A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters
Kiryl D Piatkevich, Erica E Jung, Christoph Straub, Changyang Linghu, Demian Park, Ho-Jun Suk, Daniel R Hochbaum, Daniel Goodwin, Eftychios Pnevmatikakis, Nikita Pak, Takashi Kawashima, Chao-Tsung Yang, Jeffrey L Rhoades, Or Shemesh, Shoh Asano, Young-Gyu Yoon, Limor Freifeld, Jessica L Saulnier, Clemens Riegler, Florian Engert, Thom Hughes, Mikhail Drobizhev, Balint Szabo, Misha B Ahrens, Steven W Flavell, Bernardo L Sabatini, Edward S Boyden
Nature chemical biology
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.
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