Abstract
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.
| Originalsprog | Engelsk |
|---|---|
| Artikelnummer | 9306 |
| Tidsskrift | Nature Communications |
| Vol/bind | 15 |
| Udgave nummer | 1 |
| Antal sider | 18 |
| ISSN | 2041-1723 |
| DOI | |
| Status | Udgivet - 2024 |
Bibliografisk note
Funding Information:We thank the department of Protein - DNA interactions at the Life Sciences Center at Vilnius University (Virginijus \u0160ik\u0161nys) for access to equipment and the Vilnius University cryo-EM facility (Giedrius Sasnauskas) for support. The authors thank Stephen K. Jones Jr. for critical reading and comments on the manuscript. We further thank Ning Jia for sharing the structure coordinates of the P. aeruginosa type IV-A1 system prior to release at the PDB, and the EMBL Proteomics Core Facility for MS-analysis of protein samples. P.P. and L.M. received funding from the European Regional Development Fund under grant agreement number 01.2.2-CPVA-V-716-01-0001 (P.P. and L.M.) with the Central Project Management Agency (CPVA), Lithuania. P.P. receives funding from the Research Council of Lithuania (LMTLT) under grant agreement number S-MIP-22-10 (P.P.), and from the European Molecular Biology Conference (EMBC) under EMBO Installation Grant agreement number 5342-2023 (P.P.). R.P.-R. was supported by the Lundbeck Foundation (grant R347-2020-2346, R.P.R.). F.B. was supported by the SNSF (grants P1EZP3_195539 and P500PB_210944, F.B.). This work was further supported by the DFG-SPP2141 (N.K. and L.R.), and LOEWE Research Cluster Diffusible Signals (L.R.) and the Max Planck Society (N.S. and G.K.A.H.).
Publisher Copyright:
© The Author(s) 2024.