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Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells

Abstract

Engineered genetic circuits for mammalian cells often require extensive fine-tuning to perform as intended. We present a robust, general, scalable system, called 'Boolean logic and arithmetic through DNA excision' (BLADE), to engineer genetic circuits with multiple inputs and outputs in mammalian cells with minimal optimization. The reliability of BLADE arises from its reliance on recombinases under the control of a single promoter, which integrates circuit signals on a single transcriptional layer. We used BLADE to build 113 circuits in human embryonic kidney and Jurkat T cells and devised a quantitative, vector-proximity metric to evaluate their performance. Of 113 circuits analyzed, 109 functioned (96.5%) as intended without optimization. The circuits, which are available through Addgene, include a 3-input, two-output full adder; a 6-input, one-output Boolean logic look-up table; circuits with small-molecule-inducible control; and circuits that incorporate CRISPR–Cas9 to regulate endogenous genes. BLADE enables execution of sophisticated cellular computation in mammalian cells, with applications in cell and tissue engineering.

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Figure 1: Orthogonal site-specific tyrosine recombinases and serine integrases enable implementation of multi-input AND gates in mammalian cells.
Figure 2: 2-input BLADE platform can produce four distinct output functions based on two inputs.
Figure 3: 113 distinct gene circuits with up to two inputs and two outputs implemented using the 2-input BLADE template.
Figure 4: Field-programmable storage and retrieval of logic and memory using a Boolean logic look-up table (LUT).
Figure 5: A 3-input BLADE template can be applied to create 3-input arithmetic computational circuits.
Figure 6: Interfacing BLADE with biologically relevant inputs and outputs.

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References

  1. Khalil, A.S. & Collins, J.J. Synthetic biology: applications come of age. Nat. Rev. Genet. 11, 367–379 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Wei, P. et al. Bacterial virulence proteins as tools to rewire kinase pathways in yeast and immune cells. Nature 488, 384–388 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Roybal, K.T. et al. Precision tumor recognition by T cells with combinatorial antigen-sensing circuits. Cell 164, 770–779 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Chakravarti, D. & Wong, W.W. Synthetic biology in cell-based cancer immunotherapy. Trends Biotechnol. 33, 449–461 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Xie, M. et al. β-cell-mimetic designer cells provide closed-loop glycemic control. Science 354, 1296–1301 (2016).

    Article  CAS  PubMed  Google Scholar 

  6. Slomovic, S. & Collins, J.J. DNA sense-and-respond protein modules for mammalian cells. Nat. Methods 12, 1085–1090 (2015).

    Article  CAS  PubMed  Google Scholar 

  7. Courbet, A., Endy, D., Renard, E., Molina, F. & Bonnet, J. Detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates. Sci. Transl. Med. 7, 289ra83 (2015).

    Article  CAS  PubMed  Google Scholar 

  8. Fenno, L.E. et al. Targeting cells with single vectors using multiple-feature Boolean logic. Nat. Methods 11, 763–772 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Madisen, L. et al. Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance. Neuron 85, 942–958 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Ro, D.K. et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940–943 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Bogorad, I.W., Lin, T.S. & Liao, J.C. Synthetic non-oxidative glycolysis enables complete carbon conservation. Nature 502, 693–697 (2013).

    Article  CAS  PubMed  Google Scholar 

  12. Gaber, R. et al. Designable DNA-binding domains enable construction of logic circuits in mammalian cells. Nat. Chem. Biol. 10, 203–208 (2014).

    Article  CAS  PubMed  Google Scholar 

  13. 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  PubMed  Google Scholar 

  14. Guinn, M. & Bleris, L. Biological 2-input decoder circuit in human cells. ACS Synth. Biol. 3, 627–633 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Weber, W. et al. A synthetic time-delay circuit in mammalian cells and mice. Proc. Natl. Acad. Sci. USA 104, 2643–2648 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  17. Brophy, J.A. & Voigt, C.A. Principles of genetic circuit design. Nat. Methods 11, 508–520 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Nielsen, A.A. et al. Genetic circuit design automation. Science 352, aac7341 (2016).

    Article  CAS  PubMed  Google Scholar 

  19. Appleton, E., Tao, J., Haddock, T. & Densmore, D. Interactive assembly algorithms for molecular cloning. Nat. Methods 11, 657–662 (2014).

    Article  CAS  PubMed  Google Scholar 

  20. Rodrigo, G. & Jaramillo, A. AutoBioCAD: full biodesign automation of genetic circuits. ACS Synth. Biol. 2, 230–236 (2013).

    Article  CAS  PubMed  Google Scholar 

  21. Huynh, L., Kececioglu, J., Köppe, M. & Tagkopoulos, I. Automatic design of synthetic gene circuits through mixed integer non-linear programming. PLoS One 7, e35529 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Stanton, B.C. et al. Genomic mining of prokaryotic repressors for orthogonal logic gates. Nat. Chem. Biol. 10, 99–105 (2014).

    Article  CAS  PubMed  Google Scholar 

  23. Nagy, A. Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99–109 (2000).

    Article  CAS  PubMed  Google Scholar 

  24. Lee, G. & Saito, I. Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. Gene 216, 55–65 (1998).

    Article  CAS  PubMed  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  PubMed  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  PubMed  Google Scholar 

  27. Schönhuber, N. et al. A next-generation dual-recombinase system for time- and host-specific targeting of pancreatic cancer. Nat. Med. 20, 1340–1347 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Shannon, C.E. The synthesis of two-terminal switching circuits. Bell Syst. Tech. J. 28, 59–98 (1949).

    Article  Google Scholar 

  29. Torella, J.P. et al. Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic Acids Res. 42, 681–689 (2014).

    Article  CAS  PubMed  Google Scholar 

  30. Torella, J.P. et al. Unique nucleotide sequence-guided assembly of repetitive DNA parts for synthetic biology applications. Nat. Protoc. 9, 2075–2089 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Canton, B., Labno, A. & Endy, D. Refinement and standardization of synthetic biological parts and devices. Nat. Biotechnol. 26, 787–793 (2008).

    Article  CAS  PubMed  Google Scholar 

  32. Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Jayanthi, S., Nilgiriwala, K.S. & Del Vecchio, D. Retroactivity controls the temporal dynamics of gene transcription. ACS Synth. Biol. 2, 431–441 (2013).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  35. Khalil, A.S. et al. A synthetic biology framework for programming eukaryotic transcription functions. Cell 150, 647–658 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Green, A.A., Silver, P.A., Collins, J.J. & Yin, P. Toehold switches: de-novo-designed regulators of gene expression. Cell 159, 925–939 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 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  PubMed  Google Scholar 

  38. Stricker, J. et al. A fast, robust and tunable synthetic gene oscillator. Nature 456, 516–519 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  40. Johnson, R.C. in Mobile DNA II (eds. Craig, N., Craigie, R., Gellert, M. & Lambowitz, A.) 230–271 (American Society of Microbiology, 2002).

  41. Blomfield, I.C. The regulation of pap and type 1 fimbriation in Escherichia coli . Adv. Microb. Physiol. 45, 1–49 (2001).

    Article  CAS  PubMed  Google Scholar 

  42. Roquet, N., Soleimany, A.P., Ferris, A.C., Aaronson, S. & Lu, T.K. Synthetic recombinase-based state machines in living cells. Science 353, aad8559 (2016).

    Article  CAS  PubMed  Google Scholar 

  43. Hsiao, V., Hori, Y., Rothemund, P.W. & Murray, R.M. A population-based temporal logic gate for timing and recording chemical events. Mol. Syst. Biol. 12, 869 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Branda, C.S. & Dymecki, S.M. Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice. Dev. Cell 6, 7–28 (2004).

    Article  CAS  PubMed  Google Scholar 

  45. Yang, L. et al. Permanent genetic memory with >1-byte capacity. Nat. Methods 11, 1261–1266 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Mercer, A.C., Gaj, T., Fuller, R.P. & Barbas, C.F. III. Chimeric TALE recombinases with programmable DNA sequence specificity. Nucleic Acids Res. 40, 11163–11172 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Sirk, S.J., Gaj, T., Jonsson, A., Mercer, A.C. & Barbas, C.F. III. Expanding the zinc-finger recombinase repertoire: directed evolution and mutational analysis of serine recombinase specificity determinants. Nucleic Acids Res. 42, 4755–4766 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Chaikind, B., Bessen, J.L., Thompson, D.B., Hu, J.H. & Liu, D.R. A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells. Nucleic Acids Res. 44, 9758–9770 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Shaikh, A.C. & Sadowski, P.D. Chimeras of the Flp and Cre recombinases: tests of the mode of cleavage by Flp and Cre. J. Mol. Biol. 302, 27–48 (2000).

    Article  CAS  PubMed  Google Scholar 

  50. Karpinski, J. et al. Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity. Nat. Biotechnol. 34, 401–409 (2016).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

B.H.W. acknowledges funding from the NSF Graduate Research Fellowship Program (DGE-1247312) and an NIH/NIGMS fellowship (T32-GM008764). S.B. was supported in part by the National Science Foundation Expeditions in Computing Award No. 1522074, which is part of the “Living Computing Project” (https://www.programmingbiology.org/). W.W.W. acknowledges funding from the NIH Director's New Innovator Award (1DP2CA186574), NSF Expedition in Computing (1522074), NSF CAREER (162457), NSF BBSRC (1614642), and Boston University College of Engineering Dean's Catalyst Award. We thank C. Bashor, D. Chakravarti, N. Patel, and S. Slomovic for suggestions on the manuscript; A. Belkina and T. Haddock for flow cytometry assistance; J. Torella for help with UNS-guided assembly; and M. Park and J. Eyckmans for RT-qPCR assistance. A. Nagy for the kind gift of the Dre construct.

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Contributions

B.H.W. made molecular and cellular reagents, performed experiments, analyzed data and generated all figures. S.B. conceived the vector proximity analyses for circuit performance and developed the datasheets attribution and website. B.H.W. and S.B. developed and performed the vector proximity analyses. L.D.C., N.T.H.P., T.L., and A.E. made molecular and cellular reagents and performed preliminary experiments. B.H.W. and W.W.W. conceived the project. B.H.W., S.B., and W.W.W. wrote the paper. All authors commented on and approved the paper.

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Correspondence to Wilson W Wong.

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W.W.W. and B.H.W. have a patent application pending (WO/2015/188191) whose value may be affected by the publication of this paper.

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Weinberg, B., Pham, N., Caraballo, L. et al. Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells. Nat Biotechnol 35, 453–462 (2017). https://doi.org/10.1038/nbt.3805

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