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PreprintCited by 2CRISPR and Genetic EngineeringBacterial Genetics and BiotechnologyBiochemical and Structural Characterization

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Structural insights into Cas9-mediated prespacer selection in CRISPR-Cas adaptation

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Gaizauskaite, Ugne, Tamulaitiene, Giedre, Silanskas, Arunas, Gasiunas, Giedrius, Siksnys, Virginijus, Sasnauskas, Giedrius·2025·Molecular Cell
DOI 10.1101/2025.06.12.659244

Preprint — published as 10.1016/j.molcel.2026.01.022 · Paper page

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Abstract

SUMMARY: 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 like Cas4, 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. However, the supercomplex structure and the role of the ring-like Csn2 protein remain unknown. Here, we present cryo-electron microscopy structures of the type II-A prespacer selection supercomplex in the DNA-scanning and two different PAM-bound configurations. Our study uncovers the mechanism of Cas9-mediated prespacer selection in type II-A CRISPR-Cas systems, and reveals the role of the accessory protein Csn2, which serves as a platform for the assembly of Cas9 and Cas1-Cas2 integrase on prespacer DNA, reminiscent of the sliding clamp in DNA replication. Repurposing of Cas9 by the CRISPR adaptation machinery for prespacer selection characterized here demonstrates Cas9 plasticity and expands our knowledge of the Cas9 biology.

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Metadata

Authors
Gaizauskaite, U., Tamulaitiene, G., Silanskas, A., Gasiunas, G., Siksnys, V., & Sasnauskas, G.
Affiliations
  • Vilnius University (LT)
  • Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania
Corresponding author
Sasnauskas, Giedrius
ORCID
Venue
Molecular Cell
Publisher
openRxiv
Published
2025-06-12
Volume
86
Issue
5
Pages
791-804.e9
ISSN
1097-2765 (ISSN-L)
DOI
10.1101/2025.06.12.659244
OpenAlex
W7129049198
Cited by
2
References
1
Citations by year
2026: 2
FWCI
4.13
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Cell (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
Lietuvos Mokslo Taryba
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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