A chimeric viral platform for directed evolution in mammalian cells

Area of Science:

• Biotechnology
• Molecular Biology
• Synthetic Biology

Background:

• Directed evolution is crucial for developing novel biomolecules but faces challenges in mammalian systems.
• Existing prokaryotic and yeast-based platforms are not ideal for all mammalian cell applications.
• Stable and efficient mammalian-based directed evolution systems are needed.

Purpose of the Study:

• To develop a stable and efficient platform for directed evolution in mammalian cells.
• To demonstrate the utility of the platform for engineering gene regulation tools and intracellular nanobodies.
• To overcome limitations of current directed evolution systems in mammalian contexts.

Main Methods:

• Development of the PROTein Evolution Using Selection (PROTEUS) platform using chimeric virus-like vesicles.
• Application of PROTEUS for altering doxycycline responsiveness of tetracycline-controlled transactivators.
• Utilizing PROTEUS for intracellular nanobody evolution, specifically targeting a DNA damage-responsive anti-p53 nanobody.

Main Results:

• The PROTEUS platform enables extended mammalian directed evolution campaigns without compromising system integrity.
• A more sensitive TetON-4G gene regulation tool was generated with mammalian-specific adaptations.
• Successful evolution of a DNA damage-responsive anti-p53 nanobody was achieved using the PROTEUS platform.

Conclusions:

• PROTEUS is an efficient and stable platform for directed evolution of biomolecules within mammalian cells.
• The platform facilitates the generation of novel tools for gene regulation and protein engineering in mammalian systems.
• PROTEUS represents a significant advancement for mammalian-based biotechnology and synthetic biology.