OMEGA-guided DNA polymerases enable random mutagenesis in a tunable window

Targeted random mutagenesis is crucial for breeding, directed evolution, and gene function studies, yet efficient tools remain scarce. Here, we present obligate mobile element guided activity (OMEGA)-R, an innovative targeted random mutagenesis system that integrates SpyCatcher-enIscB and PolI3M-TBD-SpyTag, outperforming existing state-of-the-art technologies in key metrics, such as protein size, mutagenesis efficiency, window length, and continuity. OMEGA-R achieves a dramatic enhancement of on-target mutagenesis, reaching a rate of 1.4 × 10^-5 base pairs (bp) per generation (bpg), with minimal off-target effects, in both Escherichia coli and Bacillus subtilis. The system also demonstrates exceptional compatibility with high-throughput screening (HTS) technologies, including fluorescence-activated droplet sorting (FADS) and phage-assisted continuous evolution (PACE). Utilizing OMEGA-R, we successfully identified a series of effective mutations within the T7 promoter (pT7), ribosome-binding site (RBS), superfolder GFP (sfGFP), and autocyclizing ribozyme (AR), which are invaluable for the development of high-performance biotechnology tools. These findings underscore the high efficiency and broad application potential of OMEGA-R.