RESUMO
RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Recent studies identified functionally divergent type V Cas12 family enzymes. Among them, Cas12c2 binds a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) and recognizes double-stranded DNA targets with a short TN PAM. Here, we report the cryo-electron microscopy structures of the Cas12c2-guide RNA binary complex and the Cas12c2-guide RNA-target DNA ternary complex. The structures revealed that the crRNA and tracrRNA form an unexpected X-junction architecture, and that Cas12c2 recognizes a single T nucleotide in the PAM through specific hydrogen-bonding interactions with two arginine residues. Furthermore, our biochemical analyses indicated that Cas12c2 processes its precursor crRNA to a mature crRNA using the RuvC catalytic site through a unique mechanism. Collectively, our findings improve the mechanistic understanding of diverse type V CRISPR-Cas effectors.