Structural variation of types IV-A1- and IV-A3-mediated CRISPR interference.
CRISPR-Cas Systems
Cryoelectron Microscopy
Gene Editing
/ methods
Clustered Regularly Interspaced Short Palindromic Repeats
/ genetics
DNA
/ genetics
DNA Helicases
/ genetics
Escherichia coli
/ genetics
R-Loop Structures
/ genetics
Escherichia coli Proteins
/ genetics
Bacterial Proteins
/ genetics
CRISPR-Associated Proteins
/ metabolism
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
29 Oct 2024
29 Oct 2024
Historique:
received:
07
03
2024
accepted:
23
10
2024
medline:
29
10
2024
pubmed:
29
10
2024
entrez:
29
10
2024
Statut:
epublish
Résumé
CRISPR-Cas mediated DNA-interference typically relies on sequence-specific binding and nucleolytic degradation of foreign genetic material. Type IV-A CRISPR-Cas systems diverge from this general mechanism, using a nuclease-independent interference pathway to suppress gene expression for gene regulation and plasmid competition. To understand how the type IV-A system associated effector complex achieves this interference, we determine cryo-EM structures of two evolutionarily distinct type IV-A complexes (types IV-A1 and IV-A3) bound to cognate DNA-targets in the presence and absence of the type IV-A signature DinG effector helicase. The structures reveal how the effector complexes recognize the protospacer adjacent motif and target-strand DNA to form an R-loop structure. Additionally, we reveal differences between types IV-A1 and IV-A3 in DNA interactions and structural motifs that allow for in trans recruitment of DinG. Our study provides a detailed view of type IV-A mediated DNA-interference and presents a structural foundation for engineering type IV-A-based genome editing tools.
Identifiants
pubmed: 39468082
doi: 10.1038/s41467-024-53778-1
pii: 10.1038/s41467-024-53778-1
doi:
Substances chimiques
DNA
9007-49-2
DNA Helicases
EC 3.6.4.-
Escherichia coli Proteins
0
Bacterial Proteins
0
CRISPR-Associated Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
9306Subventions
Organisme : European Molecular Biology Organization (EMBO)
ID : 5342-2023
Organisme : Lietuvos Mokslo Taryba (Research Council of Lithuania)
ID : S-MIP-22-10
Informations de copyright
© 2024. The Author(s).
Références
Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007).
pubmed: 17379808
doi: 10.1126/science.1138140
Wang, J. Y., Pausch, P. & Doudna, J. A. Structural biology of CRISPR–Cas immunity and genome editing enzymes. Nat. Rev. Microbiol. 20, 641–656 (2022).
pubmed: 35562427
doi: 10.1038/s41579-022-00739-4
Pinilla-Redondo, R. et al. CRISPR-Cas systems are widespread accessory elements across bacterial and archaeal plasmids. Nucleic Acids Res. 50, 4315–4328 (2021).
pmcid: 9071438
doi: 10.1093/nar/gkab859
Al-Shayeb, B. et al. Clades of huge phages from across Earth’s ecosystems. Nature 578, 425–431 (2020).
pubmed: 32051592
pmcid: 7162821
doi: 10.1038/s41586-020-2007-4
Al-Shayeb, B. et al. Diverse virus-encoded CRISPR-Cas systems include streamlined genome editors. Cell 185, 4574–4586.e16 (2022).
pubmed: 36423580
doi: 10.1016/j.cell.2022.10.020
Pinilla-Redondo, R. et al. Type IV CRISPR–Cas systems are highly diverse and involved in competition between plasmids. Nucleic Acids Res. 48, 2000–2012 (2019).
pmcid: 7038947
doi: 10.1093/nar/gkz1197
Moya-Beltrán, A. et al. Evolution of Type IV CRISPR-Cas systems: insights from CRISPR loci in integrative conjugative elements of Acidithiobacillia. CRISPR J. 4, 656–672 (2021).
pubmed: 34582696
pmcid: 8658065
doi: 10.1089/crispr.2021.0051
Newire, E., Aydin, A., Juma, S., Enne, V. I. & Roberts, A. P. Identification of a Type IV-A CRISPR-cas system located exclusively on IncHI1B/IncFIB plasmids in enterobacteriaceae. Front. Microbiol. 11, 555928 (2020).
doi: 10.3389/fmicb.2020.01937
Guo, X. et al. Characterization of the self-targeting Type IV CRISPR interference system in Pseudomonas oleovorans. Nat. Microbiol. 7, 1870–1878 (2022).
pubmed: 36175516
doi: 10.1038/s41564-022-01229-2
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).
pubmed: 31857715
doi: 10.1038/s41579-019-0299-x
Altae-Tran, H. et al. Uncovering the functional diversity of rare CRISPR-Cas systems with deep terascale clustering. Science 382, eadi1910 (2023).
pubmed: 37995242
pmcid: 10910872
doi: 10.1126/science.adi1910
Benz, F. et al. Type IV-A3 CRISPR-Cas systems drive inter-plasmid conflicts by acquiring spacers in trans. Cell Host & Microbe 32, 875–886.e9 (2024).
Özcan, A. et al. Type IV CRISPR RNA processing and effector complex formation in Aromatoleum aromaticum. Nat. Microbiol. 4, 89–96 (2019).
pubmed: 30397343
doi: 10.1038/s41564-018-0274-8
Taylor, H. N. et al. Structural basis of Type IV CRISPR RNA biogenesis by a Cas6 endoribonuclease. RNA Biol. 16, 1438–1447 (2019).
pubmed: 31232162
pmcid: 6779396
doi: 10.1080/15476286.2019.1634965
Domgaard, H. et al. CasDinG is a 5’-3’ dsDNA and RNA/DNA helicase with three accessory domains essential for type IV CRISPR immunity. Nucleic Acids Res. https://doi.org/10.1093/nar/gkad546 (2023).
Gleditzsch, D. et al. PAM identification by CRISPR-Cas effector complexes: diversified mechanisms and structures. RNA Biol. 16, 504–517 (2019).
pubmed: 30109815
doi: 10.1080/15476286.2018.1504546
Crowley, V. M. et al. A Type IV-A CRISPR-cas system in pseudomonas aeruginosa mediates RNA-guided plasmid interference in vivo. CRISPR J. 2, 434–440 (2019).
pubmed: 31809194
pmcid: 6919247
doi: 10.1089/crispr.2019.0048
Sanchez-Londono, M. et al. Visualization of Type IV-A1 CRISPR-mediated repression of gene expression and plasmid replication. Nucleic Acids Res. https://doi.org/10.1093/nar/gkae879 (2024) .
Taylor, H. N. et al. Positioning diverse type IV structures and functions within class 1 CRISPR-cas systems. Front. Microbiol. 12, 671522 (2021).
pubmed: 34093491
pmcid: 8175902
doi: 10.3389/fmicb.2021.671522
Cui, N. et al. Type IV-A CRISPR-Csf complex: assembly, dsDNA targeting, and CasDinG recruitment. Mol. Cell 83, 2493–2508.e5 (2023).
pubmed: 37343553
doi: 10.1016/j.molcel.2023.05.036
Liu, T. Y. & Doudna, J. A. Chemistry of class 1 CRISPR-cas effectors: binding, editing, and regulation. J. Biol. Chem. 295, 14473–14487 (2020).
pubmed: 32817336
pmcid: 7573268
doi: 10.1074/jbc.REV120.007034
Jackson, R. N. & Wiedenheft, B. A conserved structural chassis for mounting versatile CRISPR RNA-guided immune responses. Mol. Cell 58, 722–728 (2015).
pubmed: 26028539
pmcid: 4465340
doi: 10.1016/j.molcel.2015.05.023
Jackson, R. N. et al. Structural biology. Crystal structure of the CRISPR RNA-guided surveillance complex from Escherichia coli. Science 345, 1473–1479 (2014).
pubmed: 25103409
pmcid: 4188430
doi: 10.1126/science.1256328
Chowdhury, S. et al. Structure reveals mechanisms of viral suppressors that intercept a CRISPR RNA-guided surveillance complex. Cell 169, 47–57.e11 (2017).
pubmed: 28340349
pmcid: 5478280
doi: 10.1016/j.cell.2017.03.012
Mulepati, S., Héroux, A. & Bailey, S. Structural biology. Crystal structure of a CRISPR RNA-guided surveillance complex bound to a ssDNA target. Science 345, 1479–1484 (2014).
pubmed: 25123481
pmcid: 4427192
doi: 10.1126/science.1256996
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).
pubmed: 35835111
pmcid: 9357151
doi: 10.1016/j.molcel.2022.06.007
O’Brien, R. E. et al. Structural snapshots of R-loop formation by a type I-C CRISPR Cascade. Mol. Cell 83, 746–758.e5 (2023).
pubmed: 36805026
pmcid: 10026943
doi: 10.1016/j.molcel.2023.01.024
Schwartz, E. A. et al. Structural rearrangements allow nucleic acid discrimination by type I-D Cascade. Nat. Commun. 13, 2829 (2022).
pubmed: 35595728
pmcid: 9123187
doi: 10.1038/s41467-022-30402-8
Xiao, Y., Luo, M., Dolan, A. E., Liao, M. & Ke, A. Structure basis for RNA-guided DNA degradation by Cascade and Cas3. Science 361, eaat0839 (2018).
Hochstrasser, M. L. & Doudna, J. A. Cutting it close: CRISPR-associated endoribonuclease structure and function. Trends Biochem. Sci. 40, 58–66 (2015).
pubmed: 25468820
doi: 10.1016/j.tibs.2014.10.007
Zhou, Y. et al. Structure of a type IV CRISPR-Cas ribonucleoprotein complex. iScience 24, 102201 (2021).
pubmed: 33733066
pmcid: 7937560
doi: 10.1016/j.isci.2021.102201
Pausch, P. et al. Structural variation of Type I-F CRISPR RNA guided DNA surveillance. Mol. Cell 67, 622–632.e4 (2017).
pubmed: 28781236
doi: 10.1016/j.molcel.2017.06.036
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).
pubmed: 28196981
doi: 10.1038/nrm.2016.174
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
pubmed: 23287718
pmcid: 3795411
doi: 10.1126/science.1231143
Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233–239 (2013).
pubmed: 23360965
pmcid: 3748948
doi: 10.1038/nbt.2508
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
pubmed: 22745249
pmcid: 6286148
doi: 10.1126/science.1225829
Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).
pubmed: 24529477
pmcid: 4139937
doi: 10.1016/j.cell.2014.02.001
Swarts, D. C., van der Oost, J. & Jinek, M. Structural basis for guide RNA processing and seed-dependent DNA targeting by CRISPR-Cas12a. Mol. Cell 66, 221–233.e4 (2017).
pubmed: 28431230
pmcid: 6879319
doi: 10.1016/j.molcel.2017.03.016
Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).
pubmed: 26422227
pmcid: 4638220
doi: 10.1016/j.cell.2015.09.038
Wiedenheft, B. et al. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc. Natl Acad. Sci. USA 108, 10092–10097 (2011).
pubmed: 21536913
pmcid: 3121849
doi: 10.1073/pnas.1102716108
Semenova, E. et al. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc. Natl Acad. Sci. USA 108, 10098–10103 (2011).
pubmed: 21646539
pmcid: 3121866
doi: 10.1073/pnas.1104144108
Hayes, R. P. et al. Structural basis for promiscuous PAM recognition in type I-E Cascade from E. coli. Nature 530, 499–503 (2016).
pubmed: 26863189
pmcid: 5134256
doi: 10.1038/nature16995
Zhang, X.-H., Tee, L. Y., Wang, X.-G., Huang, Q.-S. & Yang, S.-H. Off-target effects in CRISPR/Cas9-mediated genome engineering. Mol. Ther. Nucleic Acids 4, e264 (2015).
pubmed: 26575098
pmcid: 4877446
doi: 10.1038/mtna.2015.37
Marino, N. D., Pinilla-Redondo, R., Csörgő, B. & Bondy-Denomy, J. Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies. Nat. Methods 17, 471–479 (2020).
pubmed: 32203383
pmcid: 8510557
doi: 10.1038/s41592-020-0771-6
Greber, B. J. et al. The cryo-electron microscopy structure of human transcription factor IIH. Nature 549, 414–417 (2017).
pubmed: 28902838
pmcid: 5844561
doi: 10.1038/nature23903
Peissert, S. et al. In TFIIH the Arch domain of XPD is mechanistically essential for transcription and DNA repair. Nat. Commun. 11, 1667 (2020).
pubmed: 32245994
pmcid: 7125077
doi: 10.1038/s41467-020-15241-9
Hallmark, T. et al. The N-terminal domain of Type IV-A1 CRISPR-associated DinG is vulnerable to proteolysis. MicroPubl. Biol. 2024, https://doi.org/10.17912/micropub.biology.001226 (2024).
Mayo-Muñoz, D., Pinilla-Redondo, R., Camara-Wilpert, S., Birkholz, N. & Fineran, P. C. Inhibitors of bacterial immune systems: discovery, mechanisms and applications. Nat. Rev. Genet. 25, 237–254 (2024).
pubmed: 38291236
doi: 10.1038/s41576-023-00676-9
Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 38, 824–844 (2020).
pubmed: 32572269
doi: 10.1038/s41587-020-0561-9
Dominguez, A. A., Lim, W. A. & Qi, L. S. Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat. Rev. Mol. Cell Biol. 17, 5–15 (2016).
pubmed: 26670017
doi: 10.1038/nrm.2015.2
Nakamura, M., Gao, Y., Dominguez, A. A. & Qi, L. S. CRISPR technologies for precise epigenome editing. Nat. Cell Biol. 23, 11–22 (2021).
pubmed: 33420494
doi: 10.1038/s41556-020-00620-7
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).
pubmed: 28165473
doi: 10.1038/nmeth.4169
Meng, E. C. et al. UCSF ChimeraX: tools for structure building and analysis. Protein Sci. 32, e4792 (2023).
pubmed: 37774136
pmcid: 10588335
doi: 10.1002/pro.4792
He, J., Li, T. & Huang, S.-Y. Improvement of cryo-EM maps by simultaneous local and non-local deep learning. Nat. Commun. 14, 3217 (2023).
pubmed: 37270635
pmcid: 10239474
doi: 10.1038/s41467-023-39031-1
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
pubmed: 34265844
pmcid: 8371605
doi: 10.1038/s41586-021-03819-2
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 66, 486–501 (2010).
pubmed: 20383002
pmcid: 2852313
doi: 10.1107/S0907444910007493
Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr D. Struct. Biol. 74, 519–530 (2018).
pubmed: 29872003
pmcid: 6096486
doi: 10.1107/S2059798318002425
Pettersen, E. F. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).
pubmed: 32881101
doi: 10.1002/pro.3943
Jamali, K. et al. Automated model building and protein identification in cryo-EM maps. bioRxiv https://doi.org/10.1101/2023.05.16.541002 (2023) .
Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr D. Struct. Biol. 75, 861–877 (2019).
pubmed: 31588918
pmcid: 6778852
doi: 10.1107/S2059798319011471
Kong, A. T., Leprevost, F. V., Avtonomov, D. M., Mellacheruvu, D. & Nesvizhskii, A. I. MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry-based proteomics. Nat. Methods 14, 513–520 (2017).
pubmed: 28394336
pmcid: 5409104
doi: 10.1038/nmeth.4256