Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A chemical-inducible CRISPR–Cas9 system for rapid control of genome editing

Abstract

CRISPR–Cas9 has emerged as a powerful technology that enables ready modification of the mammalian genome. The ability to modulate Cas9 activity can reduce off-target cleavage and facilitate precise genome engineering. Here we report the development of a Cas9 variant whose activity can be switched on and off in human cells with 4-hydroxytamoxifen (4-HT) by fusing the Cas9 enzyme with the hormone-binding domain of the estrogen receptor (ERT2). The final optimized variant, termed iCas, showed low endonuclease activity without 4-HT but high editing efficiency at multiple loci with the chemical. We also tuned the duration and concentration of 4-HT treatment to reduce off-target genome modification. Additionally, we benchmarked iCas against other chemical-inducible methods and found that it had the fastest on rate and that its activity could be toggled on and off repeatedly. Collectively, these results highlight the utility of iCas for rapid and reversible control of genome-editing function.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Building and testing a 4-HT-inducible Cas9.
Figure 2: Optimization of the ERT2–Cas9–ERT2 architecture.
Figure 3: Optimization of 4-HT treatment conditions.
Figure 4: Comparison of iCas with an alternative inducible-promoter-based system.
Figure 5: Comparison of iCas with intein–Cas9 and split-Cas9.
Figure 6: Toggling the activity of iCas on and off.

Similar content being viewed by others

References

  1. Cho, S.W., Kim, S., Kim, J.M. & Kim, J.S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232 (2013).

    Article  CAS  Google Scholar 

  2. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  Google Scholar 

  3. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    Article  CAS  Google Scholar 

  4. Guilinger, J.P., Thompson, D.B. & Liu, D.R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 32, 577–582 (2014).

    Article  CAS  Google Scholar 

  5. Ran, F.A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

    Article  CAS  Google Scholar 

  6. Shen, B. et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat. Methods 11, 399–402 (2014).

    Article  CAS  Google Scholar 

  7. Tsai, S.Q. et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 32, 569–576 (2014).

    Article  CAS  Google Scholar 

  8. Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).

    Article  CAS  Google Scholar 

  9. Fu, Y., Sander, J.D., Reyon, D., Cascio, V.M. & Joung, J.K. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279–284 (2014).

    Article  CAS  Google Scholar 

  10. Kleinstiver, B.P. et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016).

    Article  CAS  Google Scholar 

  11. Slaymaker, I.M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016).

    Article  CAS  Google Scholar 

  12. Kim, S., Kim, D., Cho, S.W., Kim, J. & Kim, J.S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).

    Article  CAS  Google Scholar 

  13. Ramakrishna, S. et al. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. 24, 1020–1027 (2014).

    Article  CAS  Google Scholar 

  14. Lin, S., Staahl, B.T., Alla, R.K. & Doudna, J.A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766 (2014).

    Article  Google Scholar 

  15. Zuris, J.A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2015).

    Article  CAS  Google Scholar 

  16. Davis, K.M., Pattanayak, V., Thompson, D.B., Zuris, J.A. & Liu, D.R. Small molecule-triggered Cas9 protein with improved genome-editing specificity. Nat. Chem. Biol. 11, 316–318 (2015).

    Article  CAS  Google Scholar 

  17. Shen, Z. et al. Conditional knockouts generated by engineered CRISPR-Cas9 endonuclease reveal the roles of coronin in C. elegans neural development. Dev. Cell 30, 625–636 (2014).

    Article  CAS  Google Scholar 

  18. Dow, L.E. et al. Inducible in vivo genome editing with CRISPR-Cas9. Nat. Biotechnol. 33, 390–394 (2015).

    Article  CAS  Google Scholar 

  19. González, F. et al. An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. Cell Stem Cell 15, 215–226 (2014).

    Article  Google Scholar 

  20. Wang, T., Wei, J.J., Sabatini, D.M. & Lander, E.S. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80–84 (2014).

    Article  CAS  Google Scholar 

  21. Konermann, S. et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476 (2013).

    Article  CAS  Google Scholar 

  22. Polstein, L.R. & Gersbach, C.A. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat. Chem. Biol. 11, 198–200 (2015).

    Article  CAS  Google Scholar 

  23. Nihongaki, Y., Yamamoto, S., Kawano, F., Suzuki, H. & Sato, M. CRISPR-Cas9-based photoactivatable transcription system. Chem. Biol. 22, 169–174 (2015).

    Article  CAS  Google Scholar 

  24. Nihongaki, Y., Kawano, F., Nakajima, T. & Sato, M. Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Nat. Biotechnol. 33, 755–760 (2015).

    Article  CAS  Google Scholar 

  25. Zetsche, B., Volz, S.E. & Zhang, F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 33, 139–142 (2015).

    Article  CAS  Google Scholar 

  26. Schellenberger, V. et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat. Biotechnol. 27, 1186–1190 (2009).

    Article  CAS  Google Scholar 

  27. Wang, X. et al. Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors. Nat. Biotechnol. 33, 175–178 (2015).

    Article  CAS  Google Scholar 

  28. Tsai, S.Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).

    Article  CAS  Google Scholar 

  29. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013).

    Article  CAS  Google Scholar 

  30. Xu, Q. et al. Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair. Cell 116, 883–895 (2004).

    Article  CAS  Google Scholar 

  31. Coombs, G.S. et al. WLS-dependent secretion of WNT3A requires Ser209 acylation and vacuolar acidification. J. Cell Sci. 123, 3357–3367 (2010).

    Article  CAS  Google Scholar 

  32. McCulloch, M.W. et al. Psammaplin A as a general activator of cell-based signaling assays via HDAC inhibition and studies on some bromotyrosine derivatives. Bioorg. Med. Chem. 17, 2189–2198 (2009).

    Article  CAS  Google Scholar 

  33. Tan, M.H. et al. RNA sequencing reveals a diverse and dynamic repertoire of the Xenopus tropicalis transcriptome over development. Genome Res. 23, 201–216 (2013).

    Article  CAS  Google Scholar 

  34. Inlay, M.A. et al. Synthesis of a photocaged tamoxifen for light-dependent activation of Cre-ER recombinase-driven gene modification. Chem. Commun. (Camb.) 49, 4971–4973 (2013).

    Article  CAS  Google Scholar 

  35. Suzuki, K., Bose, P., Leong-Quong, R.Y., Fujita, D.J. & Riabowol, K. REAP: A two minute cell fractionation method. BMC Res. Notes 3, 294 (2010).

    Article  Google Scholar 

Download references

Acknowledgements

We thank R. Dasgupta for helpful discussions, Y. Wan for critical reading of the manuscript, and H.H. Ng (Genome Institute of Singapore), R. Dasgupta (Genome Institute of Singapore), and B. Lim (Genome Institute of Singapore) for reagents. STF3A cells were a gift from D. Virshup (Duke-National University of Singapore Graduate Medical School). This work was supported by core funding from the Genome Institute of Singapore (M.H.T.), an Agency for Science Technology and Research Joint Council Office grant 1431AFG103 (M.H.T.), and a startup grant from Nanyang Technological University (M.H.T.).

Author information

Authors and Affiliations

Authors

Contributions

M.H.T. conceived the project, designed the experiments, performed the experiments, analyzed the data, supervised the research, and wrote the manuscript. K.I.L., M.N.B.R., C.W.A.W., Y.W., T.Z., B.Y.C., A.G., M.Z.H.C., J.J., and J.H.J.L. performed experiments and analyzed data. X.Z. analyzed deep-sequencing data. G.R.D.Y. provided input about the experimental design, performed the experiments, and analyzed the data.

Corresponding author

Correspondence to Meng How Tan.

Ethics declarations

Competing interests

The authors declare that a patent has been filed for iCas (Singapore Patent Application No. 10201509153Y). M.H.T. is an inventor on the patent.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–26 and Supplementary Tables 1–4. (PDF 2930 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Liu, K., Ramli, M., Woo, C. et al. A chemical-inducible CRISPR–Cas9 system for rapid control of genome editing. Nat Chem Biol 12, 980–987 (2016). https://doi.org/10.1038/nchembio.2179

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2179

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing