CasDinG is an ATP-dependent 5’-3’ DNA helicase with accessory domains essential for type IV CRISPR immunity

CRISPR-associated DinG protein (CasDinG) is essential to type IV-A CRISPR function. However, the enzymatic activities of CasDinG are unknown. Here we demonstrate that CasDinG from Pseudomonas aeruginosa strain 83 is an ATP- and metal-dependent 5’-3’ DNA helicase. The crystal structure of CasDinG reveals a helicase core of two RecA-like domains with three accessory domains (N-terminal, arch, and vestigial FeS). To examine the in vivo function of these CasDinG domains, we first identified the preferred PAM sequence (5’-GNAWN-3’ on the 5’-side of the target) with a plasmid library containing all combinations of the five nucleotides upstream of the target sequence. Plasmid clearance assays (using a 5’-GGAAA-3’ PAM) with CasDinG domain mutants demonstrated the vFeS and arch accessory domains are both essential for type IV immunity. These results provide a needed structural and biochemical framework for understanding the type IV-A CRISPR system. ### Competing Interest Statement Ryan Jackson has filed several pending patents related to CRISPR-associated systems.