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Nat Chem Biol DOI:10.1038/s41589-018-0004-9

A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters.

Publication TypeJournal Article
Year of Publication2018
AuthorsPiatkevich, KD, Jung, EE, Straub, C, Linghu, C, Park, D, Suk, H-J, Hochbaum, DR, Goodwin, D, Pnevmatikakis, E, Pak, N, Kawashima, T, Yang, C-T, Rhoades, JL, Shemesh, O, Asano, S, Yoon, Y-G, Freifeld, L, Saulnier, JL, Riegler, C, Engert, F, Hughes, T, Drobizhev, M, Szabo, B, Ahrens, MB, Flavell, SW, Sabatini, BL, Boyden, ES
JournalNat Chem Biol
Volume14
Issue4
Pages352-360
Date Published2018 04
ISSN1552-4469
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.

DOI10.1038/s41589-018-0004-9
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/29483642?dopt=Abstract

Alternate JournalNat. Chem. Biol.
PubMed ID29483642
PubMed Central IDPMC5866759
Grant ListU01 NS099691 / NS / NINDS NIH HHS / United States
R01 DA029639 / DA / NIDA NIH HHS / United States
R24 MH106075 / MH / NIMH NIH HHS / United States
R01 MH103910 / MH / NIMH NIH HHS / United States
R01 EY023173 / EY / NEI NIH HHS / United States
R01 GM104948 / GM / NIGMS NIH HHS / United States
R01 NS087950 / NS / NINDS NIH HHS / United States
DP1 NS087724 / NS / NINDS NIH HHS / United States
R43 MH109332 / MH / NIMH NIH HHS / United States
R01 NS104892 / NS / NINDS NIH HHS / United States