Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer.
Bottom Line: Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune 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.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.
Affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.Show MeSH
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Mentions: Five millilitres cultures of P. atrosepticum ΔHAI2 with pTRB30 (vector control) or pPF189 were grown overnight without antibiotic selection (Figure 1). Note that 10 μl were used to inoculate a fresh overnight culture and dilutions were plated onto LBA. This was repeated over 5 days and performed in triplicate. Colonies (100) from each replicate were patched onto LBA ± Km. Km sensitive (KmS) colonies were screened by PCR for new spacers as described later.
Affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.