Accelerated Engineering of Optogenetic Tools with a High-throughput Microwell Array Platform

Abstract

Optogenetics has shifted from a cutting-edge technology to a mainstay experimental technique. At its core, optogenetics is the use of light sensitive proteins to report or control biological activity. The unique spatiotemporal precision afforded by optogenetic tools has propelled the technique to the forefront of neuroscience, where it is used to investigate the complexities of the mammalian nervous system. Several technologies need to be integrated to implement an optogenetic experiment, coalescing around light delivery to a light sensitive transgene. Typically, optogenetic experiments are limited by the performance of the protein-based, light-sensitive tools. Constructing and optimizing optogenetic tools is laborious and resource intensive, prohibiting extensive tool development. Here, we present a high-throughput optogenetic tool pipeline that screens thousands of protein variants each day in mammalian cells. To showcase the pipeline, dubbed Opto-MASS, we improved a widely adopted dopamine sensor’s response to 100 nM DA >6-fold, and demonstrated the sensor could detect dopamine transients in vivo. We improved a mu opioid receptor derived sensor > 4-fold and detected morphine administration in vivo in the mammalian nervous system. To further showcase Opto-MASS, we expanded the applications to screen for ligand selectivity by making three biophysical measurements of thousands of sensors nearly simultaneously. Opto-MASS directly links the measured phenotype to the underlying genotype, reducing the time from gene identification to tool application. Opto-MASS is poised to shift the optogenetic tool engineering landscape by rapidly constructing high performance optogenetic actuators and sensors that can be quickly shared with the circuit neuroscience community.