Functional characterization of a plasmid-encoded type IV-A3 CRISPR-Cas system. In trans adaptation and RNA-guided silencing by a Klebsiella plasmid-encoded type IV-A3 CRISPR-Cas system

Type IV CRISPR-Cas systems are primarily encoded on plasmids and form RNA-guided multiprotein complexes with unknown biological functions. In contrast to other CRISPR-Cas types, they lack the archetypical spacer acquisition module and encode a DinG helicase instead of a nuclease component. Type IV-A3 systems are present in clinically relevant enterobacteria, carried by large conjugative plasmids that often harbor antibiotic-resistance genes. Although their CRISPR spacer contents suggest a role in inter-plasmid conflicts, this function and the underlying mechanisms have remained unexplored. Here, we demonstrate that a IncHI1B/FIB plasmid-encoded type IV-A3 system co-opts the I-E adaptation machinery from its clinical Klebsiella pneumoniae host to acquire new spacers. Furthermore, we demonstrate that robust interference of conjugative plasmids and phages is elicited through crRNA-dependent transcriptional repression. By targeting plasmid core functions, type IV-A3 can prevent the uptake of incoming plasmids, limit their horizontal transfer, and destabilize co-residing plasmids, altogether supporting their involvement in plasmid competition. Collectively, our findings shed light on the ecological function and molecular mechanisms of type IV-A3 systems and have broad implications for understanding and countering the spread of antibiotic resistance plasmids in clinically relevant strains.