A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters

Nature Chemical Biology
Kiryl D PiatkevichEdward S Boyden

Abstract

We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.

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Datasets Mentioned

BETA
MG250278
MG250279
MG250280
MG250281
MG250283
MG250284
MG250285

Methods Mentioned

BETA
fluorescence-activating cell sorting
transfections
PCR
FACS
light scattering
gel filtration
ISS
genotyping
transfection
electrophoresis

Software Mentioned

Elements Advance Research
CellSorter
BDFACS Diva8
ImageJ
GenScript
Arivis Vision4D
SAS
Excel
MATLAB code
NIS

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