Optogenetic Microwell Array Screening System: A High-Throughput Engineering Platform for Genetically Encoded...

Area of Science:

• Biotechnology
• Molecular Biology
• Optogenetics

Background:

• Genetically encoded fluorescent indicators (GEFIs) are crucial for monitoring cellular physiology but engineering them is slow.
• Current methods involve laborious trial-and-error mutagenesis and in vitro testing of individual variants.
• The vast protein mutational landscape hinders the development of sensitive and specific GEFIs.

Purpose of the Study:

• To develop a high-throughput platform for rapid engineering of GEFIs.
• To overcome the limitations of traditional GEFI optimization methods.
• To accelerate the discovery of novel GEFI variants with improved performance.

Main Methods:

• Developed the optogenetic microwell array screening system (Opto-MASS) for parallel screening of thousands of GEFI variants in mammalian cells.
• Applied Opto-MASS to screen over 13,000 dopamine and 21,000 opioid sensor variants.
• Utilized a multiplexed, high-throughput approach to functionally test GEFI variants.

Main Results:

• Generated a novel dopamine sensor (dMASS^1) with a >6-fold signal increase in response to dopamine.
• Developed a new opioid sensor (μMASS^1) exhibiting a ~4.6-fold signal increase to DAMGO.
• Demonstrated Opto-MASS's capability to screen large variant libraries significantly faster than existing methods.

Conclusions:

• Opto-MASS enables rapid engineering of novel GEFIs.
• The platform significantly shortens optimization time for sensors with distinct biophysical properties.
• This high-throughput system advances the development of optogenetic tools for cellular monitoring.