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Nature Methods | Article

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

Journal name:
Nature Methods
Volume:
12,
Pages:
1143–1149
Year published:
DOI:
doi:10.1038/nmeth.3630
Received
Accepted
Published online

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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  1. Silencing of downstream globin genes by dCas9-KRAB transcription factors targeted to the distal HS2 enhancer.
    Figure 1: Silencing of downstream globin genes by dCas9-KRAB transcription factors targeted to the distal HS2 enhancer.

    (a,b) A panel of 21 sgRNAs was designed to target dCas9-KRAB to the HS2 enhancer (a). Cr2, Cr4, Cr7 and Cr10 sgRNAs were selected for further study and stably delivered to K562 cells using the lentiviral vector shown in b. (c–e) Repression of the HS2 enhancer was assayed by qRT-PCR of (c) HBE1, (d) HBG1/2 and (e) HBB, and fold-changes were calculated relative to nontransduced K562 cells (mean ± s.e.m.). In c–e, groups that share the same letter (A–F) are not significantly different as determined by multiway analysis of variance followed by Tukey's post hoc test; P < 0.05 (n = 3 independent experiments).

  2. Specificity of gene regulation by dCas9-KRAB repressors targeted to the HS2 enhancer.
    Figure 2: Specificity of gene regulation by dCas9-KRAB repressors targeted to the HS2 enhancer.

    RNA-seq was performed for genome-wide analysis of HS2 sgRNA silencing specificity. (a–d) Differential-expression analyses of globin genes comparing dCas9-KRAB targeted by (a) Cr4 and (b) Cr10 versus dCas9-KRAB without sgRNA and comparing dCas9-KRAB guided by (c) Cr4 and (d) Cr10 to no-lentivirus control (no LV CTL) in K562 cells. Red data points indicate FDR < 0.01 by differential-expression analysis compared with dCas9-KRAB controls without sgRNA (n = 3 biological replicates). Blue data points indicate other globin genes.

  3. Genome-wide binding activity of dCas9 repressors targeted to the HS2 enhancer.
    Figure 3: Genome-wide binding activity of dCas9 repressors targeted to the HS2 enhancer.

    (a) ChIP-seq tracks for binding of Flag-tagged dCas9 and dCas9-KRAB to the HS2 enhancer (shaded region) of the globin locus (chr11:5,244,651–5,314,450), compared with binding of dCas9-KRAB without sgRNA. An ENCODE K562 DNase I–hypersensitivity DNase-seq track is included to highlight the globin LCR22. (b,c) Differential analyses of global binding activity including comparisons of dCas9-KRAB targeted by (b) Cr4 and (c) Cr10 versus dCas9-KRAB without sgRNA. Red data points indicate FDR < 0.05 by differential DESeq analysis (n = 3 biological replicates).

  4. Genome-wide H3K9me3 signal in K562 cells treated with dCas9-KRAB targeted to the HS2 enhancer.
    Figure 4: Genome-wide H3K9me3 signal in K562 cells treated with dCas9-KRAB targeted to the HS2 enhancer.

    (a) ChIP-seq tracks for H3K9me3 signal at the HS2 enhancer (shaded area) and flanking DHS sites in the globin LCR (chr11:5,241,410–5,317,466). An ENCODE K562 DNase I–hypersensitivity DNase-seq track is included to highlight the globin LCR22. (b,c) Global analysis of H3K9me3 patterns was performed by ChIP-seq for (b) Cr4 and (c) Cr10, comparing dCas9-KRAB with sgRNA versus dCas9-KRAB without sgRNA. Red data points indicate FDR < 0.05 by differential-expression analysis compared to dCas9-KRAB without sgRNA (n = 3 biological replicates). (d) Read counts for the HS2 enhancer (chr11:5,301,862–5,302,715) from MACS-based peak calls normalized to total counts (mean ± s.e.m.). *P < 0.05 by Student's t-test compared with dCas9-KRAB–only control.

  5. Changes in global chromatin landscape with dCas9-KRAB localized to the HS2 distal enhancer.
    Figure 5: Changes in global chromatin landscape with dCas9-KRAB localized to the HS2 distal enhancer.

    (a) Genome-browser tracks of DNase-seq alignments at the globin locus (chr11:5,244,651–5,314,450) comparing the indicated conditions. Red shading labels the HBG1 promoter, HBG2 promoter, HS2 enhancer and HS3 enhancer regions for dCas9-KRAB + Cr4 or Cr10. (b,c) Normalized DNase-seq cut counts within an 800-bp window surrounding the (b) HBG2 promoter and (c) HS2 enhancer (mean ± s.e.m., n = 3 biological replicates). *P < 0.05 compared with the dCas9-KRAB only sample (Student's t-test). (d,e) Differential genome-wide analysis of changes in chromatin accessibility induced by dCas9-KRAB targeted by (d) Cr4 and (e) Cr10 compared with dCas9-KRAB without sgRNA in K562 cells. (f,g) Volcano plots of significance (P value) versus fold change for differential DESeq expression analysis of dCas9-KRAB guided by (f) Cr4 or (g) Cr10 compared with dCas9-KRAB without sgRNAs. Red data points indicate FDR < 0.05 by DESeq analysis. Blue data points indicate other regions in the globin promoters or globin LCR.

  6. Screening single gRNAs targeted to the HS2 enhancer.
    Supplementary Fig. 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).

  7. HBG1/2 expression after transient delivery of sgRNAs.
    Supplementary Fig. 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).

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

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

  10. Genome-wide binding activity of dCas9-KRAB targeted to the HS2 enhancer.
    Supplementary Fig. 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).

  11. Effect of dCas9-KRAB localization on binding of endogenous transcription factors GATA2 and FOSL1 at HS2.
    Supplementary Fig. 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).

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

  13. ChIP-qPCR of H3K9 trimethylation at the HS2 enhancer.
    Supplementary Fig. 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).

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

  15. Changes in global chromatin accessibility with dCas9-KRAB localized to the HS2 distal enhancer.
    Supplementary Fig. 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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Author information

Affiliations

  1. Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA.

    • Pratiksha I Thakore,
    • Nishkala K Shivakumar,
    • Ami M Kabadi &
    • Charles A Gersbach

  2. Center for Genomic and Computational Biology, Duke University, Durham, North Carolina, USA.

    • Pratiksha I Thakore,
    • Anthony M D'Ippolito,
    • Lingyun Song,
    • Alexias Safi,
    • Ami M Kabadi,
    • Timothy E Reddy,
    • Gregory E Crawford &
    • Charles A Gersbach

  3. University Program in Genetics and Genomics, Duke University, Durham, North Carolina, USA.

    • Anthony M D'Ippolito

  4. Department of Pediatrics, Division of Medical Genetics, Duke University Medical Center, Durham, North Carolina, USA.

    • Lingyun Song,
    • Alexias Safi &
    • Gregory E Crawford

  5. Department of Biostatistics & Bioinformatics, Duke University Medical Center, Durham, North Carolina, USA.

    • Timothy E Reddy

  6. Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina, USA.

    • Charles A Gersbach

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.

Competing financial 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.

Corresponding authors

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Screening single gRNAs targeted to the HS2 enhancer. (257 KB)

    (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).

  2. Supplementary Figure 2: HBG1/2 expression after transient delivery of sgRNAs. (60 KB)

    (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).

  3. Supplementary Figure 3: Protein silencing of HBG1 by dCas9-KRAB transcription factors targeted to the distal HS2 enhancer. (34 KB)

    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.

  4. Supplementary Figure 4: Specificity of gene regulation by dCas9-KRAB repressors targeted to the HS2 enhancer. (160 KB)

    (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.

  5. Supplementary Figure 5: Genome-wide binding activity of dCas9-KRAB targeted to the HS2 enhancer. (55 KB)

    (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).

  6. Supplementary Figure 6: Effect of dCas9-KRAB localization on binding of endogenous transcription factors GATA2 and FOSL1 at HS2. (57 KB)

    (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).

  7. Supplementary Figure 7: Genome-wide H3K9me3 signal in K562 cells treated with dCas9-KRAB targeted to the HS2 enhancer. (176 KB)

    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.

  8. Supplementary Figure 8: ChIP-qPCR of H3K9 trimethylation at the HS2 enhancer. (104 KB)

    (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).

  9. Supplementary Figure 9: Changes in chromatin accessibility at the globin gene locus with dCas9-KRAB localized to the HS2 distal enhancer. (132 KB)

    (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).

  10. Supplementary Figure 10: Changes in global chromatin accessibility with dCas9-KRAB localized to the HS2 distal enhancer. (137 KB)

    (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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