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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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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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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.

Corresponding author

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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