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

Area of Science:

• Synthetic biology
• Molecular biology
• Genetics

Background:

• Mammalian cell-based directed evolution is crucial for synthetic biology but hindered by host genome interference and limited library sizes.
• Existing methods struggle with uncontrolled mutagenesis and scalability, restricting their application.
• Developing robust, scalable directed evolution systems in mammalian cells is a key challenge.

Purpose of the Study:

• To engineer an orthogonal alphaviral RNA replication system for directed evolution in mammalian cells.
• To establish a platform for continuous evolution of RNA-based devices and cellular adaptation.
• To overcome bottlenecks in current mammalian directed evolution methodologies.

Main Methods:

• Engineered an orthogonal alphaviral RNA replication system for RNA-based directed evolution.
• Developed RNA replicase-assisted continuous evolution (REPLACE) in proliferating mammalian cells.
• Utilized replicase-limited replication and inducible mutagenesis for continuous library diversification.

Main Results:

• Successfully generated large, continuously diversified libraries of replicative RNAs.
• Engineered functional RNA-based devices, including fluorescent proteins and transcription factors.
• Demonstrated that cells equipped with REPLACE can undergo Darwinian adaptation to environmental challenges.

Conclusions:

• REPLACE provides a powerful new platform for advancing mammalian synthetic biology and cell engineering.
• This system overcomes key limitations of existing directed evolution methods in mammalian cells.
• Enables directed evolution of RNA devices and adaptive evolution of cells for diverse applications.