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A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters
Open accessAbstract
AbstractWe 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.Directed evolution of opsins via robotic high-content screening finds a fluorescent reporter of voltage that is simultaneously optimized for brightness, localization and voltage sensitivity and is applicable in three model systems.
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Metadata
- Authors
- 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.
- Venue
- Nature Chemical Biology
- Published
- 2018-02-26
- Volume
- 14
- Issue
- 4
- Pages
- 352-360
- ISSN
- 1552-4450 (ISSN-L)
- 1552-4469
- OpenAlex
- W2792403811
- Cited by
- 319
- References
- 55
- FWCI
- 17.69
- Type
- journal article
- Language
- en
- Open access
- green
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- 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., . . . Boyden, E. S. (2018). A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters. Nature Chemical Biology. https://doi.org/10.1038/s41589-018-0004-9
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