A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters

Nat Chem Biol. 2018 Apr;14(4):352-360. doi: 10.1038/s41589-018-0004-9. Epub 2018 Feb 26.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

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*

Substances

  • Luminescent Proteins