A Type IV-A CRISPR-Cas System in Pseudomonas aeruginosa Mediates RNA-Guided Plasmid Interference In Vivo

Abstract

Bacteria and archaea use CRISPR-Cas adaptive immune systems to destroy complementary nucleic acids using RNAs derived from CRISPR loci. Here, we provide the first functional evidence for type IV CRISPR-Cas, demonstrating that the system from Pseudomonas aeruginosa strain PA83 mediates RNA-guided interference against a plasmid in vivo, both clearing the plasmid and inhibiting its uptake. This interference depends on the putative NTP-dependent helicase activity of Csf4/DinG.

FIG. 1.

Type IV-A CRISPR-Cas systems found in Pseudomonas aeruginosa. (A) Classification of type IV-A CRISPR-Cas systems found in P. aeruginosa. Conserved architectures of these four variants are observed. All four variants include csf2, csf3, csf4/dinG, and csf5/cas6. Despite the similar architecture between type IV-A1 and type-IV-A2, they represent two separate groups based on differences in direct repeat sequences and amino acid divergence (Supplementary Fig. S1). (B) Consensus sequences for direct repeats found in type IV-A CRISPR arrays in P. aeruginosa. Palindromic regions are in the center of the repeat. (C) Table of spacers found in each system. Systems are grouped by variant, with group 2 consolidated, as all their CRISPR arrays are identical. Spacers are colored based on mapping results according to the legend. A dendrogram was generated based on representative csf2 sequences to illustrate the relationship between variants.

FIG. 2.

The type IV-A1 CRISPR-Cas variant from P. aeruginosa PA83 mediates RNA-guided interference in vivo. (A) Plasmid transformation efficiency assay described in the methods section. Small arrows on plasmids indicate a T7 promoter. (B) A reduction in plasmid transformation efficiency was observed when cells harboring a CRISPR array with TS1, csf1, csf2, csf5/cas6, csf3, and csf4/dinG were transformed with the target compared to those transformed with the non-target plasmid. (C) Plasmid maintenance assay described in the Methods section. Small arrows on plasmids indicate a T7 promoter. (D) An interference defect is observed when any component is removed and when the Csf4/DinG DEAH-box is mutated (D336A/E337A). TS1 was used in each deletion line and the Csf4/DinG mutant. As each condition represents a different competent cell line, an uninduced control was always included (Supplementary Fig. S2). TS, targeting spacer; NTS, non-targeting spacer.