Multiplexed CRISPR base editing enables pulse-activated irreversible biocontainment of engineered bacteria

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.