Abstract
CRISPR–Cas systems provide bacteria with adaptive immunity against foreign nucleic acids by acquiring short, invader-derived sequences called spacers. Here, we use high-throughput sequencing to analyse millions of spacer acquisition events in wild-type populations of Pectobacterium atrosepticum. Plasmids not previously encountered, or plasmids that had escaped CRISPR–Cas targeting via point mutation, are used to provoke naive or primed spacer acquisition, respectively. The origin, location and order of spacer acquisition show that spacer selection through priming initiates near the site of CRISPR–Cas recognition (the protospacer), but on the displaced strand, and is consistent with 3′–5′ translocation of the Cas1:Cas2-3 acquisition machinery. Newly acquired spacers determine the location and strand specificity of subsequent spacers and demonstrate that interference-driven spacer acquisition (‘targeted acquisition') is a major contributor to adaptation in type I-F CRISPR–Cas systems. Finally, we show that acquisition of self-targeting spacers is occurring at a constant rate in wild-type cells and can be triggered by foreign DNA with similarity to the bacterial chromosome.
Highlights
CRISPR–Cas systems provide bacteria with adaptive immunity against foreign nucleic acids by acquiring short, invader-derived sequences called spacers
To monitor the natural process of naive and primed spacer acquisition in a bacterial population, wild-type P. atrosepticum with a plasmid lacking a protospacer, or with plasmids carrying a protospacer on either the minus (pPriming( À )) or plus (pPriming( þ )) strand were cultured for 5 days without selection (Fig. 1a)
By using a high-throughput spacer acquisition assay in a native type I-F system, we dissected important features underlying naive and primed spacer acquisition, which allowed us to form a new model for adaptation in type I-F systems (Fig. 9)
Summary
CRISPR–Cas systems provide bacteria with adaptive immunity against foreign nucleic acids by acquiring short, invader-derived sequences called spacers. Exposure to foreign genetic elements results in acquisition of short invader-derived sequences into CRISPR arrays. These arrays are composed of repeats, interrupted by the invaderderived sequences—the spacers. For type I systems, acquisition of new spacers requires a short protospacer adjacent motif (PAM) that is recognized by Cas[1] and Cas[2] (refs 14,19). We previously proposed a model for priming by type I-F systems, whereby the Cas-crRNA ribonucleoprotein complex (Csy complex) first recognizes the mutated invader, which leads to the generation of an R-loop and the recruitment of the Cas1:Cas[2,3] complex to the displaced (non-primed strand)[31]. Cas1:Cas[2,3 30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50] translocation along the displaced (non-primed) strand was proposed to unwind and expose the primed strand, allowing secondary recruitment of Cas1:Cas[2,3] and translocation on the primed strand[31]
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