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. (2018) A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters. Nature Chemical Biology. 14(4):352-360.
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.