Fig. 3: TnpB-mediated plasmid interference in vivo.

a, Experimental workflow of the plasmid interference assay in E. coli. The cleavage of a target plasmid results in loss of kanamycin (Kn) resistance. The reRNA-encoding construct contained the 16-nt guide sequence. AmpR, ampicillin/carbenicillin (Ap/Cb) resistance gene; KanR, kanamycin resistance gene. b, Plasmid interference assay. E. coli culture samples were serially diluted (10×) and the E. coli transformants were grown on the media supplemented with Cb and Kn at 25 °C for 44 h. Interference is compromised for the catalytically dead D191A and E278A TnpB variants. Target ‘+’ or ‘−’ indicates the plasmids with or without the target, respectively. For the uncropped plate image, see Supplementary Fig. 1. c, Proposed role of TnpB in transposition. The IS200/IS605 transposon circle is excised from the lagging strand during DNA replication resulting in two DNA copies: one copy that originates from the leading strand and carries an intact transposon, and another copy that originates from the lagging strand and lacks the transposon at the original site due to the strand-specific transposon excision. However, the latter DNA copy still carries the transposon ‘footprint’ in the form of the donor joint, comprised of the 5′-TTGAT sequence and the 3′-flanking DNA sequence that becomes a target to the TnpB–reRNA complex. In this case, the 5′-TTGAT sequence serves as a TAM that initiates the binding of the reRNA sequence to the matching DNA sequence followed by dsDNA cleavage. TnpB-induced DSB could facilitate homology-directed repair to reinstate the transposon at the donor joint using its intact copy on the sister chromatid, ensuring that both DNA copies have a transposon-coding gene before cell division. Red triangles indicate DNA cleavage sites.