Omega-based mini prime- and base-editing systems in Escherichia coli

Prime editing (PE) and base editing (BE) are potent gene-editing techniques that avoid introducing exogenous DNA donors and double-strand breaks, but their large molecular size hinders efficient delivery and widespread application. Here, we engineer and evolve compact obligate mobile element-guided activity (OMEGA)-based PE and BE systems, termed OMEGA-PE and OMEGA-adenine base editor, which achieve superior editing efficiency and versatility compared with existing PE and BE tools in Escherichia coli. Notably, OMEGA-PE enables rapid, programmable targeted mutagenesis with outstanding specificity and efficiency compared with current tools. Moreover, it exhibits excellent compatibility with several high-throughput screening platforms and can be used to enhance the efficiency of cofactor regeneration and protein translation. These groundbreaking advancements offer significant potential for gene editing, directed evolution, metabolic engineering, and protein expression optimization. Combining compact size with high performance, our OMEGA-based systems address key bottlenecks in current tools, offering new possibilities for basic research and industrial biotechnology.