Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 108, Issue 25, Pages 10098-10103Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1104144108
Keywords
bacteriophage; RNA interference; small RNA
Categories
Funding
- National Institutes of Health [GM59295, GM09047]
- Russian Academy of Sciences
- Federal Program Scientific and scientific-pedagogical personnel of innovative Russia [02.740.11.0771]
- Russian Foundation for Basic Research [1-04-01373-a]
- Netherlands Organisation for Scientific Research [863.08.014, 865.05.001]
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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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