Abstract
Electronic computer circuits consisting of a large number of connected logic gates of the same type, such as NOR, can be easily fabricated and can implement any logic function. In contrast, designed genetic circuits must employ orthogonal information mediators owing to free diffusion within the cell. Combinatorial diversity and orthogonality can be provided by designable DNA- binding domains. Here, we employed the transcription activator–like repressors to optimize the construction of orthogonal functionally complete NOR gates to construct logic circuits. We used transient transfection to implement all 16 two-input logic functions from combinations of the same type of NOR gates within mammalian cells. Additionally, we present a genetic logic circuit where one input is used to select between an AND and OR function to process the data input using the same circuit. This demonstrates the potential of designable modular transcription factors for the construction of complex biological information-processing devices.
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References
Tamsir, A., Tabor, J.J. & Voigt, C.A. Robust multicellular computing using genetically encoded NOR gates and chemical 'wires'. Nature 469, 212–215 (2011).
Ausländer, S., Ausländer, D., Muller, M., Wieland, M. & Fussenegger, M. Programmable single-cell mammalian biocomputers. Nature 487, 123–127 (2012).
Friedland, A.E. et al. Synthetic gene networks that count. Science 324, 1199–1202 (2009).
Elowitz, M.B. & Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335–338 (2000).
Gardner, T.S., Cantor, C.R. & Collins, J.J. Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339–342 (2000).
Tigges, M., Marquez-Lago, T.T., Stelling, J. & Fussenegger, M. A tunable synthetic mammalian oscillator. Nature 457, 309–312 (2009).
Bonnet, J., Subsoontorn, P. & Endy, D. Rewritable digital data storage in live cells via engineered control of recombination directionality. Proc. Natl. Acad. Sci. USA 109, 8884–8889 (2012).
Ye, H., Daoud-El Baba, M., Peng, R.W. & Fussenegger, M. A synthetic optogenetic transcription device enhances blood-glucose homeostasis in mice. Science 332, 1565–1568 (2011).
Hall, E.C. Journey to the Moon: the History of the Apollo Guidance Computer (American Institute of Aeronautics and Astronautics, 1996).
Boch, J. et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509–1512 (2009).
Moscou, M.J. & Bogdanove, A.J. A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 (2009).
Garg, A., Lohmueller, J.J., Silver, P.A. & Armel, T.Z. Engineering synthetic TAL effectors with orthogonal target sites. Nucleic Acids Res. 40, 7584–7595 (2012).
Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector–based constructs for DNA targeting. Nucleic Acids Res. 39, e82 (2011).
Zhang, F. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29, 149–153 (2011).
Blount, B.A., Weenink, T., Vasylechko, S. & Ellis, T. Rational diversification of a promoter providing fine-tuned expression and orthogonal regulation for synthetic biology. PLoS ONE 7, e33279 (2012).
Cong, L., Zhou, R., Kuo, Y.C., Cunniff, M. & Zhang, F. Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains. Nat. Commun. 3, 968 (2012).
Meckler, J.F. et al. Quantitative analysis of TALE-DNA interactions suggests polarity effects. Nucleic Acids Res. 41, 4118–4128 (2013).
Witzgall, R., O'Leary, E., Leaf, A., Onaldi, D. & Bonventre, J.V. The Kruppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression. Proc. Natl. Acad. Sci. USA 91, 4514–4518 (1994).
Perez-Pinera, P. et al. Synergistic and tunable human gene activation by combinations of synthetic transcription factors. Nat. Methods 10, 239–242 (2013).
Maeder, M.L. et al. Robust, synergistic regulation of human gene expression using TALE activators. Nat. Methods 10, 243–245 (2013).
Kramer, B.P., Fischer, C. & Fussenegger, M. BioLogic gates enable logical transcription control in mammalian cells. Biotechnol. Bioeng. 87, 478–484 (2004).
Moon, T.S., Lou, C.B., Tamsir, A., Stanton, B.C. & Voigt, C.A. Genetic programs constructed from layered logic gates in single cells. Nature 491, 249–253 (2012).
Fussenegger, M. et al. Streptogramin-based gene regulation systems for mammalian cells. Nat. Biotechnol. 18, 1203–1208 (2000).
Weber, W. et al. Macrolide-based transgene control in mammalian cells and mice. Nat. Biotechnol. 20, 901–907 (2002).
Siuti, P., Yazbek, J. & Lu, T.K. Synthetic circuits integrating logic and memory in living cells. Nat. Biotechnol. 31, 448–452 (2013).
Bonnet, J., Yin, P., Ortiz, M.E., Subsoontorn, P. & Endy, D. Amplifying genetic logic gates. Science 340, 599–603 (2013).
Andrianantoandro, E., Basu, S., Karig, D.K. & Weiss, R. Synthetic biology: new engineering rules for an emerging discipline. Mol. Syst. Biol. 2, 2006.0028 (2006).
Reyon, D. et al. FLASH assembly of TALENs for high-throughput genome editing. Nat. Biotechnol. 30, 460–465 (2012).
Briggs, A.W. et al. Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers. Nucleic Acids Res. 40, e117 (2012).
Schmid-Burgk, J.L., Schmidt, T., Kaiser, V., Honing, K. & Hornung, V. A ligation-independent cloning technique for high-throughput assembly of transcription activator-like effector genes. Nat. Biotechnol. 31, 76–81 (2013).
Weber, E., Gruetzner, R., Werner, S., Engler, C. & Marillonnet, S. Assembly of designer TAL effectors by Golden Gate cloning. PLoS ONE 6, e19722 (2011).
Lohmueller, J.J., Armel, T.Z. & Silver, P.A. A tunable zinc finger–based framework for Boolean logic computation in mammalian cells. Nucleic Acids Res. 40, 5180–5187 (2012).
Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).
Gilbert, L.A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).
Qi, L.S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).
Regot, S. et al. Distributed biological computation with multicellular engineered networks. Nature 469, 207–211 (2011).
Greber, D., El-Baba, M.D. & Fussenegger, M. Intronically encoded siRNAs improve dynamic range of mammalian gene regulation systems and toggle switch. Nucleic Acids Res. 36, e101 (2008).
Xie, Z., Wroblewska, L., Prochazka, L., Weiss, R. & Benenson, Y. Multi-input RNAi-based logic circuit for identification of specific cancer cells. Science 333, 1307–1311 (2011).
Nissim, L. & Bar-Ziv, R.H. A tunable dual-promoter integrator for targeting of cancer cells. Mol. Syst. Biol. 6, 444 (2010).
Acknowledgements
This research study was supported by the program and projects from the Slovenian Research Agency (P4-0176 and N5-0003 to R.J.) and the EN-FIST Centre of Excellence financed in part by the European structural funds. We acknowledge the members and mentors of the 2012 Slovenian International Genetically Engineered Machine (iGEM) Team (U. Bezeljak, V. Forstnerič, A. Golob, M. Jerala, L. Kadunc, J. Lonzarić, Z. Lužnik, A. Oblak, F. Pavlovec, B. Pirč, A. Smole, M. Somrak, M. Stražar, D. Vučko and U. Zupančič) for their inspiration and help in the development of TALE-based regulation. We thank M. Fussenegger (Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Zurich) for plasmids for erythromycin- and pristinamycin-inducible systems.
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R.G., T.L., M.B. and A.M. performed and analyzed the experiments; B.Š., A.D. and R.G. designed the logic gates and analyzed the triple gate circuits; R.J. designed the study and wrote the manuscript; and M.B., R.G., B.Š. and A.D. helped in writing the manuscript.
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Supplementary Results, Supplementary Figures 1–14, Supplementary Note and Supplementary Tables 1–11. (PDF 2980 kb)
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Gaber, R., Lebar, T., Majerle, A. et al. Designable DNA-binding domains enable construction of logic circuits in mammalian cells. Nat Chem Biol 10, 203–208 (2014). https://doi.org/10.1038/nchembio.1433
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DOI: https://doi.org/10.1038/nchembio.1433
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