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Nucleic Acids Res., Apr 23 2026, 54 (8) gkag422 | https://doi.org/10.1093/nar/gkag422 Multiplexed CRISPR base editing enables pulse-activated irreversible biocontainment of engineered bacteria

Sung Won Cho^1,†, TaeHyun Kim^1,†, Jina Yang², Gibyuck Byun¹, Sang Woo Seo^1,3,4,5,6,*

¹Department of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-Gu, Seoul 08826, Republic of Korea
²Department of Chemical Engineering, Jeju National University, 102 Jejudaehak-ro, Jeju-si, Jeju-do 63243, South Korea
³Institute of Chemical Processes, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, South Korea
⁴Bio-MAX Institute, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, South Korea
⁵Institute of Bio Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, South Korea
⁶Interdisciplinary Program in Bioengineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, South Korea

^∗To whom correspondence should be addressed.: Sang Woo Seo
^†The first two authors should be regarded as Joint First Authors.

Abstract

The environmental and therapeutic application of genetically engineered microorganisms necessitates the development of robust, irreversible biocontainment systems. In this study, we present an eEGM (editing-driven essential gene multiplex inactivation) module that utilizes CRISPR-mediated cytidine base editing to induce permanent self-killing via a single transient induction. By targeting the start codons of essential genes, we achieved an irreversible translational blockade that avoids the fitness costs associated with basal toxicity in nuclease-based systems. Multiplexed targeting of non-redundant essential loci (holA, ftsB, and dfp) yielded escape frequencies at or below the NIH guideline criterion (10-8) within 1 h of pulse induction. Furthermore, the eEGM system exhibited robust functional orthogonality and portability across laboratory, industrial, and therapeutic Escherichia coli strains, including MG1655, W3110, and Nissle 1917, without detectable interference with heterologous protein expression. This work establishes base editing as a cleavage-free CRISPR effector for pulse-activated, irreversible biocontainment and provides a practical framework for safer deployment of engineered microbes.

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