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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Packer, M. S. & Liu, D. R. Methods for the directed evolution of proteins. Nat. Rev. Genet. 16, 379–394 (2015).
Subach, F. V., Piatkevich, K. D. & Verkhusha, V. V. Directed molecular evolution to design advanced red fluorescent proteins. Nat. Methods 8, 1019–1026 (2011).
Chen, T.-W. et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).
Klapoetke, N. C. et al. Independent optical excitation of distinct neural populations. Nat. Methods 11, 338–346 (2014).
Zhao, Y. et al. Microfluidic cell sorter-aided directed evolution of a protein-based calcium ion indicator with an inverted fluorescent response. Integr. Biol. (Camb) 6, 714–725 (2014).
Dean, K. M. et al. Microfluidics-based selection of red-fluorescent proteins with decreased rates of photobleaching. Integr. Biol. (Camb) 7, 263–273 (2015).
Fiedler, B. L. et al. Droplet microfluidic flow cytometer for sorting on transient cellular responses of genetically-encoded sensors. Anal. Chem. 89, 711–719 (2017).
Lin, M. Z. & Schnitzer, M. J. Genetically encoded indicators of neuronal activity. Nat. Neurosci. 19, 1142–1153 (2016).
Boyden, E. S. Optogenetics and the future of neuroscience. Nat. Neurosci. 18, 1200–1201 (2015).
Ai, H.-W., Baird, M. A., Shen, Y., Davidson, M. W. & Campbell, R. E. Engineering and characterizing monomeric fluorescent proteins for live-cell imaging applications. Nat. Protoc. 9, 910–928 (2014).
Chow, B. Y., Chuong, A. S., Klapoetke, N. C. & Boyden, E. S. Synthetic physiology strategies for adapting tools from nature for genetically targeted control of fast biological processes. Methods. Enzymol. 497, 425–443 (2011).
Hochbaum, D. R. et al. All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins. Nat. Methods 11, 825–833 (2014).
Flytzanis, N. C. et al. Archaerhodopsin variants with enhanced voltage-sensitive fluorescence in mammalian and Caenorhabditis elegans neurons. Nat. Commun. 5, 4894 (2014).
St-Pierre, F. et al. High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor. Nat. Neurosci. 17, 884–889 (2014).
Gong, Y., Wagner, M. J., Zhong Li, J. & Schnitzer, M. J. Imaging neural spiking in brain tissue using FRET-opsin protein voltage sensors. Nat. Commun. 5, 3674 (2014).
Gong, Y. et al. High-speed recording of neural spikes in awake mice and flies with a fluorescent voltage sensor. Science 350, 1361–1366 (2015).
Környei, Z. et al. Cell sorting in a Petri dish controlled by computer vision. Sci. Rep. 3, 1088 (2013).
Salánki, R. et al. Automated single cell sorting and deposition in submicroliter drops. Appl. Phys. Lett. 105, 83703 (2014).
Giraud, E. et al. Bacteriophytochrome controls photosystem synthesis in anoxygenic bacteria. Nature 417, 202–205 (2002).
He, L., Friedman, A. M. & Bailey-Kellogg, C. A divide-and-conquer approach to determine the Pareto frontier for optimization of protein engineering experiments. Proteins 80, 790–806 (2012).
Knowles, J. D. & Corne, D. W. Approximating the nondominated front using the Pareto Archived Evolution Strategy. Evol. Comput. 8, 149–172 (2000).
Currin, A., Swainston, N., Day, P. J. & Kell, D. B. Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently. Chem. Soc. Rev. 44, 1172–1239 (2015).
Grigoryan, G., Reinke, A. W. & Keating, A. E. Design of protein-interaction specificity gives selective bZIP-binding peptides. Nature 458, 859–864 (2009).
McIsaac, R. S. et al. Directed evolution of a far-red fluorescent rhodopsin. Proc. Natl. Acad. Sci. USA 111, 13034–13039 (2014).
Kotnik, T., Pucihar, G. & Miklavčič, D. Induced transmembrane voltage and its correlation with electroporation-mediated molecular transport. J. Membr. Biol. 236, 3–13 (2010).
Del, Re,A. M. & Woodward, J. J. Inhibition of gap junction currents by the abused solvent toluene. Drug. Alcohol. Depend. 78, 221–224 (2005).
Gradinaru, V. et al. Molecular and cellular approaches for diversifying and extending optogenetics. Cell 141, 154–165 (2010).
Chuong, A. S. et al. Noninvasive optical inhibition with a red-shifted microbial rhodopsin. Nat. Neurosci. 17, 1123–1129 (2014).
Shemesh, O. A. et al. Temporally precise single-cell-resolution optogenetics. Nat. Neurosci. 20, 1796–1806 (2017).
Friedrich, R. W., Jacobson, G. A. & Zhu, P. Circuit neuroscience in zebrafish. Curr. Biol. 20, R371–R381 (2010).
Stewart, A. M., Braubach, O., Spitsbergen, J., Gerlai, R. & Kalueff, A. V. Zebrafish models for translational neuroscience research: from tank to bedside. Trends. Neurosci. 37, 264–278 (2014).
Ahrens, M. B. et al. Brain-wide neuronal dynamics during motor adaptation in zebrafish. Nature 485, 471–477 (2012).
Ahrens, M. B., Orger, M. B., Robson, D. N., Li, J. M. & Keller, P. J. Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nat. Methods 10, 413–420 (2013).
Wyart, C. et al. Optogenetic dissection of a behavioural module in the vertebrate spinal cord. Nature 461, 407–410 (2009).
Gordus, A., Pokala, N., Levy, S., Flavell, S. W. & Bargmann, C. I. Feedback from network states generates variability in a probabilistic olfactory circuit. Cell 161, 215–227 (2015).
Dobes, N. C. et al. Laser-based directed release of array elements for efficient collection into targeted microwells. Analyst 138, 831–838 (2013).
Bouchard, M. B. et al. Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms. Nat. Photonics 9, 113–119 (2015).
Prevedel, R. et al. Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy. Nat. Methods 11, 727–730 (2014).
Krzywinski, M. & Altman, N. Visualizing samples with box plots. Nat. Methods 11, 119–120 (2014).
Subedi, A. et al. Adoption of the Q transcriptional regulatory system for zebrafish transgenesis. Methods 66, 433–440 (2014).
Piatkevich, K. D., Subach, F. V. & Verkhusha, V. V. Far-red light photoactivatable near-infrared fluorescent proteins engineered from a bacterial phytochrome. Nat. Commun. 4, 2153 (2013).
Filonov, G. S. et al. Bright and stable near-infrared fluorescent protein for in vivo imaging. Nat. Biotechnol. 29, 757–761 (2011).
Makarov, N. S., Drobizhev, M. & Rebane, A. Two-photon absorption standards in the 550-1600 nm excitation wavelength range. Opt. Express. 16, 4029–4047 (2008).
Drobizhev, M., Makarov, N. S., Tillo, S. E., Hughes, T. E. & Rebane, A. Two-photon absorption properties of fluorescent proteins. Nat. Methods 8, 393–399 (2011).
Lebkowski, J. S., DuBridge, R. B., Antell, E. A., Greisen, K. S. & Calos, M. P. Transfected DNA is mutated in monkey, mouse, and human cells. Mol. Cell. Biol. 4, 1951–1960 (1984).
Mahon, M. J. Vectors bicistronically linking a gene of interest to the SV40 large T antigen in combination with the SV40 origin of replication enhance transient protein expression and luciferase reporter activity. Biotechniques 51, 119–128 (2011).
Qin, J. Y. et al. Systematic comparison of constitutive promoters and the doxycycline-inducible promoter. PLoS One 5, e10611 (2010).
Chen, C. & Okayama, H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7, 2745–2752 (1987).
Okazaki, M., Yoshida, Y., Yamaguchi, S., Kaneno, M. & Elliott, J. C. Affinity binding phenomena of DNA onto apatite crystals. Biomaterials 22, 2459–2464 (2001).
Pucihar, G., Kotnik, T. & Miklavcic, D. Measuring the induced membrane voltage with Di-8-ANEPPS. J. Vis. Exp. 88, 4–6 (2009).
Chow, B. Y. et al. High-performance genetically targetable optical neural silencing by light-driven proton pumps. Nature 463, 98–102 (2010).
Jiang, M. & Chen, G. High Ca2+ -phosphate transfection efficiency in low-density neuronal cultures. Nat. Protoc. 1, 695–700 (2006).
Kimura, Y., Satou, C. & Higashijima, S. V2a and V2b neurons are generated by the final divisions of pair-producing progenitors in the zebrafish spinal cord. Development 135, 3001–3005 (2008).
Kwan, K. M. et al. The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev. Dyn. 236, 3088–3099 (2007).
Fisher, S. et al. Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish. Nat. Protoc. 1, 1297–1305 (2006).
Renaud, O., Herbomel, P. & Kissa, K. Studying cell behavior in whole zebrafish embryos by confocal live imaging: application to hematopoietic stem cells. Nat. Protoc. 6, 1897–1904 (2011).
Brenner, S. The genetics of Caenorhabditis elegans. Genetics. 77, 71–94 (1974).
Kralj, J. M., Douglass, A. D., Hochbaum, D. R., Maclaurin, D. & Cohen, A. E. Optical recording of action potentials in mammalian neurons using a microbial rhodopsin. Nat. Methods 9, 90–95 (2011).
Lee, D. D. & Seung, H. S. Learning the parts of objects by non-negative matrix factorization. Nature 401, 788–791 (1999).
Dell, R. B., Holleran, S. & Ramakrishnan, R. Sample size determination. ILAR. J. 43, 207–213 (2002).
We thank G. Paradis and M. Saturno-Condon for help with flow cytometry, F. Chen and L. Kang for help with confocal imaging, N. Ji for assistance with C. elegans imaging, and B. Trout and C. Sudrik for help with spectroscopic analysis of iRFPs. We are grateful to X. Han and K. Hansen (Boston University) for the pCAG-WPRE expression vector, and F. Subach (Moscow Institute of Physics and Technology) for the pWA23h plasmid. We are grateful to E. Costa, D. Estandian, A. Wassie and L. Cai for useful discussions. C.S. acknowledges the Lefler Center for the Study of Neurodegenerative Disorders for support. E.S.B. was supported by the HHMI-Simons Faculty Scholars Program, the IET Harvey Prize, the MIT Media Lab, the New York Stem Cell Foundation-Robertson Award, the Open Philanthropy Project, Human Frontier Science Program RGP0015/2016, and NIH grants 1R43MH109332, 1R24MH106075, 2R01DA029639, 1R01EY023173, 1R01NS087950, 1R01MH103910 and 1R01GM104948, and NIH Director’s Pioneer Award 1DP1NS087724. O.S. was supported by a Simons Fellowship. H.-J.S. was supported by a Samsung Fellowship. D.G. was supported by an NSF Fellowship. Y.-G.Y. was supported by a Samsung Fellowship. L.F. was supported by a Simons Fellowship.
B.S. is a founder of the CellSorter startup company. K.D.P., E.E.J. and E.S.B. are inventors on patent applications regarding the molecules described here. B.S., K.D.P., E.E.J. and E.S.B. are inventors on a patent application regarding the screening method developed here.
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A correction to this article is available online at https://doi.org/10.1038/s41589-018-0023-6.
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Piatkevich, K.D., Jung, E.E., Straub, C. et al. A robotic multidimensional directed evolution approach applied to fluorescent voltage reporters. Nat Chem Biol 14, 352–360 (2018). https://doi.org/10.1038/s41589-018-0004-9
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