Evans, SamuelLu, ZeyuMcDonnell, LiamAnderson, WillPeralta, FranciscoWatkins, TysonAhmed, HafnaLuna-Flores, Carlos HoracioLoan, ThomasNavone, LauraTrau, MattScott, ColinSpeight, Robert E.Vickers, Claudia E.Peng, Bingyin2026-07-032026-07-03ORCID:/0000-0001-8129-9777/work/219175276https://hdl.handle.net/1885/733812858Background: Tandem gene repeats naturally occur as important genomic features and determine many traits in living organisms, like human diseases and microbial productivities of target bioproducts. Methods: Here, we developed a bacterial type-II toxin-antitoxin-mediated method to manipulate genomic integration of tandem gene repeats in Saccharomyces cerevisiae and further visualised the evolutionary trajectories of gene repeats. We designed a tri-vector system to introduce toxin-antitoxin-driven gene amplification modules. Results: This system delivered multi-copy gene integration in the form of tandem gene repeats spontaneously and independently from toxin-antitoxin-mediated selection. Inducing the toxin (RelE) expressing via a copper (II)-inducible CUP1 promoter successfully drove the in-situ gene amplification of the antitoxin (RelB) module, resulting in ~40 copies of a green fluorescence reporter gene per copy of genome. Copy-number changes, copy-number increase and copy-number decrease, and stable maintenance were visualised using the green fluorescence protein and blue chromoprotein AeBlue as reporters. Copy-number increases happened spontaneously and independent on a selection pressure. Increased copy number was quickly enriched through toxin-antitoxin-mediated selection. Conclusion: In summary, the bacterial toxin-antitoxin systems provide a flexible mechanism to manipulate gene copy number in eukaryotic cells and can be exploited for synthetic biology and metabolic engineering applications.This research was supported partially by the Australian Government through the Australian Research Council Centres of Excellence funding scheme (project CE200100029). The views expressed herein are those of the authors and are not necessarily those of the Australian Government or Australian Research Council. This work is supported by Galaxy Australia, a service provided by the Australian Biocommons and its partners.12en© 2026 By Author(s).gene amplificationgenetic dosagegenome evolutiontandem repeatstoxin-antitoxin2026-01-1610.53388/LR20260002105029004720