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CRISPR transcriptional repression devices and layered circuits in mammalian cells

Nature Methods volume 11, pages 723–726 (2014)Cite this article

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Abstract

A key obstacle to creating sophisticated genetic circuits has been the lack of scalable device libraries. Here we present a modular transcriptional repression architecture based on clustered regularly interspaced palindromic repeats (CRISPR) system and examine approaches for regulated expression of guide RNAs in human cells. Subsequently we demonstrate that CRISPR regulatory devices can be layered to create functional cascaded circuits, which provide a valuable toolbox for engineering purposes.

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Figure 1: Design and experimental analysis in human cells of CRISPR repression devices and circuits based on the RNA Pol III U6 promoter.
Figure 2: Design and experimental analysis in human cells of CRISPR repression devices and circuits based on RNA Pol II promoters.

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Acknowledgements

This work was supported by US National Institutes of Health grants 5R01CA155320-04 and P50 GM098792. We thank L. Wrobleska and P. Guye (Massachusetts Institute of Technology) for providing the initial intronic miRNA–based plasmid and the primary Cas9 construct, and for helpful discussions, and M. Graziano (Massachusetts Institute of Technology) for providing us the HEK293 cell lines that constitutively express rtTA3. J.H. was partially supported by the Intelligent Synthetic Biology Center of Global Frontier Project (2013M3A6A8073557) funded by the Ministry of Science, Information and Communication Technology and Future Planning of Korea.

Author information

Authors and Affiliations

  1. Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Samira Kiani, Jin Huh, Richard N Hall & Ron Weiss

  2. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Samira Kiani, Mohammad R Ebrahimkhani, Jin Huh, Richard N Hall & Ron Weiss

  3. Raytheon BBN Technologies, Cambridge, Massachusetts, USA

    Jacob Beal

  4. Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, South Korea

    Jin Huh

  5. Bioinformatics Division, Center for Synthetic & Systems Biology, Tsinghua National Laboratory for Information Science and Technology, Beijing, China

    Zhen Xie

  6. Department of Automation, Tsinghua University, Beijing, China

    Zhen Xie

  7. Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Yinqing Li & Ron Weiss

Authors
  1. Samira Kiani
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  2. Jacob Beal
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  3. Mohammad R Ebrahimkhani
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  4. Jin Huh
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  5. Richard N Hall
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  6. Zhen Xie
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  7. Yinqing Li
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  8. Ron Weiss
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Contributions

R.W. and S.K. conceived the idea. S.K., R.W., M.R.E. and J.B. designed experiments. S.K. performed the majority of experiments. M.R.E. and R.N.H. helped with flow cytometry and transfections. Y.L. and Z.X. built initial versions of CRP-a and CRP-b promoters. J.H. helped with DNA constructions. J.B. developed and applied computational analysis techniques. J.B. and S.K. performed the flow cytometry and statistical analysis. S.K. wrote the manuscript. R.W., J.B. and M.R.E. edited the manuscript.

Corresponding author

Correspondence to Ron Weiss.

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The authors declare no competing financial interests.

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Supplementary Text and Figures

Supplementary Figures 1–15, Supplementary Discussion and Supplementary Note (PDF 31793 kb)

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Kiani, S., Beal, J., Ebrahimkhani, M. et al. CRISPR transcriptional repression devices and layered circuits in mammalian cells. Nat Methods 11, 723–726 (2014). https://doi.org/10.1038/nmeth.2969

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