{"title":"Orthogonal RNA replication enables directed evolution and Darwinian adaptation in mammalian cells","authors":"Liang Ma, Yihan Lin","doi":"10.1038/s41589-024-01783-2","DOIUrl":null,"url":null,"abstract":"<p>Directed evolution in mammalian cells offers a powerful approach for advancing synthetic biology applications. However, existing mammalian-based directed evolution methods face substantial bottlenecks, including host genome interference, small library size and uncontrolled mutagenesis. Here we engineered an orthogonal alphaviral RNA replication system to evolve RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in proliferating mammalian cells. This system generates a large, continuously diversified library of replicative RNAs through replicase-limited mode of replication and inducible mutagenesis. Using REPLACE, we engineered fluorescent proteins and transcription factors. Notably, cells equipped with REPLACE can undergo Darwinian adaptation, allowing them to evolve in response to both cell-extrinsic and cell-intrinsic challenges. Collectively, this work establishes a powerful platform for advancing mammalian synthetic biology and cell engineering applications through directed evolution.</p><figure></figure>","PeriodicalId":18832,"journal":{"name":"Nature chemical biology","volume":"17 1","pages":""},"PeriodicalIF":12.9000,"publicationDate":"2025-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Nature chemical biology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1038/s41589-024-01783-2","RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Directed evolution in mammalian cells offers a powerful approach for advancing synthetic biology applications. However, existing mammalian-based directed evolution methods face substantial bottlenecks, including host genome interference, small library size and uncontrolled mutagenesis. Here we engineered an orthogonal alphaviral RNA replication system to evolve RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in proliferating mammalian cells. This system generates a large, continuously diversified library of replicative RNAs through replicase-limited mode of replication and inducible mutagenesis. Using REPLACE, we engineered fluorescent proteins and transcription factors. Notably, cells equipped with REPLACE can undergo Darwinian adaptation, allowing them to evolve in response to both cell-extrinsic and cell-intrinsic challenges. Collectively, this work establishes a powerful platform for advancing mammalian synthetic biology and cell engineering applications through directed evolution.
期刊介绍：
Nature Chemical Biology stands as an esteemed international monthly journal, offering a prominent platform for the chemical biology community to showcase top-tier original research and commentary. Operating at the crossroads of chemistry, biology, and related disciplines, chemical biology utilizes scientific ideas and approaches to comprehend and manipulate biological systems with molecular precision.
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