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As emphasized by Varma and colleagues, traditional single-layer kill-switch mechanisms often exhibit instability and may lose functionality within days, whereas multilayered systems—particularly those combining CRISPR-inducible modules, toxin–antitoxin systems, and synthetic auxotrophies—demonstrate markedly greater robustness, maintaining containment across hundreds of generations ( Saccharomyces cerevisiae and S

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doi:10.1016/j.fct.2013.03.024