Repurposing Cas12a2-mediated abortive infection for efficient and low-toxicity microbial genome editing

Abstract

CRISPR-Cas systems have revolutionized microbial genome engineering, yet counter-selection strategies typically rely on targeted DNA cleavage, where the severe cytotoxicity of double-strand breaks restricts transformation efficiency and clonal recovery. Here, we present an innovative strategy, SuCas12a2-Universal Recombination Editing (SURE), that circumvents these limitations by repurposing the RNA-targeting CRISPR-Cas12a2 system. Instead of directly attacking the genome, SURE utilizes Cas12a2’s RNA-triggered collateral activity to induce an abortive infection-like state, providing a potent yet recoverable selective pressure. This strategy increases transformation efficiency by up to 1,000-fold compared to Cas9- or Cas12-based systems while achieving over 90% editing efficiency for gene knockout, insertion, and replacement in Escherichia coli MG1655. We validate the SURE platform’s broad utility by extending it to diverse species, including Bacillus subtilis BSZR and Shewanella oneidensis MR-1. Mechanistically, we identify collateral double-stranded DNA degradation as the decisive factor for driving efficient selection. Finally, we apply this system to engineer E. coli MG1655 for efficient assimilation of the plastic monomer ethylene glycol, demonstrating its practical utility. Collectively, this work establishes a universal and robust RNA-guided editing framework that resolves the toxicity-efficiency trade-off inherent to the conventional DNA-targeting methods. Microbial genome engineering is often limited by toxicity from existing selection methods. Here, the authors develop a Cas12a2-based RNA-guided editing strategy that improves efficiency and reduces cellular damage, providing a robust tool for microbial engineering.