Brief Communication | Published:

Riboregulated toehold-gated gRNA for programmable CRISPR–Cas9 function

Abstract

Predictable control over gene expression is essential to elicit desired synthetic cellular phenotypes. Although CRISPR–Cas9 offers a simple RNA-guided method for targeted transcriptional control, it lacks the ability to integrate endogenous cellular information for efficient signal processing. Here, we present a new class of riboregulators termed toehold-gated gRNA (thgRNA) by integrating toehold riboswitches into sgRNA scaffolds, and demonstrate their programmability for multiplexed regulation in Escherichia coli with minimal cross-talks.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

Sequences of all thgRNAs and trigger strands studied are included in the Supplementary Information. Additional data that support the findings of this study are available from the authors on reasonable request.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Khalil, A. S. & Collins, J. J. Nat. Rev. Genet. 11, 367–379 (2010).

  2. 2.

    Lim, W. A. Nat. Rev. Mol. Cell Biol. 11, 393–403 (2010).

  3. 3.

    Taylor, N. D. et al. Nat. Methods 13, 177–183 (2016).

  4. 4.

    Tang, S. Y. & Cirino, P. C. Angew. Chem. Int. Ed. Engl. 50, 1084–1086 (2011).

  5. 5.

    Serganov, A. & Nudler, E. Cell 152, 17–24 (2013).

  6. 6.

    Chappell, J., Watters, K. E., Takahashi, M. K. & Lucks, J. B. Curr. Opin. Chem. Biol. 28, 47–56 (2015).

  7. 7.

    Callura, J. M., Cantor, C. R. & Collins, J. J. Proc. Natl. Acad. Sci. USA 109, 5850–5855 (2012).

  8. 8.

    Mutalik, V. K., Qi, L., Guimaraes, J. C., Lucks, J. B. & Arkin, A. P. Nat. Chem. Biol. 8, 447–454 (2012).

  9. 9.

    Horvath, P. & Barrangou, R. Science 327, 167–170 (2010).

  10. 10.

    Wiedenheft, B., Sternberg, S. H. & Doudna, J. A. Nature 482, 331–338 (2012).

  11. 11.

    Qi, L. S. et al. Cell 152, 1173–1183 (2013).

  12. 12.

    Tang, W., Hu, J. H. & Liu, D. R. Nat. Commun. 8, 15939 (2017).

  13. 13.

    Liu, Y. et al. Nat. Methods 13, 938–944 (2016).

  14. 14.

    Davis, K. M., Pattanayak, V., Thompson, D. B., Zuris, J. A. & Liu, D. R. Nat. Chem. Biol. 11, 316–318 (2015).

  15. 15.

    Zhang, D. Y. & Winfree, E. J. Am. Chem. Soc. 131, 17303–17314 (2009).

  16. 16.

    Green, A. A., Silver, P. A., Collins, J. J. & Yin, P. Cell 159, 925–939 (2014).

  17. 17.

    Zadeh, J. N. et al. J. Comput. Chem. 32, 170–173 (2011).

  18. 18.

    Mekler, V., Minakhin, L., Semenova, E., Kuznedelov, K. & Severinov, K. Nucleic Acids Res. 44, 2837–2845 (2016).

  19. 19.

    Xu, P., Vansiri, A., Bhan, N. & Koffas, M. A. G. ACS Synth. Biol. 1, 256–266 (2012).

  20. 20.

    Lutz, R. & Bujard, H. Nucleic Acids Res. 25, 1203–1210 (1997).

  21. 21.

    Green, A. A. et al. Nature 548, 117–121 (2017).

  22. 22.

    Groves, B. et al. Nat. Nanotechnol. 11, 287–294 (2016).

  23. 23.

    Altuvia, S., Zhang, A., Argaman, L., Tiwari, A. & Storz, G. EMBO J. 17, 6069–6075 (1998).

  24. 24.

    Massé, E., Escorcia, F. E. & Gottesman, S. Genes Dev. 17, 2374–2383 (2003).

  25. 25.

    Ran, F. A. et al. Nat. Protoc. 8, 2281–2308 (2013).

  26. 26.

    Gagnon, J. A. et al. PLoS One 9, e98186 (2014).

  27. 27.

    Summer, H., Grämer, R. & Dröge, P. J. Vis. Exp. 32, e1485 (2009).

Download references

Acknowledgements

This work was supported by grants to W.C. from the National Science Foundation (MCB1615731 and MCB1817675). We thank D. Liu (Harvard University), T. Pederson (University of Massachusetts Medical School), and M. Koffas (Rensselaer Polytechnic Institute) for their generous gifts of plasmids as noted in the manuscript.

Author information

K-H.S. and W.C. conceived the project. K-H.S. designed experiments, performed the experiments, analyzed the data, and wrote the manuscript. W.C. designed experiments, analyzed the data, and wrote the manuscript. Both authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing interests.

Correspondence to Wilfred Chen.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–10, Supplementary Tables 1–2

  2. Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark
Fig. 1: Design and screening of toehold-gated guide RNAs (thgRNAs).
Fig. 2: thgRNAs can be selectively activated intracellularly by induced expression of cognate trigger RNAs.
Fig. 3: thgRNA can be activated specifically by endogenous RNAs.