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Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements

Abstract

Epigenome editing with the CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 platform is a promising technology for modulating gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of nuclease-inactive dCas9 to the Krüppel-associated box (KRAB) repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB are not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates the expression of multiple globin genes, and observed highly specific induction of H3K9 trimethylation (H3K9me3) at the enhancer and decreased chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in global gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome.

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Figure 1: Silencing of downstream globin genes by dCas9-KRAB transcription factors targeted to the distal HS2 enhancer.
Figure 2: Specificity of gene regulation by dCas9-KRAB repressors targeted to the HS2 enhancer.
Figure 3: Genome-wide binding activity of dCas9 repressors targeted to the HS2 enhancer.
Figure 4: Genome-wide H3K9me3 signal in K562 cells treated with dCas9-KRAB targeted to the HS2 enhancer.
Figure 5: Changes in global chromatin landscape with dCas9-KRAB localized to the HS2 distal enhancer.

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Acknowledgements

We thank the Duke Genome Sequencing & Analysis Core for sequencing the RNA-seq, ChIP-seq and DNase-seq libraries. This work was supported by US National Institutes of Health (NIH) grants R01DA036865, U01HG007900, R21AR065956 and P30AR066527; an NIH Director's New Innovator Award (DP2OD008586); a US National Science Foundation (NSF) Faculty Early Career Development (CAREER) Award (CBET-1151035); and an American Heart Association Scientist Development grant (10SDG3060033) to C.A.G. P.I.T. was supported by an NSF Graduate Research Fellowship and an American Heart Association Mid-Atlantic Affiliate Predoctoral Fellowship.

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Contributions

P.I.T., G.E.C., T.E.R. and C.A.G. designed experiments. P.I.T., A.M.D., A.S., N.K.S. and A.M.K. performed the experiments. P.I.T., A.M.D., L.S., A.M.K., G.E.C., T.E.R. and C.A.G. analyzed the data. P.I.T., G.E.C., T.E.R. and C.A.G. wrote the manuscript. All authors contributed to editing of the manuscript.

Corresponding authors

Correspondence to Timothy E Reddy, Gregory E Crawford or Charles A Gersbach.

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Competing interests

C.A.G., G.E.C., T.E.R., P.I.T. and A.M.K. are inventors on patent applications related to genome engineering with the CRISPR-Cas9 system. C.A.G. is a scientific advisor to Editas Medicine, a company engaged in therapeutic development of genome engineering technologies.

Integrated supplementary information

Supplementary Figure 1 Screening single gRNAs targeted to the HS2 enhancer.

(a) K562 cells that were not transduced, transduced with dCas9 lentivirus, and transduced with dCas9-KRAB were transfected with a panel of 21 HS2 sgRNAs and assayed by qRT-PCR at 3 days post-transfection. (b-d) Gene expression of single gRNAs targeted to the HS2 enhancer for silencing of (d) HBE1, (c) HBG1 and HBG2 (HBG1/2), and (d) HBB by (mean ± s.e.m, n = 3 – 4 independent experiments).

Source data

Supplementary Figure 2 HBG1/2 expression after transient delivery of sgRNAs.

(a,b) Gene expression of HBG1 and HBG2 (HBG1/2) from (a) 3 to (b) 6 days after transient electroporation of sgRNA plasmids in K562 cells expressing dCas9-KRAB (mean ± s.e.m, n = 2 independent experiments).

Source data

Supplementary Figure 3 Protein silencing of HBG1 by dCas9-KRAB transcription factors targeted to the distal HS2 enhancer.

Western blot for γ-globin and GAPDH demonstrate globin silencing in K562 cells treated with dCas9-KRAB (dCK) and sgRNAs compared to non-transduced controls (No LV), no sgRNA controls, IL1RN-targeted sgRNA controls, and dCas9 (dC) with sgRNA (cropped from representative images from n = 3 biological replicates). Western blot for the FLAG epitope show dCas9 and dCas9-KRAB expression in transduced K562 cells.

Supplementary Figure 4 Specificity of gene regulation by dCas9-KRAB repressors targeted to the HS2 enhancer.

(a-e) Differential analysis was performed to evaluate the genome-wide effects of lentiviral transduction of dCas9-KRAB guided by (a) Cr4 and (b) Cr10 compared to dCas9 with the same gRNA, dCas9 guided by (c) Cr4 and (d) Cr10 compared to non-treated K562s (No LV CTL), and (e) dCas9-KRAB without gRNA compared to No LV CTL K562s.. Red data points indicate FDR < 0.01 by differential expression analysis compared to dCas9-KRAB only controls. Points labeled in blue indicate other globin genes.

Source data

Supplementary Figure 5 Genome-wide binding activity of dCas9-KRAB targeted to the HS2 enhancer.

(a,b) Differential analyses of global binding activity include comparisons of dCas9-KRAB versus dCas9 targeted by (a) Cr4 and (b) Cr10. Points labeled in red indicate FDR < 0.05 by differential DESeq analysis (n = 3 biological replicates).

Source data

Supplementary Figure 6 Effect of dCas9-KRAB localization on binding of endogenous transcription factors GATA2 and FOSL1 at HS2.

(a) The HS2 regulatory element contains a GATA2 binding site and two adjacent FOSL1 binding sites proximal to Cr4 and Cr10 target sites. (b,c) ChIP-qPCR demonstrates reduced (b) GATA2 and (c) FOSL1 binding when dCas9-KRAB was targeted to the HS2 enhancer (mean ± s.e.m). * indicates p<0.05 by Student’s t-test compared to dCas9-KRAB only control (n = 3 independent experiments).

Source data

Supplementary Figure 7 Genome-wide H3K9me3 signal in K562 cells treated with dCas9-KRAB targeted to the HS2 enhancer.

Global analysis of H3K9me3 patterns was assessed by ChIP-seq. (a,b) Volcano plots demonstrate significance (p-value) versus fold-change for dCas9-KRAB with (a) Cr4 or (b) Cr10 compared to dCas9-KRAB without sgRNA. (c-f) H3K9me3 ChIP-seq differential analysis was also performed for dCas9-KRAB versus dCas9 guided by (c,e) Cr4 or (d,f) Cr10. Points labeled red indicate FDR < 0.05 by differential expression analysis compared to dCas9-KRAB without sgRNA or dCas9 + Cr4/10.

Source data

Source data

Source data

Supplementary Figure 8 ChIP-qPCR of H3K9 trimethylation at the HS2 enhancer.

(a) ChIP-seq tracks show increased H3K9me3 signal at the HS2 enhancer (shaded area, magnified inset). An ENCODE K562 DNase I hypersensitivity DNase-seq track is included to highlight the globin LCR49. Two primer sets were designed for the HS2 enhancer for ChIP-qPCR of H3K9me3. (b) ChIP-qPCR demonstrates increased H3K9me3 when dCas9-KRAB was targeted to the HS2 enhancer (mean ± s.e.m., n = 3 independent experiments).

Source data

Supplementary Figure 9 Changes in chromatin accessibility at the globin gene locus with dCas9-KRAB localized to the HS2 distal enhancer.

(a-h) Normalized DNase-seq cut counts within an 800 bp window surrounding the (a) HS1 enhancer, (b) HS3 enhancer, (c) HS4 enhancer, (d) HS5 enhancer, (e) HBE1 promoter, (f) HBG1 promoter, (g) HBD promoter, and (h) HBB promoter are shown (mean ± s.e.m, n = 3 independent experiments). * indicates p <0.05 compared to the dCas9-KRAB only sample (Student’s t-test).

Source data

Supplementary Figure 10 Changes in global chromatin accessibility with dCas9-KRAB localized to the HS2 distal enhancer.

(a,b) Differential genome-wide analysis of changes in chromatin accessibility induced by dCas9-KRAB versus dCas9 guided by (a) Cr4 and (b) Cr10. (c,d) Volcano plots of significance (p-value) versus fold change for differential expression analysis of dCas9-KRAB compared to dCas9 guided by (c) Cr4 or (d) Cr10. Points labeled red indicate FDR < 0.05 by DESeq analysis. Points labeled in blue indicate other regions in the globin promoters or globin LCR.

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Thakore, P., D'Ippolito, A., Song, L. et al. Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nat Methods 12, 1143–1149 (2015). https://doi.org/10.1038/nmeth.3630

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