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Small molecule–triggered Cas9 protein with improved genome-editing specificity


Directly modulating the activity of genome-editing proteins has the potential to increase their specificity by reducing activity following target locus modification. We developed Cas9 nucleases that are activated by the presence of a cell-permeable small molecule by inserting an evolved 4-hydroxytamoxifen–responsive intein at specific positions in Cas9. In human cells, conditionally active Cas9s modify target genomic sites with up to 25-fold higher specificity than wild-type Cas9.

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Figure 1: Insertion of an evolved ligand-dependent intein enables small-molecule control of Cas9.
Figure 2: Genomic DNA modification by intein-Cas9(S219), intein-Cas9(C574) and wild-type Cas9.


  1. Jinek, M. et al. Science 337, 816–821 (2012).

    Article  CAS  Google Scholar 

  2. Mali, P. et al. Science 339, 823–826 (2013).

    Article  CAS  Google Scholar 

  3. Cong, L. et al. Science 339, 819–823 (2013).

    Article  CAS  Google Scholar 

  4. Jinek, M. et al. eLife 2, e00471 (2013).

    Article  Google Scholar 

  5. Pattanayak, V. et al. Nat. Biotechnol. 31, 839–843 (2013).

    Article  CAS  Google Scholar 

  6. Fu, Y. et al. Nat. Biotechnol. 31, 822–826 (2013).

    Article  CAS  Google Scholar 

  7. Hsu, P.D. et al. Nat. Biotechnol. 31, 827–832 (2013).

    Article  CAS  Google Scholar 

  8. Cho, S.W. et al. Genome Res. 24, 132–141 (2014).

    Article  CAS  Google Scholar 

  9. Tsai, S.Q. et al. Nat. Biotechnol. 33, 187–197 (2015).

    Article  CAS  Google Scholar 

  10. Mali, P. et al. Nat. Biotechnol. 31, 833–838 (2013).

    Article  CAS  Google Scholar 

  11. Ran, F.A. et al. Cell 154, 1380–1389 (2013).

    Article  CAS  Google Scholar 

  12. Guilinger, J.P., Thompson, D.B. & Liu, D.R. Nat. Biotechnol. 32, 577–582 (2014).

    Article  CAS  Google Scholar 

  13. Tsai, S.Q. et al. Nat. Biotechnol. 32, 569–576 (2014).

    Article  CAS  Google Scholar 

  14. Fu, Y., Sander, J.D., Reyon, D., Cascio, V.M. & Joung, J.K. Nat. Biotechnol. 32, 279–284 (2014).

    Article  CAS  Google Scholar 

  15. Kim, S., Kim, D., Cho, S.W., Kim, J. & Kim, J.S. Genome Res. 24, 1012–1019 (2014).

    Article  CAS  Google Scholar 

  16. Ramakrishna, S. et al. Genome Res. 24, 1020–1027 (2014).

    Article  CAS  Google Scholar 

  17. Zuris, J.A. et al. Nat. Biotechnol. 33, 73–80 (2015).

    Article  CAS  Google Scholar 

  18. Wang, T., Wei, J.J., Sabatini, D.M. & Lander, E.S. Science 343, 80–84 (2014).

    Article  CAS  Google Scholar 

  19. González, F. et al. Cell Stem Cell 15, 215–226 (2014).

    Article  Google Scholar 

  20. Pruett-Miller, S.M., Reading, D.W., Porter, S.N. & Porteus, M.H. PLoS Genet. 5, e1000376 (2009).

    Article  Google Scholar 

  21. Buskirk, A.R., Ong, Y.C., Gartner, Z.J. & Liu, D.R. Proc. Natl. Acad. Sci. USA 101, 10505–10510 (2004).

    Article  CAS  Google Scholar 

  22. Yuen, C.M., Rodda, S.J., Vokes, S.A., McMahon, A.P. & Liu, D.R. J. Am. Chem. Soc. 128, 8939–8946 (2006).

    Article  CAS  Google Scholar 

  23. Peck, S.H., Chen, I. & Liu, D.R. Chem. Biol. 18, 619–630 (2011).

    Article  CAS  Google Scholar 

  24. Jinek, M. et al. Science 343, 1247997 (2014).

    Article  Google Scholar 

  25. Danielian, P.S., White, R., Hoare, S.A., Fawell, S.E. & Parker, M.G. Mol. Endocrinol. 7, 232–240 (1993).

    CAS  PubMed  Google Scholar 

  26. Zetsche, B., Volz, S.E. & Zhang, F. Nat. Biotechnol. 33, 139–142 (2015).

    Article  CAS  Google Scholar 

  27. Shcherbakova, D.M. & Verkhusha, V.V. Nat. Methods 10, 751–754 (2013).

    Article  CAS  Google Scholar 

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This work was supported by National Institutes of Health R01 GM095501, Defense Advanced Research Projects Agency HR0011-11-2-0003 and N66001-12-C-4207 and the Howard Hughes Medical Institute. K.M.D. acknowledges the Natural Sciences and Engineering Research Council of Canada (NSERC) for a Postgraduate Scholarship–Doctoral (PGS D) award. J.A.Z. is a Ruth L. Kirchstein National Research Service Awards Postdoctoral Fellow (F32 GM 106601-2). We are grateful to J. Doudna, S. Sternberg, D. Taylor, M. Jinek and F. Jiang for providing the structural coordinates of Cas9 and to J. Guilinger, Y. Kim, M. Li and A. Badran for helpful discussions.

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Authors and Affiliations



K.M.D., V.P., D.B.T. and D.R.L. designed the research. K.M.D. performed the experiments, and J.A.Z. assisted with high-throughput sequencing. K.M.D., V.P., D.B.T. and J.A.Z. analyzed the data. D.R.L. supervised the research. All authors wrote the manuscript.

Corresponding author

Correspondence to David R Liu.

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Competing interests

The co-authors have filed a provisional patent application related to this work. D.R.L is a consultant for Editas Medicine, a company that applies genome-editing technologies.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–8, Supplementary Tables 1–7 and Supplementary Notes. (PDF 1802 kb)

Supplementary Data Set 1

Indel sequences used to calculate modification frequencies in Figure 2 and Supplementary Figures 3 and 4 (XLSX 171 kb)

Supplementary Data Set 2

Indel sequences used to calculate modification frequencies in Fig. 2b–e and Supplementary Figures 5–7 (XLSX 126 kb)

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Davis, K., Pattanayak, V., Thompson, D. et al. Small molecule–triggered Cas9 protein with improved genome-editing specificity. Nat Chem Biol 11, 316–318 (2015).

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