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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.

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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.


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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.

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Authors and Affiliations



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.

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Correspondence to Jian Jin or Nathaniel A. Hathaway.

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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.”

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

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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).

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