Brief Communication | Published:

Cas9 gRNA engineering for genome editing, activation and repression

Nature Methods 12, 1051–1054 (2015) | Download Citation

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Abstract

We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.

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Acknowledgements

We thank K. Esvelt, J. Scheiman, J. Huh, J. Aach, M.K. Cromer, S. Haque, M. Tung and all the other members of the Church, Collins and Weiss labs for their assistance and insightful discussions. We also thank P. Mali at University of California San Diego (UCSD; San Diego, California, USA) for the HEK293T cells. The dSaCas9 plasmid was a generous gift from F.A. Ran, W.X. Yan and F. Zhang (Broad Institute–MIT, Boston, Massachusetts, USA). This work was supported by US National Institutes of Health National Human Genome Research Institute grant P50 HG005550, US Department of Energy grant DE-FG02-02ER63445, the Wyss Institute for Biologically Inspired Engineering, the US Army Research Office (DARPA W911NF-11-2-0054), the National Science Foundation (Emerging Frontiers in Research and Innovation Award in Engineering New Technologies Based on Multicellular and Inter-kingdom Signaling) and US National Institutes of Health grants 5R01CA155320-04 and P50 GM098792. A.C. acknowledges funding by the National Cancer Institute (grant 5T32CA009216-34). S.V. acknowledges funding by the National Science Foundation Graduate Research Fellowship Program, the Department of Biological Engineering at MIT and the Department of Genetics at Harvard Medical School. R.C. was funded by a Banting postdoctoral fellowship from the Canadian Institutes of Health Research. J.J.C. acknowledges funding from Defense Threat Reduction Agency grant HDTRA1-14-1-0006.

Author information

    • Samira Kiani
    •  & Alejandro Chavez

    These authors contributed equally to this work.

Affiliations

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

    • Samira Kiani
    • , Richard N Hall
    • , Suhani Vora
    • , Mohammad R Ebrahimkhani
    • , James J Collins
    •  & Ron Weiss

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

    • Samira Kiani
    • , Richard N Hall
    • , James J Collins
    •  & Ron Weiss

  3. Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.

    • Alejandro Chavez
    • , Marcelle Tuttle
    • , Dmitry Ter-Ovanesyan
    • , Jason Qian
    • , Benjamin W Pruitt
    • , Suhani Vora
    • , Joanna Buchthal
    • , Emma J K Kowal
    • , James J Collins
    •  & George Church

  4. Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA.

    • Alejandro Chavez

  5. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

    • Alejandro Chavez
    • , Raj Chari
    • , Dmitry Ter-Ovanesyan
    • , Jason Qian
    • , Suhani Vora
    •  & George Church

  6. Raytheon BBN Technologies, Cambridge, Massachusetts, USA.

    • Jacob Beal

  7. Center for Emergent Behaviors of Integrated Cellular Systems (EBICS), Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Mohammad R Ebrahimkhani
    •  & Ron Weiss

  8. Institute for Medical Engineering & Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • James J Collins

  9. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

    • James J Collins

  10. Harvard-MIT Program in Health Sciences and Technology, Cambridge, Massachusetts, USA.

    • James J Collins

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Contributions

S.K. and A.C. designed and performed experiments and analyzed data. M.T., R.N.H., R.C., D.T.-O. and J.Q. performed experiments and interpreted data. S.V. and B.W.P. developed tools and contributed expertise. E.J.K.K. contributed to the analysis of RNA-Seq data. J. Beal helped with the initial design of repression promoter constructs and analysis of data via the TASBE method. M.R.E. helped with the design of experiments and interpretation of data. J. Buchthal analyzed data. J.J.C., R.W. and G.C. supervised the study. A.C., S.K., M.T. and M.R.E. wrote the manuscript with the support of all the other authors.

Competing interests

G.C. is a cofounder of Editas Medicine, a company that uses CRISPR-Cas9 technology.

Corresponding authors

Correspondence to Ron Weiss or George Church.

Integrated supplementary information

Supplementary figures

  1. 1.

    Activation and cutting of a transcriptional reporter using gRNAs with progressively shorter 5ʹ end lengths.

  2. 2.

    Activation and cutting of a transcriptional reporter using orthogonal Cas9 proteins.

  3. 3.

    Activation and cutting of endogenous HBG1 gene.

  4. 4.

    Mismatch comparison between 20-nt and 14-nt sgRNA activation using Cas9-VPR.

  5. 5.

    Off-target expression analysis.

  6. 6.

    Pictorial representation of Figure 1d.

  7. 7.

    gRNA-mediated recruitment of an activator using Cas9.

  8. 8.

    Simplified schematics of circuits in Figure 2.

  9. 9.

    Building different promoter architectures to analyze Cas9-VPR–mediated transcriptional repression.

  10. 10.

    Analysis of dynamics of a genetic kill-switch circuit.

  11. 11.

    Understanding the design rules based on the concentrations of Cas9-VPR, 14-nt gRNA and 20-nt gRNA.

  12. 12.

    Design and analysis of a genetic kill switch that functions based on DNA cleavage in the Cas9-VPR coding sequence.

  13. 13.

    Design and analysis of a genetic kill switch that operates based on DNA cleavage in the TALER coding sequence and reversal of a transcriptional repression device.

  14. 14.

    Design and analysis of a genetic kill switch that operates based on DNA cleavage in the TALER coding sequence and reversal of a transcriptional repression device.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–14 and Supplementary Notes 1–4

Excel files

  1. 1.

    Supplementary Data

    Figure data.

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DOI

https://doi.org/10.1038/nmeth.3580

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