OMEGA-guided DNA polymerases enable random mutagenesis in a tunable window.
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| Autores principales: | , , , , , , , , , , , , |
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| Formato: | Artículo científico |
| Lenguaje: | en |
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Trends in biotechnology
2025
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| _version_ | 1868266232026759169 |
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| author | Liu, Li-Hua Lei, Wei Zhang, Zhiqian Lai, Shijing Xu, Bo Ge, Qijun Luo, Jiawen Yang, Min Zhang, Yang Chen, Jinde Zhong, Qiuru Wu, Yi-Rui Jiang, Ao |
| author_facet | Liu, Li-Hua Lei, Wei Zhang, Zhiqian Lai, Shijing Xu, Bo Ge, Qijun Luo, Jiawen Yang, Min Zhang, Yang Chen, Jinde Zhong, Qiuru Wu, Yi-Rui Jiang, Ao Liu, Li-Hua Lei, Wei Zhang, Zhiqian Lai, Shijing Xu, Bo Ge, Qijun Luo, Jiawen Yang, Min Zhang, Yang Chen, Jinde Zhong, Qiuru Wu, Yi-Rui Jiang, Ao |
| collection | PubMed - marine biology |
| contents | OMEGA-guided DNA polymerases enable random mutagenesis in a tunable window. Liu, Li-Hua Lei, Wei Zhang, Zhiqian Lai, Shijing Xu, Bo Ge, Qijun Luo, Jiawen Yang, Min Zhang, Yang Chen, Jinde Zhong, Qiuru Wu, Yi-Rui Jiang, Ao Mutagenesis Escherichia coli Bacillus subtilis Directed Molecular Evolution DNA-Directed DNA Polymerase Promoter Regions, Genetic High-Throughput Screening Assays 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 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. |
| format | Artículo científico |
| id | pubmed_40074636 |
| institution | PubMed |
| language | en |
| publishDate | 2025 |
| publisher | Trends in biotechnology |
| record_format | pubmed |
| spellingShingle | OMEGA-guided DNA polymerases enable random mutagenesis in a tunable window. Liu, Li-Hua Lei, Wei Zhang, Zhiqian Lai, Shijing Xu, Bo Ge, Qijun Luo, Jiawen Yang, Min Zhang, Yang Chen, Jinde Zhong, Qiuru Wu, Yi-Rui Jiang, Ao Mutagenesis Escherichia coli Bacillus subtilis Directed Molecular Evolution DNA-Directed DNA Polymerase Promoter Regions, Genetic High-Throughput Screening Assays OMEGA-guided DNA polymerases enable random mutagenesis in a tunable window. Liu, Li-Hua Lei, Wei Zhang, Zhiqian Lai, Shijing Xu, Bo Ge, Qijun Luo, Jiawen Yang, Min Zhang, Yang Chen, Jinde Zhong, Qiuru Wu, Yi-Rui Jiang, Ao Mutagenesis Escherichia coli Bacillus subtilis Directed Molecular Evolution DNA-Directed DNA Polymerase Promoter Regions, Genetic High-Throughput Screening Assays 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 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. |
| title | OMEGA-guided DNA polymerases enable random mutagenesis in a tunable window. |
| topic | Mutagenesis Escherichia coli Bacillus subtilis Directed Molecular Evolution DNA-Directed DNA Polymerase Promoter Regions, Genetic High-Throughput Screening Assays |
| url | https://pubmed.ncbi.nlm.nih.gov/40074636/ |