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

Area of Science:

• Molecular Biology
• Biotechnology
• Synthetic Biology

Background:

• Targeted random mutagenesis is essential for advancing breeding, directed evolution, and gene function studies.
• Existing mutagenesis tools often lack efficiency and exhibit significant off-target effects, limiting their application.
• There is a critical need for innovative and highly efficient targeted random mutagenesis systems.

Purpose of the Study:

• To introduce OMEGA-R (obligate mobile element guided activity-R), a novel system for targeted random mutagenesis.
• To evaluate the performance of OMEGA-R compared to existing state-of-the-art mutagenesis technologies.
• To demonstrate the utility of OMEGA-R in high-throughput screening and the development of biotechnology tools.

Main Methods:

• Integration of SpyCatcher-enIscB and PolI3M-TBD-SpyTag components to create the OMEGA-R system.
• Assessment of mutagenesis efficiency, on-target rates, and off-target effects in *Escherichia coli* and *Bacillus subtilis*.
• Evaluation of OMEGA-R compatibility with high-throughput screening (HTS) technologies like FADS and PACE.

Main Results:

• OMEGA-R significantly outperforms current technologies in protein size, mutagenesis efficiency, window length, and continuity.
• Achieved a high on-target mutagenesis rate of 1.4 × 10^-5 base pairs per generation (bpg) with minimal off-target mutations.
• Demonstrated successful identification of beneficial mutations in T7 promoter, RBS, sfGFP, and AR, valuable for biotechnology.

Conclusions:

• OMEGA-R represents a significant advancement in targeted random mutagenesis technology.
• The system offers enhanced efficiency, reduced off-target effects, and broad compatibility with HTS platforms.
• OMEGA-R holds substantial potential for applications in synthetic biology, protein engineering, and biotechnology tool development.