Mechanistic insights into transposon cleavage and integration by TnsB of ShCAST system.

The type V-K CRISPR-associated transposons (CASTs) allow RNA-guided DNA integration and have great potential as a programmable site-specific gene insertion tool. Although all core components have been independently characterized structurally, the mechanism of how the transposase TnsB associates with AAA+ ATPase TnsC and catalyzes donor DNA cleavage and integration remains ambiguous. In this study, we demonstrate that TniQ-dCas9 fusion can direct site-specific transposition by TnsB/TnsC in ShCAST. TnsB is a 3'-5' exonuclease that specifically cleaves donor DNA at the end of the terminal repeats and integrates the left end prior to the right end. The nucleotide preference and the cleavage site of TnsB are markedly different from those of the well-documented MuA. We also find that TnsB/TnsC association is enhanced in a half-integration state. Overall, our results provide valuable insights into the mechanism and application expansion of CRISPR-mediated site-specific transposition by TnsB/TnsC.