A native single-transcript TnpB architecture enables efficient virus-induced genome editing and visual screening of heritable progeny harboring phenotypically silent edits

Abstract

TnpB are ultra-compact RNA-guided nucleases, yet most reported systems require separate expression of the nuclease and cognate reRNA, eroding their size advantage and restricting viral delivery in plants. Here we demonstrate that ISDra2 TnpB harnesses its native overlapping architecture in which the reRNA sequence resides within the 3' coding region of TnpB, processing its own mRNA to generate functional reRNA and enabling single-transcript genome editing in plants. Only a 7-bp conserved motif followed by a 16-20 bp spacer directly downstream of the coding sequence suffices for editing, requiring no exogenous ribozymes or independent guide promoters. This ultra-compact design enables simultaneous packaging of the editing system and an NbPDS silencing module into a single TRV vector, achieving robust editing in systemic Nicotiana benthamiana tissues and establishing a dual VIGE-VIGS strategy for direct visual selection of heritable, transgene-free edited progeny via distinct albino phenotypes, reducing laborious large-scale genotyping even for targets lacking inherent visible traits.