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The type V-K CRISPR-Cas system, featured by Cas12k effector with a naturally inactivated RuvC domain and associated with Tn7-like transposon for RNA-guided DNA transposition, is a promising tool for precise DNA insertion. To reveal the mechanism underlying target DNA recognition, we determined a cryoelectron microscopy (cryo-EM) structure of Cas12k from cyanobacteria Scytonema hofmanni in complex with a single guide RNA (sgRNA) and a double-stranded target DNA. Coupled with mutagenesis and in vitro DNA transposition assay, our results revealed mechanisms for the recognition of the GGTT protospacer adjacent motif (PAM) sequence and the structural elements of Cas12k critical for RNA-guided DNA transposition. These structural and mechanistic insights should aid in the development of type V-K CRISPR-transposon systems as tools for genome editing. [Display omitted] • Cryo-EM structure of the Cas12k-sgRNA-target DNA complex is presented at 3.6 Å • Cas12k uses an unusually large sgRNA for RNA-guided DNA transposition • Identification of key residues for recognition of the PAM sequence • Cryo-EM structure of the Cas12k-sgRNA complex is solved at 3.8 Å CRISPR-associated transposase (CAST) systems allow for RNA-guided DNA insertion at precise locations in genomes and are promising tools for genome editing. Xiao et al. report the structure of Cas12k, the CRISPR effector protein of the type V-K CAST system, and present mechanistic insights into target DNA recognition and RNA-guided transposition. [ABSTRACT FROM AUTHOR]