title: Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer.
abstract: 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 ∼ 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.
authors.affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
authors.forename: Corinna
authors.initials: C
authors.lastname: Richter
authors.affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
authors.forename: Ron L
authors.initials: RL
authors.lastname: Dy
authors.affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
authors.forename: Rebecca E
authors.initials: RE
authors.lastname: McKenzie
authors.affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
authors.forename: Bridget N J
authors.initials: BN
authors.lastname: Watson
authors.affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
authors.forename: Corinda
authors.initials: C
authors.lastname: Taylor
authors.affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
authors.forename: James T
authors.initials: JT
authors.lastname: Chang
authors.affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: McNeil
authors.affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand.
authors.forename: Raymond H J
authors.initials: RH
authors.lastname: Staals
authors.affiliation: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand peter.fineran@otago.ac.nz.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Fineran
mesh_terms: D064130:CRISPR-Associated Proteins / Q000235:genetics; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D003227:Conjugation, Genetic; D044043:Pectobacterium / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D064130:CRISPR-Associated Proteins