Structures of the holo CRISPR RNA-guided transposon integration complex

CRISPR-associated transposons (CAST) are programmable mobile elements that insert large DNA cargo by an RNA-guided mechanism. Multiple conserved components act in concert at the target site through formation of an integration complex (transpososome). We reconstituted the type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined the structure using cryo-EM. Transpososome architecture ensures orientation-specific association: AAA+ regulator TnsC has defined polarity and length, with dedicated interaction interfaces for other CAST components. Interestingly, transposase (TnsB)-TnsC interactions we observe contribute to stimulating TnsC’s ATP hydrolysis activity. TnsC deviates from previously observed helical configurations of TnsC, and target DNA does not track with TnsC protomers. Consequently, TnsC makes new, functionally important protein-DNA interactions throughout the transpososome. Finally, two distinct transpososome populations suggests that associations with the CRISPR effector are flexible. These ShCAST transpososome structures significantly enhances our understanding of CAST transposition systems and suggests avenues for improving CAST transposition for precision genome-editing applications.