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Insertion sequences are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance 1 . IS200- and IS605-family transposons undergo 'peel-and-paste' transposition catalysed by a TnpA transposase 2 , but they also encode diverse, TnpB- and IscB-family proteins that are evolutionarily related to the CRISPR-associated effectors Cas12 and Cas9, respectively 3,4 . Recent studies have demonstrated that TnpB and IscB function as RNA-guided DNA endonucleases 5,6 , but the broader biological role of this activity has remained enigmatic. Here we show that TnpB and IscB are essential to prevent permanent transposon loss as a consequence of the TnpA transposition mechanism. We selected a family of related insertion sequences from Geobacillus stearothermophilus that encode several TnpB and IscB orthologues, and showed that a single TnpA transposase was broadly active for transposon mobilization. The donor joints formed upon religation of transposon-flanking sequences were efficiently targeted for cleavage by RNA-guided TnpB and IscB nucleases, and co-expression of TnpB and TnpA led to substantially greater transposon retention relative to conditions in which TnpA was expressed alone. Notably, TnpA and TnpB also stimulated recombination frequencies, surpassing rates observed with TnpB alone. Collectively, this study reveals that RNA-guided DNA cleavage arose as a primal biochemical activity to bias the selfish inheritance and spread of transposable elements, which was later co-opted during the evolution of CRISPR-Cas adaptive immunity for antiviral defence., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)