Characterization of the self-targeting Type IV CRISPR interference system in Pseudomonas oleovorans

Bacterial Type IV CRISPR-Cas systems are thought to rely on multi-subunit ribonucleoprotein complexes to interfere with mobile genetic elements, but the substrate requirements and potential DNA nuclease activities for many systems within this type are uncharacterized. Here we show that the native Pseudomonas oleovorans Type IV-A CRISPR-Cas system targets DNA in a PAM-dependent manner and elicits interference without showing DNA nuclease activity. We found that the first crRNA of P. oleovorans contains a perfect match in the host gene coding for the Type IV pilus biogenesis protein PilN. Deletion of the native Type IV CRISPR array resulted in upregulation of pilN operon transcription in the absence of genome cleavage, indicating that Type IV-A CRISPR-Cas systems can function in host gene regulation. These systems resemble CRISPR interference (CRISPRi) methodology but represent a natural CRISPRi-like system that is found in many Pseudomonas and Klebsiella species and allows for gene silencing using engineered crRNAs.