De novo engineered guide RNA-directed transposition with TnpB-family proteins reveals features of naturally evolved systems.

Programmable DNA integration using CRISPR-associated transposons (CASTs) offers powerful capabilities for genome engineering. The single effector Cas12k CAST examples evolved from a fixed guide TnpB nuclease protein. Here, we engineer de novo RNA-guided transposition systems, where the single guide RNA effector components are repurposed nuclease-dead TnpB-family proteins. These compact systems mediate high-efficiency guide RNA-directed DNA insertion with preserved orientation control and target immunity, reduced off-site targeting, release of a host factor requirement, and can be paired with an exonuclease domain to mediate cut-and-paste transposition. In this engineered context, the TnpB derivatives show features not predicted from the original enzymes suggesting untapped avenues for improvement. In parallel, we show that mutations at the TniQ-TnsC interface in the Cas12k CAST system selectively attenuate off-site insertions while enhancing on-site activity. These results establish how Cas12 proteins and antecedent TnpB proteins can be engineered for high performance and specificity with guide RNA directed systems.