryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017). · DOI · Paper page
  61. Punjani, A., Zhang, H. & Fleet, D. J. Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction. Nat. Methods 17, 1214–1221 (2020). · DOI
  62. Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021). · DOI
  63. Goddard, T. D. et al. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018). · DOI · Paper page
  64. Kidmose, R. T. et al. Namdinator–automatic molecular dynamics flexible fitting of structural models into cryo-EM and crystallography experimental maps. IUCrJ 6, 526–531 (2019). · DOI
  65. Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr. Sect. D. 74, 519–530 (2018). · DOI
  66. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr D. Struct. Biol. 74, 531–544 (2018). · DOI
  67. Emsley, P. & Cowtan, K. Coot: Model-building tools for molecular graphics. Acta Crystallogr D. Biol. Crystallogr 60, 2126–2132 (2004). · DOI · Paper page
  68. Zgarbová, M. et al. Refinement of the Cornell et al. Nucleic acids force field based on reference quantum chemical calculations of glycosidic torsion profiles. J. Chem. Theory Comput. 7, 2886–2902 (2011). · DOI
  69. Tian, C. et al. Ff19SB: amino-acid-specific protein backbone parameters trained against quantum mechanics energy surfaces in solution. J. Chem. Theory Comput. 16, 528–552 (2020). · DOI
  70. Li, P., Roberts, B. P., Chakravorty, D. K. & Merz, K. M. Rational design of particle mesh ewald compatible lennard-jones parameters for +2 metal cations in explicit solvent. J. Chem. Theory Comput. 9, 2733–2748 (2013). · DOI
  71. Li, P., Song, L. F. & Merz, K. M. Systematic parameterization of monovalent ions employing the nonbonded model. J. Chem. Theory Comput. 11, 1645–1657 (2015). · DOI
  72. Panteva, M. T., Giambaşu, G. M. & York, D. M. Force field for Mg2+, Mn2+, Zn2+, and Cd2+ ions that have balanced interactions with nucleic acids. J. Phys. Chem. B 119, 15460–15470 (2015). · DOI
  73. Essmann, U. et al. A smooth particle mesh Ewald method. J. Chem. Phys. 103, 8577–8593 (1995). · DOI
  74. Becke, A. D. Density-functional exchange-energy approximation with correct asymptotic behavior. Phys. Rev. A 38, 3098 (1988). · DOI
  75. Lee, C., Yang, W. & Parr, R. G. Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys. Rev. B 37, 785 (1988). · DOI · Paper page
  76. Troullier, N. & Martins, J. L. Efficient pseudopotentials for plane-wave calculations. II. Operators for fast iterative diagonalization. Phys. Rev. B 43, 8861 (1991). · DOI
  77. Martyna, G. J. & Tuckerman, M. E. A reciprocal space based method for treating long range interactions in ab initio and force-field-based calculations in clusters. J. Chem. Phys. 110, 2810–2821 (1999). · DOI
  78. Laio, A., VandeVondele, J. & Rothlisberger, U. A Hamiltonian electrostatic coupling scheme for hybrid Car–Parrinello molecular dynamics simulations. J. Chem. Phys. 116, 6941–6947 (2002). · DOI
  79. Nosé, S. An extension of the canonical ensemble molecular dynamics method. Mol. Phys. 57, 187–191 (2006). · DOI
  80. Car, R. & Parrinello, M. Unified approach for molecular dynamics and density-functional theory. Phys. Rev. Lett. 55, 2471 (1985). · DOI
  81. Martick, M., Lee, T. S., York, D. M. & Scott, W. G. Solvent structure and hammerhead ribozyme catalysis. Chem. Biol. 15, 332 (2008). · DOI
  82. Lee, T. S. et al. Role of Mg2+ in hammerhead ribozyme catalysis from molecular simulation. J. Am. Chem. Soc. 130, 3053 (2008). · DOI
  83. Ren, A. et al. Pistol ribozyme adopts a pseudoknot fold facilitating site-specific in-line cleavage. Nat. Chem. Biol. 12, 702 (2016). · DOI
  84. Kostenbader, K. & York, D. M. Molecular simulations of the pistol ribozyme: unifying the interpretation of experimental data and establishing functional links with the hammerhead ribozyme. RNA 25, 1439–1456 (2019). · DOI
  85. Dasgupta, S., Suslov, N. B. & Piccirilli, J. A. Structural basis for substrate helix remodeling and cleavage loop activation in the Varkud satellite ribozyme. J. Am. Chem. Soc. 139, 9591–9597 (2017). · DOI

Metadata

Authors
Schwartz, E. A., Bravo, J. P. K., Ahsan, M., Macias, L. A., McCafferty, C. L., Dangerfield, T. L., Walker, J. N., Brodbelt, J. S., Palermo, G., Fineran, P. C., Fagerlund, R. D., & Taylor, D. W.
Affiliations
  • The University of Texas at Austin (US)
  • University of California, Riverside (US)
  • University of Otago (NZ)
Corresponding author
  • Palermo, Giulia
  • Fagerlund, Robert D.
  • Taylor, David W.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2024-04-18
Volume
15
Issue
1
Pages
3324-3324
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-024-47506-y
PMC
PMC11026444
OpenAlex
W4394927053
Cited by
30
References
5
Citations by year
2026: 6 · 2025: 9 · 2024: 13
FWCI
3.80
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion, cc-by)
  • PubMed Central (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
U.S. Department of Health & Human Services | National Institutes of Health (R35GM138348)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
RNA-guided RNA cleavage, RNA endonuclease, Synechocystis, adaptive immunity, catalytic mechanism, ribozyme evolution, molecular dynamics simulation

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