Engineering TnpB as a compact RNA-guided genome editor from molecular constraints to design principles.

Recent discoveries of non-CRISPR RNA-guided nucleases have expanded the landscape of programmable genome editing beyond canonical Cas systems. Among these, TnpB proteins derived from IS200/IS605-family transposable elements are ultra-compact RNA-guided DNA endonucleases with potential for delivery-constrained genome engineering. Here, we review the structural and mechanistic basis of TnpB function, including protein architecture, transposon-associated motif (TAM) recognition, ωRNA/reRNA scaffold organization, RNA-DNA heteroduplex formation, and RuvC-mediated DNA cleavage. We highlight that TnpB activity emerges from coordinated interactions between the nuclease and its RNA scaffold, which jointly influence ribonucleoprotein assembly, target recognition, catalytic activation, and specificity. We further discuss engineering strategies involving ortholog selection, protein and TAM-recognition engineering, RNA scaffold optimization, delivery, and specificity control, and relate these approaches to current applications across biological systems. Although TnpB remains less mature than established CRISPR-Cas platforms, its compact architecture and growing engineering toolkit support its development as a complementary genome editing platform. Future progress will benefit from establishing predictive models that integrate target accessibility, RNA-protein compatibility, activity, and fidelity.