Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Designable DNA-binding domains enable construction of logic circuits in mammalian cells

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Design of single-layer genetic NOR gates based on orthogonal designed TALE repressors in mammalian cells.
Figure 2: Construction of the XOR logic gate from NOR gates based on the orthogonal designed TALE repressors.
Figure 3: Implementation of all 16 two-input Boolean logic functions constructed from combinations of designed TALE repressor-based NOR gates.
Figure 4: Design of the genetic circuit that allows selection of logic function processing data inputs.

References

  1. Tamsir, A., Tabor, J.J. & Voigt, C.A. Robust multicellular computing using genetically encoded NOR gates and chemical 'wires'. Nature 469, 212–215 (2011).

    Article  CAS  Google Scholar 

  2. Ausländer, S., Ausländer, D., Muller, M., Wieland, M. & Fussenegger, M. Programmable single-cell mammalian biocomputers. Nature 487, 123–127 (2012).

    Article  Google Scholar 

  3. Friedland, A.E. et al. Synthetic gene networks that count. Science 324, 1199–1202 (2009).

    Article  CAS  Google Scholar 

  4. Elowitz, M.B. & Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335–338 (2000).

    Article  CAS  Google Scholar 

  5. Gardner, T.S., Cantor, C.R. & Collins, J.J. Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339–342 (2000).

    Article  CAS  Google Scholar 

  6. Tigges, M., Marquez-Lago, T.T., Stelling, J. & Fussenegger, M. A tunable synthetic mammalian oscillator. Nature 457, 309–312 (2009).

    Article  CAS  Google Scholar 

  7. 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).

    Article  CAS  Google Scholar 

  8. 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).

    Article  CAS  Google Scholar 

  9. Hall, E.C. Journey to the Moon: the History of the Apollo Guidance Computer (American Institute of Aeronautics and Astronautics, 1996).

  10. Boch, J. et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509–1512 (2009).

    Article  CAS  Google Scholar 

  11. Moscou, M.J. & Bogdanove, A.J. A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 (2009).

    Article  CAS  Google Scholar 

  12. 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).

    Article  CAS  Google Scholar 

  13. 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).

    Article  CAS  Google Scholar 

  14. Zhang, F. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29, 149–153 (2011).

    Article  Google Scholar 

  15. 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).

    Article  CAS  Google Scholar 

  16. 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).

    Article  Google Scholar 

  17. Meckler, J.F. et al. Quantitative analysis of TALE-DNA interactions suggests polarity effects. Nucleic Acids Res. 41, 4118–4128 (2013).

    Article  CAS  Google Scholar 

  18. 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).

    Article  CAS  Google Scholar 

  19. Perez-Pinera, P. et al. Synergistic and tunable human gene activation by combinations of synthetic transcription factors. Nat. Methods 10, 239–242 (2013).

    Article  CAS  Google Scholar 

  20. Maeder, M.L. et al. Robust, synergistic regulation of human gene expression using TALE activators. Nat. Methods 10, 243–245 (2013).

    Article  CAS  Google Scholar 

  21. Kramer, B.P., Fischer, C. & Fussenegger, M. BioLogic gates enable logical transcription control in mammalian cells. Biotechnol. Bioeng. 87, 478–484 (2004).

    Article  CAS  Google Scholar 

  22. 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).

    Article  CAS  Google Scholar 

  23. Fussenegger, M. et al. Streptogramin-based gene regulation systems for mammalian cells. Nat. Biotechnol. 18, 1203–1208 (2000).

    Article  CAS  Google Scholar 

  24. Weber, W. et al. Macrolide-based transgene control in mammalian cells and mice. Nat. Biotechnol. 20, 901–907 (2002).

    Article  CAS  Google Scholar 

  25. Siuti, P., Yazbek, J. & Lu, T.K. Synthetic circuits integrating logic and memory in living cells. Nat. Biotechnol. 31, 448–452 (2013).

    Article  CAS  Google Scholar 

  26. Bonnet, J., Yin, P., Ortiz, M.E., Subsoontorn, P. & Endy, D. Amplifying genetic logic gates. Science 340, 599–603 (2013).

    Article  CAS  Google Scholar 

  27. 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).

    Article  Google Scholar 

  28. Reyon, D. et al. FLASH assembly of TALENs for high-throughput genome editing. Nat. Biotechnol. 30, 460–465 (2012).

    Article  CAS  Google Scholar 

  29. 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).

    Article  CAS  Google Scholar 

  30. 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).

    Article  CAS  Google Scholar 

  31. Weber, E., Gruetzner, R., Werner, S., Engler, C. & Marillonnet, S. Assembly of designer TAL effectors by Golden Gate cloning. PLoS ONE 6, e19722 (2011).

    Article  CAS  Google Scholar 

  32. 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).

    Article  CAS  Google Scholar 

  33. Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).

    Article  CAS  Google Scholar 

  34. Gilbert, L.A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

    Article  CAS  Google Scholar 

  35. Qi, L.S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    Article  CAS  Google Scholar 

  36. Regot, S. et al. Distributed biological computation with multicellular engineered networks. Nature 469, 207–211 (2011).

    Article  CAS  Google Scholar 

  37. 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).

    Article  Google Scholar 

  38. 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).

    Article  CAS  Google Scholar 

  39. Nissim, L. & Bar-Ziv, R.H. A tunable dual-promoter integrator for targeting of cancer cells. Mol. Syst. Biol. 6, 444 (2010).

    Article  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Roman Jerala.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–14, Supplementary Note and Supplementary Tables 1–11. (PDF 2980 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.1433

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing