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. [Display omitted] • Cryo-EM structures of the Cas12c2–guide RNA and Cas12c2–guide RNA–target DNA complexes • The crRNA and tracrRNA of Cas12c2 form an unexpected X-junction structure • Cas12c2 uses two arginine residues to recognize a single T nucleotide in the PAM • Cas12c2 processes its pre-crRNA at the RuvC active site via a unique mechanism Kurihara et al. report the cryo-EM structures of the Cas12c2–guide RNA complex and the Cas12c2–guide RNA–target DNA complex, providing mechanistic insights into the TN PAM recognition by Cas12c2. Furthermore, biochemical analysis revealed that Cas12c2 processes its pre-crRNA through a unique mechanism. [ABSTRACT FROM AUTHOR]