Journal
NUCLEIC ACIDS RESEARCH
Volume 42, Issue 13, Pages 8516-8526Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gku527
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Funding
- Otago School of Medical Sciences Dean's Strategic Fund
- Royal Society of New Zealand
- University of Otago Doctoral Scholarship
- University of Otago Division of Health Sciences Career Development Postdoctoral Fellowships
- Otago School of Medical Sciences Summer Research Scholarship
- Dean's Prestigious Summer Scholarship
- Rutherford Discovery Fellowship
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Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed similar to 350 new spacers acquired in priming events and identified a 5'-protospacer-GG-3' protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2-3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.
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