Efficient transgene-free multiplexed germline editing via viral delivery of an engineered TnpB

Abstract Virus-induced genome editing (VIGE) using compact RNA-guided endonucleases is a transformational new approach in plant biotechnology, enabling tissue-culture-independent and transgene-free genome editing (Hu et al. 2025; Liu et al. 2025; Weiss et al. 2025). We recently established a VIGE approach for heritable editing at single loci in Arabidopsis by delivering the compact genome editor ISYmu1 TnpB (Ymu1) and its guide RNA (gRNA) via Tobacco Rattle Virus (TRV) (Weiss et al. 2025). Here, we greatly improved this approach by devising a multiple gRNA expression system and by utilizing an engineered high-activity Ymu1 variant (Ymu1-WFR) (Zhou et al. 2026) to develop an efficient multiplexed genome editing platform. Competing Interest Statement T.W., M.K, H.S., J.A.D. and S.E.J. have filed a patent covering aspects of this work. S.E.J. is a cofounder and consultant for Inari Agriculture and a consultant for Terrana Biosciences, Invaio Sciences, Sail Biomedicines and Zymo Research. J.A.D. is a cofounder of Azalea Therapeutics, Caribou Biosciences, Editas Medicine, Evercrisp, Scribe Therapeutics and Mammoth Biosciences. J.A.D. is a scientific advisory board member at Evercrisp, Caribou Biosciences, Scribe Therapeutics, Mammoth Biosciences, The Column Group and Inari. She is also an advisor for Aditum Bio. J.A.D. is Chief Science Advisor to Sixth Street, a Director at Johnson & Johnson, Altos and Tempus, and has a research project sponsored by Apple Tree Partners. The other authors declare no competing interests. Footnotes germline transmission of multiplexed editing using the Ymu1-WFR TnpB.