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.