Ma Liang, Lin Yihan
Center for Quantitative Biology, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.
Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.
Nat Chem Biol. 2025 Mar;21(3):451-463. doi: 10.1038/s41589-024-01783-2. Epub 2025 Jan 3.
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.
哺乳动物细胞中的定向进化为推进合成生物学应用提供了一种强大的方法。然而，现有的基于哺乳动物的定向进化方法面临着重大瓶颈，包括宿主基因组干扰、文库规模小和诱变不受控制。在这里，我们设计了一个正交的α病毒RNA复制系统来进化基于RNA的装置，从而在增殖的哺乳动物细胞中实现RNA复制酶辅助的连续进化（REPLACE）。该系统通过复制酶限制的复制模式和诱导诱变产生一个庞大的、不断多样化的复制RNA文库。使用REPLACE，我们设计了荧光蛋白和转录因子。值得注意的是，配备REPLACE的细胞可以进行达尔文式适应，使其能够应对细胞外和细胞内的挑战而进化。总的来说，这项工作建立了一个强大的平台，通过定向进化推进哺乳动物合成生物学和细胞工程应用。
