Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer

TLDR

CRISPR‑Cas systems provide bacterial adaptive immunity by acquiring short nucleic acid sequences from invaders and inserting them as spacers, yet the spacer acquisition step remains poorly understood. This study used *Pectobacterium atrosepticum* to examine spacer acquisition in Type I‑F CRISPR‑Cas systems. Analysis of ~350 primed spacers revealed a 5′‑protospacer‑GG‑3′ PAM and suggested a bi‑directional translocation of the Cas1:Cas2‑3 complex. Primed acquisition was highly active, incorporating up to nine new spacers across all three arrays, requiring endogenous cas expression, and producing spacers that inhibited conjugation and transformation, with interference strength increasing with spacer number and showing strand‑independent, biased clustering near the primed protospacer, indicating system‑specific mechanisms.

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.