A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters

Area of Science:

• Biotechnology
• Molecular Biology
• Neuroscience

Background:

• Engineering complex proteins with multiple specifications is challenging.
• Directed evolution offers a powerful approach for protein optimization.
• High-performance fluorescent voltage indicators are crucial for neuroscience research.

Purpose of the Study:

• To develop a scalable directed evolution strategy for engineering complex proteins.
• To create a genetically encoded fluorescent voltage indicator with optimized brightness and membrane localization.
• To demonstrate the utility of the engineered protein in various biological systems.

Main Methods:

• Utilized a microscopy-guided, robotic cell-picking strategy for high-throughput screening of protein libraries.
• Applied directed evolution to simultaneously optimize multiple protein properties, including fluorescence and localization.
• Developed a novel opsin-based fluorescent voltage reporter, Archon1.

Main Results:

• Successfully screened hundreds of thousands of proteins in hours, evaluating multiple performance axes.
• Engineered Archon1, a high-performance fluorescent voltage reporter with enhanced brightness and membrane localization.
• Demonstrated Archon1's utility in imaging neural activity in mouse brain slices, zebrafish, and C. elegans.

Conclusions:

• The developed directed evolution strategy is effective for engineering complex proteins with multidimensional specifications.
• Archon1 is a valuable tool for high-resolution imaging of neural activity across different model organisms.
• This approach has broad implications for protein engineering and neuroscience research.