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Directing cellular information flow via CRISPR signal conductors

Abstract

The complex phenotypes of eukaryotic cells are controlled by decision-making circuits and signaling pathways. A key obstacle to implementing artificial connections in signaling networks has been the lack of synthetic devices for efficient sensing, processing and control of biological signals. By extending sgRNAs to include modified riboswitches that recognize specific signals, we can create CRISPR–Cas9-based 'signal conductors' that regulate transcription of endogenous genes in response to external or internal signals of interest. These devices can be used to construct all the basic types of Boolean logic gates that perform logical signal operations in mammalian cells without needing the layering of multiple genetic circuits. They can also be used to rewire cellular signaling events by constructing synthetic links that couple different signaling pathways. Moreover, this approach can be applied to redirect oncogenic signal transduction by controlling simultaneous bidirectional (ON–OFF) gene transcriptions, thus enabling reprogramming of the fate of cancer cells.

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Figure 1: Design of the CRISPR–Cas9-based signal conductor that links one signal with another.
Figure 2: Ligand-regulated transcriptional activation or inactivation.
Figure 3: Logic connections were constructed using the signal conductors in HEK-293T cells.
Figure 4: The CRISPR–Cas9-based signal conductors redirect the oncogenic signaling to the antioncogenic pathway.
Figure 5: The CRISPR–Cas9-based signal-conductor induces simultaneous activation and repression of cellular genes in response to oncogenic signals.
Figure 6: Apoptosis levels of several cell lines treated with the AND gate circuit.

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Acknowledgements

We are indebted to the donors whose names were not included in the author list, but who participated in this program. This work was supported by the National Key Basic Research Program of China (973 Program) (2014CB745201 to Z.C.), National Natural Science Foundation of China (81402103 to Y.L.), the Shenzhen Municipal Government of China (ZDSYS201504301722174, JCYJ20150330102720130 and GJHZ20150316154912494 to W.H.), Special Support Funds of Shenzhen for Introduced High-Level Medical Team to W.H., and Shenzhen High Level Medical Discipline Development Program (2016031638 to W.H. and Z.C.).

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Contributions

Y.L., Y.Z., Z.C., A.H., J.L., H.W., L.L., C.Z., J.L., X.G. and Q.Z. performed experiments and data analysis. Y.L., W.H. and Z.C. designed and supervised the project and wrote the paper. W.H. and Z.C. provided financial support for the project.

Corresponding authors

Correspondence to Weiren Huang or Zhiming Cai.

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The authors declare no competing financial interests.

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Supplementary Figures 1–36 and Supplementary Tables 1–5. (PDF 5395 kb)

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Liu, Y., Zhan, Y., Chen, Z. et al. Directing cellular information flow via CRISPR signal conductors. Nat Methods 13, 938–944 (2016). https://doi.org/10.1038/nmeth.3994

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