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Modular construction of mammalian gene circuits using TALE transcriptional repressors

Abstract

An important goal of synthetic biology is the rational design and predictable implementation of synthetic gene circuits using standardized and interchangeable parts. However, engineering of complex circuits in mammalian cells is currently limited by the availability of well-characterized and orthogonal transcriptional repressors. Here, we introduce a library of 26 reversible transcription activator–like effector repressors (TALERs) that bind newly designed hybrid promoters and exert transcriptional repression through steric hindrance of key transcriptional initiation elements. We demonstrate that using the input-output transfer curves of our TALERs enables accurate prediction of the behavior of modularly assembled TALER cascade and switch circuits. We also show that TALER switches using feedback regulation exhibit improved accuracy for microRNA-based HeLa cancer cell classification versus HEK293 cells. Our TALER library is a valuable toolkit for modular engineering of synthetic circuits, enabling programmable manipulation of mammalian cells and helping elucidate design principles of coupled transcriptional and microRNA-mediated post-transcriptional regulation.

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Figure 1: Design and construction of TALE repressors for mammalian cells.
Figure 2: Design and construction of TALER cascade.
Figure 3: Design and construction of TALER sensory switches.
Figure 4: Response of TALER sensory switches to shRNA inputs.
Figure 5: Connecting microRNAs to regulate TALER sensory switches enables cell-type classification.

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Acknowledgements

We thank members of the Xie lab and the Weiss lab for helpful discussions. We thank H. Huang and T. Wang for technical support. We thank C. Le and F. Zhang (both at Department of Biological Engineering, Massachusetts Institute of Technology) for providing TALE1 and TALE2 as gifts. We thank J. Beal and M.Q. Zhang for insightful discussions. The research is supported by Beijing Natural Science Foundation (5152013 to Z.X.), National Key Basic Research Program of China (2014CB745200 to Z.X.), Junior '1,000 Plan' Program (to Z.X.), Cross-strait Tsinghua University Joint Grant (2012THZ0 to Z.X.) and US National Institutes of Health Grants (5R01CA155320-04, P50GM098792 and 1R01CA173712-01 to R.W.).

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Authors and Affiliations

Authors

Contributions

Z.X., R.W. and Y.L. conceived the ideas implemented in this work. Y.L. and Z.X. performed TALER promoter optimization experiments. Y.L. constructed the TALER library and circuits for stable cell lines and performed orthogonality analysis and initial experiments for transfer function curves, TALER switches and stable cell lines. Y.J. performed experiments for transfer function curves, color models, TALER switches and cell classification. H.C. performed initial experiments for TALER switches and cell classification. W.L. performed experiments for stable cell lines and cell classification. Z.L. performed initial tests for TALE transfer function curves. Z.X., Y.L., W.L. and R.W. performed data analysis. Z.X. and R.W. supervised the project. Z.X., Y.L. and R.W. wrote the paper.

Corresponding authors

Correspondence to Ron Weiss or Zhen Xie.

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Competing interests

Three pending patent applications recently submitted to Sate Intellectual Property Office of P.R. China (application nos. 201410341963.8, 201410342334.7 and 201410342350.6) by the authors are related to TALER circuit engineering described in this manuscript.

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Supplementary Results, Supplementary Figures 1–11 and Supplementary Tables 1–4. (PDF 3780 kb)

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Li, Y., Jiang, Y., Chen, H. et al. Modular construction of mammalian gene circuits using TALE transcriptional repressors. Nat Chem Biol 11, 207–213 (2015). https://doi.org/10.1038/nchembio.1736

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