Structural basis for the assembly of the type V CRISPR-associated transposon complex

Cell. 2022 Dec 22;185(26):4999-5010.e17. doi: 10.1016/j.cell.2022.11.009. Epub 2022 Nov 25.


CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprised of RNA-guided Cas12k, TniQ, a polymeric TnsC filament and, unexpectedly, the ribosomal protein S15. Complex assembly, mediated by a network of interactions involving the guide RNA, TniQ, and S15, results in R-loop completion. TniQ contacts two TnsC protomers at the Cas12k-proximal filament end, likely nucleating its polymerization. Transposition activity assays corroborate our structural findings, implying that S15 is a bona fide component of the type V crRNA-guided transposon machinery. Altogether, our work uncovers key mechanistic aspects underpinning RNA-mediated assembly of CRISPR-associated transposons to guide their development as programmable tools for site-specific insertion of large DNA payloads.

Keywords: CAST; CRISPR; CRISPR-Cas system; CRISPR-associated transposons; Cas12k; S15; TniQ; TnsB; TnsC; transposon.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • CRISPR-Associated Proteins* / genetics
  • CRISPR-Cas Systems
  • DNA Transposable Elements* / genetics
  • DNA-Binding Proteins / metabolism
  • RNA
  • Transposases / genetics


  • DNA Transposable Elements
  • Transposases
  • DNA-Binding Proteins
  • RNA
  • Bacterial Proteins
  • CRISPR-Associated Proteins