A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters.
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.
|Year of Publication||
Nat Chem Biol
|PubMed Central ID||
U01 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