Cited by 22CRISPR and Genetic EngineeringBacterial Genetics and BiotechnologyBiochemical and Structural Characterization

PAM binding ensures orientational integration during Cas4-Cas1-Cas2-mediated CRISPR adaptation

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Dhingra, Yukti, Suresh, Shravanti K., Juneja, Puneet, Sashital, Dipali G.·2022·Molecular Cell

Published version of a preprint

Abstract

Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of DNA segments containing PAM sequences prior to integration of these "prespacer" substrates as spacers in the CRISPR array. We determined cryo-EM structures of the Cas4-Cas1-Cas2 adaptation complex from the type I-C system that encodes standalone Cas1 and Cas4 proteins. The structures reveal how Cas4 specifically reads out bases within the PAM sequence and how interactions with both Cas1 and Cas2 activate Cas4 endonuclease activity. The Cas4-PAM interaction ensures tight binding between the adaptation complex and the prespacer, significantly enhancing integration of the non-PAM end into the CRISPR array and ensuring correct spacer orientation. Corroborated with our biochemical results, Cas4-Cas1-Cas2 structures with substrates representing various stages of CRISPR adaptation reveal a temporally resolved mechanism for maturation and integration of functional spacers into the CRISPR array.

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Crossref · 10.1016/j.molcel.2022.09.030

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Authors
Dhingra, Y., Suresh, S. K., Juneja, P., & Sashital, D. G.
Affiliations
Iowa State University (US)
Corresponding author
Sashital, Dipali G.
Venue
Molecular Cell
Publisher
Elsevier BV
Published
2022-11
Volume
82
Issue
22
Pages
4353-4367.e6
ISSN
1097-2765 (ISSN-L)
OpenAlex
W4307115370
Cited by
21
References
67
Citations by year
2026: 5 · 2025: 6 · 2024: 3 · 2023: 6 · 2022: 1
FWCI
1.30
Type
journal article
Language
en
Open access
Locations
  • Molecular Cell (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • Simons Foundation (SF349247)
  • Roy J. Carver Charitable Trust
  • National Institutes of Health (GM140876, GM115874, U24 GM129539)
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
CRISPR adaptation, PAM sequence, cryo-EM structure, mobile genetic elements
MeSH terms
DNA, CRISPR-Cas Systems, CRISPR-Associated Proteins

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