An orthogonal transcription mutation system generating all transition mutations for accelerated protein evolution in vivo
Abstract Targeted mutagenesis systems are critical for protein evolution. Current deaminase-T7 RNA polymerase fusion systems enable gene-specific mutagenesis but remain limited to certain model organisms. Here, we develop an orthogonal transcription mutation system for in vivo hypermutation in both...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-07-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-61354-4 |
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| _version_ | 1849334458133512192 |
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| author | Mingwei Shao Zhongnan Zhang Xiaofan Jin Jun Ding Guo-Qiang Chen |
| author_facet | Mingwei Shao Zhongnan Zhang Xiaofan Jin Jun Ding Guo-Qiang Chen |
| author_sort | Mingwei Shao |
| collection | DOAJ |
| description | Abstract Targeted mutagenesis systems are critical for protein evolution. Current deaminase-T7 RNA polymerase fusion systems enable gene-specific mutagenesis but remain limited to certain model organisms. Here, we develop an orthogonal transcription mutation system for in vivo hypermutation in both non-model organism Halomonas bluephagenesis and E. coli, achieving >1,500,000-fold increased mutation rates. By fusing deaminases with three phage RNA polymerases, this system uniformly introduces C:G to T:A and A:T to G:C mutations across target genes. The system demonstrates high specificity, minimal off-target effects, and high orthogonality between phage polymerases. We apply this system to rapidly evolve fluorescent proteins, chromoproteins, cytoskeletal proteins, cell division-related proteins, global sigma factor, and the LysE exporter within a single day of the mutagenesis process. Overall, the orthogonal transcription mutation system is a modular and versatile platform that accelerates protein evolution in the shortest period reported so far. |
| format | Article |
| id | doaj-art-51aad04fec8649059cba63ce28ce124e |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-51aad04fec8649059cba63ce28ce124e2025-08-20T03:45:33ZengNature PortfolioNature Communications2041-17232025-07-0116111910.1038/s41467-025-61354-4An orthogonal transcription mutation system generating all transition mutations for accelerated protein evolution in vivoMingwei Shao0Zhongnan Zhang1Xiaofan Jin2Jun Ding3Guo-Qiang Chen4School of Life Sciences, Tsinghua UniversitySchool of Life Sciences, Tsinghua UniversitySchool of Life Sciences, Tsinghua UniversitySchool of Life Sciences, Tsinghua UniversitySchool of Life Sciences, Tsinghua UniversityAbstract Targeted mutagenesis systems are critical for protein evolution. Current deaminase-T7 RNA polymerase fusion systems enable gene-specific mutagenesis but remain limited to certain model organisms. Here, we develop an orthogonal transcription mutation system for in vivo hypermutation in both non-model organism Halomonas bluephagenesis and E. coli, achieving >1,500,000-fold increased mutation rates. By fusing deaminases with three phage RNA polymerases, this system uniformly introduces C:G to T:A and A:T to G:C mutations across target genes. The system demonstrates high specificity, minimal off-target effects, and high orthogonality between phage polymerases. We apply this system to rapidly evolve fluorescent proteins, chromoproteins, cytoskeletal proteins, cell division-related proteins, global sigma factor, and the LysE exporter within a single day of the mutagenesis process. Overall, the orthogonal transcription mutation system is a modular and versatile platform that accelerates protein evolution in the shortest period reported so far.https://doi.org/10.1038/s41467-025-61354-4 |
| spellingShingle | Mingwei Shao Zhongnan Zhang Xiaofan Jin Jun Ding Guo-Qiang Chen An orthogonal transcription mutation system generating all transition mutations for accelerated protein evolution in vivo Nature Communications |
| title | An orthogonal transcription mutation system generating all transition mutations for accelerated protein evolution in vivo |
| title_full | An orthogonal transcription mutation system generating all transition mutations for accelerated protein evolution in vivo |
| title_fullStr | An orthogonal transcription mutation system generating all transition mutations for accelerated protein evolution in vivo |
| title_full_unstemmed | An orthogonal transcription mutation system generating all transition mutations for accelerated protein evolution in vivo |
| title_short | An orthogonal transcription mutation system generating all transition mutations for accelerated protein evolution in vivo |
| title_sort | orthogonal transcription mutation system generating all transition mutations for accelerated protein evolution in vivo |
| url | https://doi.org/10.1038/s41467-025-61354-4 |
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