VEGAS as a Platform for Facile Directed Evolution in Mammalian Cells

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• Directed evolution is crucial for developing molecular tools and therapeutics.
• Current methods often rely on unicellular organisms, which can limit the evolution of certain proteins.
• A need exists for efficient directed evolution systems in mammalian cells.

Purpose of the Study:

• To develop a facile system for directed evolution in mammalian cells.
• To enable rapid selection and evolution of proteins within their native cellular environment.
• To demonstrate the system's utility for evolving diverse protein classes.

Main Methods:

• Development of the viral evolution of genetically actuating sequences (VEGAS) system.
• Utilizing the Sindbis virus as an RNA vector for heredity and diversity.
• Implementing genetically actuated sequences for selection within host cells.
• Achieving rapid selection cycles with high mutation rates ( > 10^-3 mutations per base) within 24 hours.

Main Results:

• Demonstrated facile directed evolution in mammalian cells using the VEGAS system.
• Achieved rapid selection cycles (24 hours) with high mutation rates.
• Successfully evolved transcription factors, GPCRs, and allosteric nanobodies.
• Completed evolution of target proteins in under one week.

Conclusions:

• The VEGAS system provides a powerful and rapid method for directed evolution in mammalian cells.
• This system overcomes limitations of unicellular-based evolution for certain protein targets.
• VEGAS accelerates the development of novel molecular tools and therapeutics by enabling evolution in a native cellular context.