PUBLICATION

A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters

Authors
Piatkevich, K.D., Jung, E.E., Straub, C., Linghu, C., Park, D., Suk, H.J., Hochbaum, D.R., Goodwin, D., Pnevmatikakis, E., Pak, N., Kawashima, T., Yang, C.T., Rhoades, J.L., Shemesh, O., Asano, S., Yoon, Y.G., Freifeld, L., Saulnier, J.L., Riegler, C., Engert, F., Hughes, T., Drobizhev, M., Szabo, B., Ahrens, M.B., Flavell, S.W., Sabatini, B.L., Boyden, E.S.
ID
ZDB-PUB-180228-6
Date
2018
Source
Nature Chemical Biology   14(4): 352-360 (Journal)
Registered Authors
Ahrens, Misha, Engert, Florian, Kawashima, Takashi, Yang, Chao-Tsung
Keywords
none
MeSH Terms
  • Animals
  • Brain/diagnostic imaging
  • Caenorhabditis elegans
  • Cell Separation
  • Directed Molecular Evolution/methods*
  • Female
  • Flow Cytometry
  • Fluorescence
  • Gene Library
  • Genes, Reporter
  • HEK293 Cells
  • Hippocampus/cytology
  • Humans
  • Luminescent Proteins/chemistry*
  • Male
  • Mice
  • Microscopy, Fluorescence
  • Neurons/cytology
  • Optogenetics
  • Protein Engineering/methods*
  • Robotics*
  • Zebrafish/embryology*
PubMed
29483642 Full text @ Nat. Chem. Biol.
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.
Errata / Notes
This article is corrected by ZDB-PUB-220906-96 .
Genes / Markers
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping