Structure–function insights into the initial step of DNA integration by a CRISPR–Cas–Transposon complex

CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated genes) surveillance complexes are RNA-based adaptive immune systems employed by prokaryotes against invading nucleic acids from bacteriophages and plasmids. 1 , 2 The CRISPR-derived RNAs (crRNAs) guide the Cas effector complex to target and degrade the invading nucleic acids. Recently, bioinformatics analyses have revealed the presence of CRISPR–Cas loci in bacterial Tn7-like transposons, thereby implicating a functional relationship between RNA-guided DNA targeting and transposition, with the latter representing a new role unrelated to host defense. 3 Support for this concept has emerged from recent functional studies on type I-F and type V-K effectors involved in sequence-specific DNA transposition, 4 , 5 thereby significantly broadening the potential biological applications of CRISPR–Cas technology. To complement the available functional studies, our efforts have focused on structural studies of the Vibrio cholerae Tn6677 multi-subunit type I-F Cascade crRNA –TniQ complex, whereby transposition subunit TniQ initiates DNA transposition with the eventual help of other transposition-associated proteins TnsA, TnsB and TnsC in the gene cluster (Fig. 1a ). Fig. 1: Cryo-EM structures of type I-F Cascade crRNA –TniQ complexes. a The cas and transposition-related genes in the Vibrio cholerae Tn6677 type I-F CRISPR–transposon system. b , c Schematic ( c ) and ribbon ( b ) representations of cryo-EM structure of Cascade crRNA –TniQ–dsDNA ternary complex. d PAM recognition by Cas8. Detailed interactions are shown in the expanded panels. e Interactions between the individual transposition protein TniQ and Cas6 and Cas7.1. Inset panels pointed by arrows provide detailed interactions. f Structure comparison between Cascade crRNA –TniQ binary complex and Cascade crRNA –TniQ–dsDNA ternary complex based on alignment of the Cas8 subunit. Vector length correlates with the domain movement scale. g Superposition of crRNA in the binary (in silver) and ternary (in cyan) complexes. Full size image