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CRISPR transcriptional repression devices and layered circuits in mammalian cells

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.

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Authors

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 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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