Structural insights into Cas9-mediated prespacer selection in CRISPR-Cas adaptation
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Abstract
During CRISPR-Cas adaptation, prokaryotic cells become immunized by the insertion of foreign DNA fragments, termed spacers, into the host genome to serve as templates for RNA-guided immunity. Spacer acquisition relies on the Cas1-Cas2 integrase and accessory proteins, which select DNA sequences flanked by the protospacer adjacent motif (PAM) and insert them into the CRISPR array. It has been shown that in type II-A systems, selection of PAM-proximal prespacers is mediated by the effector nuclease Cas9, which forms a "supercomplex" with the Cas1-Cas2 integrase and the Csn2 protein. Here, we present cryo-electron microscopy structures of the Streptococcus thermophilus type II-A prespacer selection supercomplex in the DNA-scanning and two distinct PAM-bound configurations, providing insights into the mechanism of Cas9-mediated prespacer selection in type II-A CRISPR-Cas systems. Repurposing Cas9 by the CRISPR adaptation machinery for prespacer selection, as characterized here, demonstrates Cas9 plasticity and expands our knowledge of Cas9 biology.
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Cites
Highly accurate protein structure prediction with AlphaFold
Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A., et al. (2021) · Nature · doi:10.1038/s41586-021-03819-2 · Cited by 43,764 · see citing works
Accurate structure prediction of biomolecular interactions with AlphaFold 3
Abramson, J., Adler, J., Dunger, J., Evans, R., Green, T., Pritzel, A., Ronneberger, O., Willmore, L., Ballard, A. J., Bambrick, J., et al. (2024) · Nature · doi:10.1038/s41586-024-07487-w · Cited by 15,861 · see citing works
PAM binding ensures orientational integration during Cas4-Cas1-Cas2-mediated CRISPR adaptation
Dhingra, Y., Suresh, S. K., Juneja, P., & Sashital, D. G. (2022) · Molecular Cell · doi:10.1016/j.molcel.2022.09.030 · Cited by 21 · see citing works
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- Authors
- Gaizauskaite, U., Tamulaitiene, G., Silanskas, A., Gasiunas, G., Siksnys, V., & Sasnauskas, G.
- Affiliations
- Vilnius University (LT)
- Corresponding author
- Sasnauskas, Giedrius
- Venue
- Molecular Cell
- Publisher
- Elsevier BV
- Published
- 2026-03
- Volume
- 86
- Issue
- 5
- Pages
- 791-804.e9
- ISSN
- 1097-2765 (ISSN-L)
- OpenAlex
- W7129049198
- Cited by
- 2
- References
- 3
- Citations by year
- 2026: 2
- FWCI
- 4.13
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- Locations
- Molecular Cell (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Funders
- Research Council of Lithuania
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
- Keywords
- Cas9, β-casein, Streptococcus thermophilus, protospacer adjacent motif, DNA scanning, spacer acquisition
- MeSH terms
- Gene Editing, CRISPR-Associated Protein 9, RNA, Guide, CRISPR-Cas Systems, Adaptation, Physiological, Bacterial Proteins, DNA, Bacterial, Models, Molecular, Nucleic Acid Conformation, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Integrases, Cryoelectron Microscopy, Protein Structure, Quaternary, Streptococcus thermophilus, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems, CRISPR-Associated Proteins
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