Skip to main content

Thank you for visiting 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.

Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery


Gene expression can be activated or suppressed using CRISPR­–Cas9 systems. However, tools that enable dose-dependent activation of gene expression without the use of exogenous transcription regulatory proteins are lacking. Here we describe chemical epigenetic modifiers (CEMs) designed to activate the expression of target genes by recruiting components of the endogenous chromatin-activating machinery, eliminating the need for exogenous transcriptional activators. The system has two parts: catalytically inactive Cas9 (dCas9) in complex with FK506-binding protein (FKBP) and a CEM consisting of FK506 linked to a molecule that interacts with cellular epigenetic machinery. We show that CEMs upregulate gene expression at target endogenous loci up to 20-fold or more depending on the gene. We also demonstrate dose-dependent control of transcriptional activation, function across multiple diverse genes, reversibility of CEM activity and specificity of our best-in-class CEM across the genome.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Using CEMs to increase gene expression.
Fig. 2: Evaluating the dCas9–CEMa system over time and a dose range, and benchmarking the system against current dCas9 activating technologies.
Fig. 3: Whole-genome analysis of the dCas9–CEMa system.

Data availability

Genome-wide data generated herein are publicly available through the Gene Expression Omnibus with accession number GSE129407. All data presented in this manuscript are available from the corresponding authors upon reasonable request.

Code availability

Readers can view our code through the public link ( There are no access restrictions.


  1. 1.

    Dawson, M. A. The cancer epigenome: concepts, challenges, and therapeutic opportunities. Science 355, 1147–1152 (2017).

    CAS  Article  Google Scholar 

  2. 2.

    MacDonald, I. A. & Hathaway, N. A. Epigenetic roots of immunologic disease and new methods for examining chromatin regulatory pathways. Immunol. Cell Biol. 93, 261–270 (2015).

    CAS  Article  Google Scholar 

  3. 3.

    Zeng, L. & Zhou, M. M. Bromodomain: an acetyl-lysine binding domain. FEBS Lett. 513, 124–128 (2002).

    CAS  Article  Google Scholar 

  4. 4.

    de Ruijter, A. J. M., van Gennip, A. H., Caron, H. N., Kemp, S. & van Kuilenburg, A. B. P. Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem. J. 370, 737–749 (2003).

    Article  Google Scholar 

  5. 5.

    Gao, Y. et al. Complex transcriptional modulation with orthogonal and inducible dCas9 regulators. Nat. Methods 13, 1043–1049 (2016).

    CAS  Article  Google Scholar 

  6. 6.

    Chen, T. et al. Chemically controlled epigenome editing through an inducible dCas9 system. J. Am. Chem. Soc. 139, 11337–11340 (2017).

    CAS  Article  Google Scholar 

  7. 7.

    Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017).

    Article  Google Scholar 

  8. 8.

    Gao, D. & Liang, F. S. Chemical inducible dCas9-guided editing of H3K27 acetylation in mammalian cells. Methods Mol. Biol. 1767, 429–445 (2018).

  9. 9.

    Ma, D., Peng, S. & Xie, Z. Integration and exchange of split dCas9 domains for transcriptional controls in mammalian cells. Nat. Commun. 7, 13056 (2016).

    CAS  Article  Google Scholar 

  10. 10.

    Shrimp, J. H. et al. Chemical control of a CRISPR–Cas9 acetyltransferase. ACS Chem. Biol. 13, 455–460 (2018).

    CAS  Article  Google Scholar 

  11. 11.

    Hilton, I. B. et al. Epigenome editing by a CRISPR–Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015).

    CAS  Article  Google Scholar 

  12. 12.

    Liszczak, G. P. et al. Genomic targeting of epigenetic probes using a chemically tailored Cas9 system. Proc. Natl Acad. Sci. USA 114, 681–686 (2017).

    CAS  Article  Google Scholar 

  13. 13.

    Erwin, G. S. et al. Synthetic transcription elongation factors license transcription across repressive chromatin. Science 6370, 1617–1622 (2017).

    Article  Google Scholar 

  14. 14.

    Butler, K. V., Chiarella, A. M., Jin, J. & Hathaway, N. A. Targeted gene repression using novel bifunctional molecules to harness endogenous histone deacetylation activity. ACS Synth. Biol. 7, 38–45 (2018).

    CAS  Article  Google Scholar 

  15. 15.

    Chung, C. et al. Discovery and characterization of small molecule inhibitors of the BET family bromodomains. J. Med. Chem. 54, 3827–3838 (2011).

    CAS  Article  Google Scholar 

  16. 16.

    Demont, E. H. et al. 1,3-Dimethyl benzimidazolones are potent, selective inhibitors of the BRPF1 bromodomain. ACS Med. Chem. Lett. 5, 1190–1195 (2014).

    CAS  Article  Google Scholar 

  17. 17.

    Hay, D. A. et al. Discovery and optimization of small-molecule ligands for the CBP/p300 bromodomain. J. Am. Chem. Soc. 26, 9308–9319 (2014).

    Article  Google Scholar 

  18. 18.

    Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).

    CAS  Article  Google Scholar 

  19. 19.

    Morita, S. et al. Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat. Biotechnol. 34, 1060–1065 (2016).

    CAS  Article  Google Scholar 

  20. 20.

    Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014).

    CAS  Article  Google Scholar 

  21. 21.

    Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR–Cas9 complex. Nature 517, 583–588 (2015).

    CAS  Article  Google Scholar 

  22. 22.

    Lim, F. et al. Altering the RNA binding specificity of a translational repressor. J. Biol. Chem. 12, 9006–9010 (1994).

    Google Scholar 

  23. 23.

    Chavez, A. et al. Comparison of Cas9 activators in multiple species. Nat. Methods 13, 563–567 (2016).

    CAS  Article  Google Scholar 

  24. 24.

    Begley, C. G. & Ellis, L. M. Raise standards for preclinical cancer research. Nature 483, 531–533 (2012).

    CAS  Article  Google Scholar 

  25. 25.

    Hay, M., Thomas, D. W., Craighead, J. L., Economides, C. & Rosenthal, J. Clinical development success rates for investigational drugs. Nat. Biotechnol. 32, 40–51 (2014).

    CAS  Article  Google Scholar 

  26. 26.

    Gryder, B. E. et al. PAX3–FOXO1 establishes myogenic super enhancers and confers BET bromodomain vulnerability. Cancer Discov. 7, 884–899 (2017).

    CAS  Article  Google Scholar 

  27. 27.

    Lovén, J. et al. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 153, 320–334 (2013).

    Article  Google Scholar 

  28. 28.

    Lin, X. et al. HEXIM1 as a robust pharmacodynamic marker for monitoring target engagement of BET family bromodomain inhibitors in tumors and surrogate tissues. Mol. Cancer Ther. 16, 388–396 (2017).

    CAS  Article  Google Scholar 

  29. 29.

    Hathaway, N. A. et al. Dynamics and memory of heterochromatin in living cells. Cell 149, 1447–1460 (2012).

    CAS  Article  Google Scholar 

  30. 30.

    Thakore, P. I. et al. RNA-guided transcriptional silencing in vivo with S. aureus CRISPR–Cas9 repressors. Nat. Commun. 9, 1674 (2018).

    Article  Google Scholar 

  31. 31.

    Liu, S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247 (2016).

    CAS  Article  Google Scholar 

  32. 32.

    Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    CAS  Article  Google Scholar 

  33. 33.

    Chiarella, A. M. et al. Cavitation enhancement increases the efficiency and consistency of chromatin fragmentation from fixed cells for downstream quantitative applications. Biochemistry 57, 2756–2761 (2018).

    CAS  Article  Google Scholar 

Download references


We thank the members of the Hathaway, Jin and Khan laboratories for many helpful discussions. We acknowledge E. Chory (Stanford University), S. Braun (Stanford University), G. Crabtree (Stanford University), R. Vale (University of California, San Francisco), F. Zhang (Massachusetts Institute of Technology), Z. Xie (Tsinghua University) and C. Gersbach (Duke University) for sharing plasmids used or adapted in this study. This work was supported in part by grant R01GM118653 from the US National Institutes of Health (to N.A.H.) and by grants R01GM122749 and R01HD088626 from the US National Institutes of Health (to J.J.). This work was further supported by a Tier 3 grant and a Student Grant from the UNC Eshelman Institute for Innovation (to N.A.H. and A.M.C., respectively). T32GM007092 from the US National Institutes of Health (to A.M.C.) also supported early portions of this work. Additional support was provided by American Cancer Society postdoctoral fellowship PF-14-021-01-CDD (to K.V.B.). Flow cytometry data were obtained at the UNC Flow Cytometry Core Facility, funded by the P30CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center.

Author information




N.A.H., J.J., K.V.B. and A.M.C. conceived the project. N.A.H., J.J., K.V.B., A.M.C., D.L., B.E.G. and J.K. contributed to the experimental design. N.A.H., J.J., K.V.B., A.M.C., D.L., B.E.G., J.K., T.A.W., X.Y. and S.P. contributed experimentally. K.V.B. synthesized the compounds. B.E.G. and J.K. carried out ChIP–seq and RNA-seq experiments and analysis. N.A.H., J.J., K.V.B., A.M.C., D.L. and B.E.G. wrote the manuscript. All authors edited the manuscript.

Corresponding authors

Correspondence to Jian Jin or Nathaniel A. Hathaway.

Ethics declarations

Competing interests

N.A.H., J.J., K.V.B. and A.M.C. are inventors on U.S. provisional patent application no. 62/654,958, “Bifunctional chemical epigenetic modifiers and methods of use.”

Additional information

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

Supplementary information

Supplementary Materials

Supplementary Figures 1–5, Supplementary Tables 1–3 and Supplementary Note 1.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Chiarella, A.M., Butler, K.V., Gryder, B.E. et al. Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery. Nat Biotechnol 38, 50–55 (2020).

Download citation

Further reading


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