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: The Type I-F system of P. atrosepticum contains CRISPR-4 (cluster 4) repeats (34), which were proposed in a bioinformatic study to possess a 5′-protospacer-GG-3′ PAM (21) (Figure 6A). However, no study has analyzed Type I-F PAMs from a large number of experimental acquisition events. Therefore, we combined our data to determine the Type I-F PAM for new spacers incorporated through priming (n = 351). Of the new spacers, 87% were 32 nt, 12% were 33 nt, <1% were 34 nt and one case of 31 nt was detected (Figure 6B). The 5′ and 3′ flanks of all protospacers were aligned and sequence logos generated (Figure 6C). No conservation was detected in the 5′ flanking sequence or in the protospacer (Supplementary Figure S5), but a GG PAM was detected in the –1 and –2 positions of the 3′ flanking sequence (i.e. a 5′-protospacer-GG-3′ PAM). When protospacer flanks were analyzed in separate groups based on length, the 32 and 33 nt groups displayed the same overall GG PAM (Figure 6D and Supplementary Figure S5A and B).
Affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.