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Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence

Semenova, Ekaterina, Jore, Matthijs M., Datsenko, Kirill A., Semenova, Anna, Westra, Edze R., Wanner, Barry, van der Oost, John, Brouns, Stan J. J., Severinov, Konstantin·2011·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.1104144108
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Abstract

Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)/Cas (CRISPR-associated sequences) systems provide adaptive immunity against viruses when a spacer sequence of small CRISPR RNA (crRNA) matches a protospacer sequence in the viral genome. Viruses that escape CRISPR/Cas resistance carry point mutations in protospacers, though not all protospacer mutations lead to escape. Here, we show that in the case of Escherichia coli subtype CRISPR/Cas system, the requirements for crRNA matching are strict only for a seven-nucleotide seed region of a protospacer immediately following the essential protospacer-adjacent motif. Mutations in the seed region abolish CRISPR/Cas mediated immunity by reducing the binding affinity of the crRNA-guided Cascade complex to protospacer DNA. We propose that the crRNA seed sequence plays a role in the initial scanning of invader DNA for a match, before base pairing of the full-length spacer occurs, which may enhance the protospacer locating efficiency of the E. coli Cascade complex. In agreement with this proposal, single or multiple mutations within the protospacer but outside the seed region do not lead to escape. The relaxed specificity of the CRISPR/Cas system limits escape possibilities and allows a single crRNA to effectively target numerous related viruses.

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  • As Cas9-gRNA screens for a potential target, the first 8-12 PAM-proximal bases of gRNA's targeting sequence (the seed sequence) begin pairing with the target DNA in the 3′-5′ direction, provided a PAM site is recognised.

    “As Cas9-gRNA screens for a potential target, the first 8–12 PAM-proximal bases of gRNA’s targeting sequence, also known as the seed sequence, will begin pairing with the target DNA in the 3′-5′ direction, provided a PAM site is recognised [66].”

    From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [66]

    The Discussion describes PAM-dependent seed pairing as a possible starting point for target recognition, but it concerns the E. coli Cascade–crRNA complex, not Cas9–gRNA. The Results identify a noncontiguous seven-nucleotide seed (positions 1–5, 7, and 8), rather than 8–12 bases; the cited passages do not establish pairing in a 3′-to-5′ direction. (LLM, conf=0.90)

    Experimental — accuracy not yet measured

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Research works printed but not linked: 27

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Metadata

Authors
Semenova, E., Jore, M. M., Datsenko, K. A., Semenova, A., Westra, E. R., Wanner, B., van der Oost, J., Brouns, S. J. J., & Severinov, K.
Affiliations
  • Waksman Institute, Piscataway, NJ 08854;
  • Laboratory of Microbiology, Wageningen University, 6703 HB, Wageningen, The Netherlands;
  • Purdue University, West Lafayette, IN 47907;
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2011-06-06
Volume
108
Issue
25
Pages
10098-10103
ISSN
  • 0027-8424
  • 1091-6490
DOI
10.1073/pnas.1104144108
Cited by
726
References
0
Language
en

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