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  3. Orthogonal RNA replication enables directed evolution and Darwinian adaptation in mammalian cells

Orthogonal RNA replication enables directed evolution and Darwinian adaptation in mammalian cells

  • Nat Chem Biol. 2025 Jan 3. doi: 10.1038/s41589-024-01783-2.
Liang Ma 1 2 3,  Yihan Lin 4 5 6 7
Affiliations

Affiliations

  • 1 Center for Quantitative Biology, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.
  • 2 Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.
  • 3 The MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China.
  • 4 Center for Quantitative Biology, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China. yihan.lin@pku.edu.cn.
  • 5 Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China. yihan.lin@pku.edu.cn.
  • 6 The MOE Key Laboratory of Cell Proliferation and Differentiation, School of Life Sciences, Peking University, Beijing, China. yihan.lin@pku.edu.cn.
  • 7 Chengdu Academy for Advanced Interdisciplinary Biotechnologies, Peking University, Chengdu, China. yihan.lin@pku.edu.cn.
PMID: 39753704 DOI: 10.1038/s41589-024-01783-2
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.

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