Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers

Journal name:
Nature Biotechnology
Volume:
33,
Pages:
510–517
Year published:
DOI:
doi:10.1038/nbt.3199
Received
Accepted
Published online

Abstract

Technologies that enable targeted manipulation of epigenetic marks could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control. Here we describe a programmable, CRISPR-Cas9-based acetyltransferase consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. The fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, leading to robust transcriptional activation of target genes from promoters and both proximal and distal enhancers. Gene activation by the targeted acetyltransferase was highly specific across the genome. In contrast to previous dCas9-based activators, the acetyltransferase activates genes from enhancer regions and with an individual guide RNA. We also show that the core p300 domain can be fused to other programmable DNA-binding proteins. These results support targeted acetylation as a causal mechanism of transactivation and provide a robust tool for manipulating gene regulation.

At a glance

Figures

  1. The dCas9p300 Core fusion protein activates transcription of endogenous genes from proximal promoter regions.
    Figure 1: The dCas9p300 Core fusion protein activates transcription of endogenous genes from proximal promoter regions.

    (a) Schematic of dCas9 fusion proteins dCas9VP64, dCas9FLp300 and dCas9p300 Core. S. pyogenes dCas9 contains nuclease-inactivating mutations D10A and H840A. The D1399 catalytic residue in the p300 HAT domain is indicated. (b) Western blot showing expression of dCas9 fusion proteins and GAPDH in co-transfected cells (full blot shown in Supplementary Fig. 1c). (c) Relative mRNA expression of IL1RN, MYOD and OCT4, determined by qRT-PCR, by the indicated dCas9 fusion protein co-transfected with four gRNAs targeted to each promoter region (Tukey-test, *P < 0.05, n = 3 independent experiments each; error bars, s.e.m.). Numbers above bars indicate mean relative expression. FLAG, epitope tag; NLS, nuclear localization signal; HA, hemagglutinin epitope tag; CH, cysteine-histidine-rich region; Bd, bromodomain; HAT, histone acetyltransferase domain.

  2. The dCas9p300 Core fusion protein activates transcription of endogenous genes from distal enhancer regions.
    Figure 2: The dCas9p300 Core fusion protein activates transcription of endogenous genes from distal enhancer regions.

    (a) Relative MYOD mRNA production in cells co-transfected with a pool of gRNAs targeted to either the proximal or distal regulatory regions and dCas9VP64 or dCas9p300 Core; promoter data from Figure 1c (Tukey-test, *P < 0.05 compared to mock-transfected cells; Tukey test P < 0.05 between dCas9p300 Core and dCas9VP64, n = 3 independent experiments; error bars, s.e.m.). The human MYOD locus is schematically depicted with corresponding gRNA locations in red. CE, MyoD core enhancer; DRR, MyoD distal regulatory region. (b) Relative OCT4 mRNA production in cells co-transfected with a pool of gRNAs targeted to the proximal and distal regulatory regions and dCas9VP64 or dCas9p300 Core; promoter data from Figure 1c (Tukey-test, *P < 0.05 compared to mock-transfected cells, Tukey test P < 0.05 between dCas9p300 Core and dCas9VP64, n = 3 independent experiments; error bars, s.e.m.). The human OCT4 locus is schematically depicted with corresponding gRNA locations in red. DE, Oct4 distal enhancer; PE, Oct4 proximal enhancer. (c) The human β-globin locus is schematically depicted with approximate locations of the hypersensitive site 2 (HS2) enhancer region and downstream genes (HBE, HBG, HBD and HBB). Corresponding HS2 gRNA locations are shown in red. Relative mRNA production from distal genes in cells co-transfected with four gRNAs targeted to the HS2 enhancer and the indicated dCas9 proteins. Numbers above bars indicate mean relative expression. Note logarithmic y axis and dashed red line indicating background expression (Tukey test among conditions for each β-globin gene, *P < 0.05, n = 3 independent experiments, error bars, s.e.m.). n.s., not significant.

  3. dCas9p300 Core-targeted transcriptional activation is specific and robust.
    Figure 3: dCas9p300 Core-targeted transcriptional activation is specific and robust.

    (ac) MA plots generated from DEseq2 analysis of genome-wide RNA-seq data from HEK293T cells transiently co-transfected with dCas9VP64 (a) dCas9p300 Core (b) or dCas9p300 Core (D1399Y) (c) and four IL1RN promoter-targeting gRNAs compared to HEK293T cells transiently co-transfected with dCas9 and four IL1RN promoter-targeting gRNAs. mRNAs corresponding to IL1RN isoforms are shown in blue and circled in each panel. Red-labeled points in b and c correspond to off-target transcripts significantly enriched after multiple hypothesis testing (KDR (FDR = 1.4 × 10−3); FAM49A (FDR = 0.04); p300 (FDR = 1.7 × 10−4) in b; and p300 (FDR = 1.5 × 10−5) in c).

  4. The dCas9p300 Core fusion protein acetylates chromatin at a targeted enhancer and corresponding downstream genes.
    Figure 4: The dCas9p300 Core fusion protein acetylates chromatin at a targeted enhancer and corresponding downstream genes.

    (a) The region encompassing the human β-globin locus on chromosome 11 (5,304,000–5,268,000; GRCh37/hg19 assembly) is shown. HS2 gRNA target locations are indicated in red and ChIP-qPCR amplicon regions are depicted in black with corresponding green numbers. ENCODE/Broad Institute H3K27ac enrichment signal in K562 cells is shown for comparison. Magnified insets for the HS2 enhancer, HBE promoter and HBG1/2 promoter regions are displayed below. (bd) H3K27ac ChIP-qPCR enrichment (relative to dCas9; red dashed line) at the HS2 enhancer (b), HBE promoter (c) and HBG1/2 promoters (d) in cells co-transfected with four gRNAs targeted to the HS2 enhancer and the indicated dCas9 fusion protein. HBG ChIP amplicons 1 and 2 amplify redundant sequences at the HBG1 and HBG2 promoters (denoted by ). Tukey test among conditions for each ChIP-qPCR region, *P < 0.05 (n = 3 independent experiments; error bars, s.e.m.).

  5. The dCas9p300 Core fusion protein activates transcription of endogenous genes from regulatory regions with a single gRNA.
    Figure 5: The dCas9p300 Core fusion protein activates transcription of endogenous genes from regulatory regions with a single gRNA.

    (ac) Relative IL1RN (a), MYOD (b) or OCT4 (c) mRNA produced from cells co-transfected with dCas9p300 Core or dCas9VP64 and gRNAs targeting respective promoters (n = 3 independent experiments, error bars, s.e.m.). (d,e) Relative MYOD (d) or OCT4 (e) mRNA produced from cells co-transfected with dCas9p300 Core and indicated gRNAs targeting the indicated MYOD or OCT4 enhancers (n = 3 independent experiments; error bars, s.e.m.). DRR, MYOD distal regulatory region; CE, MYOD core enhancer; PE, OCT4 proximal enhancer; DE, OCT4 distal enhancer. (Tukey test between dCas9p300 Core and single OCT4 DE gRNAs compared to mock-transfected cells, *P < 0.05; Tukey test among dCas9p300 Core and OCT4 DE gRNAs compared to All, P < 0.05). (f,g) Relative HBE (f) or HBG (g) mRNA production in cells co-transfected with dCas9p300 Core and the indicated gRNAs targeted to the HS2 enhancer (Tukey test between dCas9p300 Core and single HS2 gRNAs compared to mock-transfected cells, *P < 0.05; Tukey test among dCas9p300 Core and HS2 single gRNAs compared to All, P < 0.05, n = 3 independent experiments; error bars, s.e.m.). HS2, β-globin locus control region hypersensitive site 2; n.s., not significant using Tukey test.

  6. The p300 Core can be targeted to genomic loci by diverse programmable DNA-binding proteins.
    Figure 6: The p300 Core can be targeted to genomic loci by diverse programmable DNA-binding proteins.

    (a) Schematic of the Neisseria meningitidis (Nm) dCas9 fusion proteins Nm-dCas9VP64 and Nm-dCas9p300 Core. Neisseria meningitidis dCas9 contains nuclease-inactivating mutations D16A, D587A, H588A and N611A. (b,c) Relative HBE (b) or HBG (c) mRNA in cells co-transfected with five individual or pooled (A–E) Nm gRNAs targeted to the HBE or HBG promoter and Nm-dCas9VP64 or Nm-dCas9p300 Core. (d,e) Relative HBE (d) or HBG (e) mRNA in cells co-transfected with five individual or pooled (A–E) Nm gRNAs targeted to the HS2 enhancer and Nm-dCas9VP64 or Nm-dCas9p300 Core. (f) Schematic of TALEs with domains containing IL1RN-targeted repeat variable di-residues (repeat domain). (g) Relative IL1RN mRNA in cells transfected with individual or pooled (A–D) IL1RN TALEVP64 or IL1RN TALEp300 Core encoding plasmids. Numbers on top of bars indicate mean relative expression. (h) Schematic of ZF fusion proteins with zinc finger helices 1–6 (F1–F6) targeting the ICAM1 promoter. (i) Relative ICAM1 mRNA in cells transfected with ICAM1 ZFVP64 or ICAM1 ZFp300 Core. Tukey-test, *P < 0.05 compared to mock-transfected control, n = 3 independent experiments each; error bars, s.e.m. NLS, nuclear localization signal; HA, hemagglutinin tag; Bd, bromodomain; CH, cysteine-histidine-rich region; HAT, histone acetyltransferase domain.

  7. dCas9p300 Core mutant fusion protein activities.
    Supplementary Fig. 1: dCas9p300 Core mutant fusion protein activities.

    (a) Schematic depiction of the WT dCas9p300 Core fusion protein and p300 Core mutant derivatives. Relative locations of mutated amino acids are displayed as yellow bars within the p300 Core effector domain. (b) dCas9p300 Core variants were transiently co-transfected with four IL1RN promoter gRNAs and were screened for hyperactivity1 (amino acid 1645/1646 RR/EE and C1204R mutations) or hypoactivity1, 2 (denoted by ) via mRNA production from the IL1RN locus (top panel, n=2 independent experiments, error bars: s.e.m.). Experiments were performed in duplicate with one well used for RNA isolation and the other for western blotting to validate expression (bottom panels). The nitrocellulose membrane was cut and incubated with α-FLAG primary antibody (top, Sigma-Aldrich cat.# F7425) or α-GAPDH (bottom, Cell Signaling Technology cat.# 14C10) then α-Rabbit HRP secondary antibody (Sigma-Aldrich cat.# A6154). (c) Full membranes from western blot shown in main text (Figure 1b). The nitrocellulose membrane was cut and incubated with α-FLAG primary antibody (top, Sigma-Aldrich cat.# F7425) or α-GAPDH (bottom, Cell Signaling Technology cat.# 14C10) then α-Rabbit HRP secondary antibody (Sigma-Aldrich cat.# A6154). Membrane was imaged for the indicated durations after careful re-alignment of trimmed pieces.

  8. Target gene activation is unaffected by overexpression of synthetic dCas9 fusion proteins.
    Supplementary Fig. 2: Target gene activation is unaffected by overexpression of synthetic dCas9 fusion proteins.

    dCas9 fusion proteins were transiently co-transfected with an empty gRNA vector backbone and mRNA expression of IL1RN, MYOD, and OCT4 was assayed as in the main text. Red dashed line indicates background expression level from No DNA-transfected cells. n=2 independent experiments, error bars: s.e.m., no significant activation was observed for any target gene assayed.

  9. Comparison of Sp. dCas9 and Nm. dCas9 gene induction from the HS2 enhancer with individual and pooled gRNAs.
    Supplementary Fig. 3: Comparison of Sp. dCas9 and Nm. dCas9 gene induction from the HS2 enhancer with individual and pooled gRNAs.

    (a) Schematic display of the human β-globin locus including Streptococcus pyogenes dCas9 (Sp. dCas9) and Neisseria meningitidis dCas9 (Nm. dCas9) gRNA locations at the HS2 enhancer. Layered transcription profiles scaled to a vertical viewing range of 8 from nine ENCODE cell lines (GM12878, H1-hESC, HeLa-S3, HepG2, HSMM, HUVEC, K562, NHEK, and NHLF) is shown in addition to ENCODE p300 binding peaks in K562, A549 (EtOH.02), HeLA-S3, and SKN_SH_RA cell lines. An ENCODE HEK293T DNase hypersensitive site (HEK293T DHS) is shown in the HS2 Enhancer inset. (b–e) Relative transcriptional induction of HBE, HBG, HBD, and HBD transcripts from single and pooled Sp. dCas9 gRNAs (A–D) or single and pooled Nm. dCas9 gRNAs (A–E) in response to co-transfection with Sp. dCas9p300 Core or Nm. dCas9p300 Core respectively. gRNAs are tiled for each dCas9 ortholog corresponding to their location in GRCh37/hg19. Gray dashed line indicates background expression level in transiently co-transfected HEK293T cells. Note shared logarithmic scale among panels b–e. Numbers above bars in b–e indicate mean expression (n = at least 3 independent experiments, error bars: s.e.m.).

  10. dCas9VP64 and dCas9p300 Core induce H3K27ac enrichment at IL1RN gRNA-targeted chromatin.
    Supplementary Fig. 4: dCas9VP64 and dCas9p300 Core induce H3K27ac enrichment at IL1RN gRNA-targeted chromatin.

    The IL1RN locus on GRCh37/hg19 is shown along with IL1RN gRNA target sites. In addition layered ENCODE H3K27ac enrichment from seven cell lines (GM12878, H1-hESC, HSMM, HUVEC, K562, NHEK, and NHLF) is indicated with the vertical range setting set to 50. Tiled IL1RN ChIP qPCR amplicons (1–13) are also shown in corresponding locations on GRCh37/hg19. H3K27ac enrichment for dCas9VP64 and dCas9p300 Core co-transfected with four IL1RN-targeted gRNAs and normalized to dCas9 co-transfected with four IL1RN gRNAs is indicated for each ChIP qPCR locus assayed. 5ng of ChIP-prepared DNA was used for each reacton (n = 3 independent experiments, error bars: s.e.m.)

  11. Direct comparison of VP64 and p300 Core effector domains between TALE and dCas9 programmable DNA binding proteins.
    Supplementary Fig. 5: Direct comparison of VP64 and p300 Core effector domains between TALE and dCas9 programmable DNA binding proteins.

    (a) The GRCh37/hg19 region encompassing the IL1RN transcription start site is shown schematically along with IL1RN TALE binding sites and dCas9 IL1RN gRNA target sites. (b) Direct comparison of IL1RN activation in HEK293T cells when transfected with individual or pooled (A–D) IL1RN TALEVP64 fusion proteins or when co-transfected with dCas9VP64 and individual or pooled (A–D) IL1RN-targeting gRNAs. (c) Direct comparison of IL1RN activation in HEK293T cells when transfected with individual or pooled (A–D) IL1RN TALEp300 Core fusion proteins or when co-transfected with dCas9p300 Core and individual or pooled (A–D) IL1RN-targeting gRNAs. Note shared logarithmic scale between panels b and c. Numbers above bars in panels b and c indicate mean values. Tukey test, *P-value <0.05, n = at least 3 independent experiments, error bars: s.e.m.

  12. TALE and ZF fusion protein expression.
    Supplementary Fig. 6: TALE and ZF fusion protein expression.

    (a) Western blotting was carried out on cells transiently transfected with individual or pooled IL1RN TALE proteins. Nitrocellulose membranes were cut and probed with α-HA primary antibody (1:1000 dilution in TBST + 5% Milk, top, Covance cat.# MMS-101P) or α-GAPDH (bottom, Cell Signaling Technology cat.# 14C10) then α-Mouse HRP (Santa Cruz, sc-2005) or α-Rabbit HRP (Sigma-Aldrich cat.# A6154) secondary antibody, respectively. (b) Western blotting was carried out on cells transiently transfected with ICAM1 ZF-effector proteins and nitrocellulose membranes were cut and probed with α-FLAG primary antibody (top, Sigma-Aldrich cat.# F7425) or α-GAPDH (bottom, Cell Signaling Technology cat.# 14C10) then α-Rabbit HRP secondary antibody (Sigma-Aldrich cat.# A6154). Red asterisk indicates non-specific band.

  13. dCas9p300 Core and dCas9VP64 do not display synergy in transactivation.
    Supplementary Fig. 7: dCas9p300 Core and dCas9VP64 do not display synergy in transactivation.

    (a) dCas9p300 Core was co-transfected at a 1:1 mass ratio to PL-SIN-EF1α-EGFP3 (GFP), dCas9, or dCas9VP64 with four IL1RN promoter gRNAs as indicated (n = 2 independent experiments, error bars: s.e.m.). (b) dCas9p300 Core was co-transfected at a 1:1 mass ratio to GFP, dCas9, or dCas9VP64 with four MYOD promoter gRNAs as indicated (n = 2 independent experiments, error bars: s.e.m.). No significant differences were observed using Tukey’s test (n.s).

  14. Underlying chromatin context of dCas9p300 Core target loci.
    Supplementary Fig. 8: Underlying chromatin context of dCas9p300 Core target loci.

    (a–d) Indicated loci are shown along with associated Streptococcus pyogenes gRNAs used in this study at corresponding genomic locations in GRCh37/hg19. ENCODE HEK293T DNase hypersensitivity enrichment is shown (note changes in scale) along with regions of significant DNase hypersensitivity in HEK293T cells (“DHS”). In addition ENCODE master DNase clusters across 125 cell types are shown. Layered ENCODE H3K27ac and H3K4me3 enrichment across seven cell lines (GM12878, H1-hESC, HSMM, HUVEC, K562, NHEK, and NHLF) is also displayed and scaled to a vertical viewing range of 50 and 150 respectively. Endogenous p300 binding profiles are also indicated for each locus and respective cell line. (e) An overview of the information provided in a–d.

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Author information

Affiliations

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

    • Isaac B Hilton,
    • Pratiksha I Thakore &
    • Charles A Gersbach
  2. Center for Genomic & Computational Biology, Duke University, Durham, North Carolina, USA.

    • Isaac B Hilton,
    • Anthony M D'Ippolito,
    • Christopher M Vockley,
    • Pratiksha I Thakore,
    • Gregory E Crawford,
    • Timothy E Reddy &
    • Charles A Gersbach
  3. University Program in Genetics and Genomics, Duke University Medical Center, Durham, North Carolina, USA.

    • Anthony M D'Ippolito
  4. Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, USA.

    • Christopher M Vockley
  5. Department of Pediatrics, Division of Medical Genetics, Duke University Medical Center, Durham, North Carolina, USA.

    • Gregory E Crawford
  6. Department of Biostatistics & Bioinformatics, Duke University Medical Center, Durham, North Carolina, USA.

    • Timothy E Reddy
  7. Department of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina, USA.

    • Charles A Gersbach

Contributions

I.B.H., A.M.D., C.M.V., P.I.T., G.E.C., T.E.R. and C.A.G. designed experiments. I.B.H., A.M.D. and C.M.V. performed the experiments. I.B.H., A.M.D., C.M.V., P.I.T., G.E.C., T.E.R. and C.A.G. analyzed the data. I.B.H. and C.A.G. wrote the manuscript with contributions by all authors.

Competing financial interests

C.A.G., I.B.H. and P.I.T. have filed patent applications related to genome engineering technologies. 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: dCas9p300 Core mutant fusion protein activities. (155 KB)

    (a) Schematic depiction of the WT dCas9p300 Core fusion protein and p300 Core mutant derivatives. Relative locations of mutated amino acids are displayed as yellow bars within the p300 Core effector domain. (b) dCas9p300 Core variants were transiently co-transfected with four IL1RN promoter gRNAs and were screened for hyperactivity1 (amino acid 1645/1646 RR/EE and C1204R mutations) or hypoactivity1, 2 (denoted by ) via mRNA production from the IL1RN locus (top panel, n=2 independent experiments, error bars: s.e.m.). Experiments were performed in duplicate with one well used for RNA isolation and the other for western blotting to validate expression (bottom panels). The nitrocellulose membrane was cut and incubated with α-FLAG primary antibody (top, Sigma-Aldrich cat.# F7425) or α-GAPDH (bottom, Cell Signaling Technology cat.# 14C10) then α-Rabbit HRP secondary antibody (Sigma-Aldrich cat.# A6154). (c) Full membranes from western blot shown in main text (Figure 1b). The nitrocellulose membrane was cut and incubated with α-FLAG primary antibody (top, Sigma-Aldrich cat.# F7425) or α-GAPDH (bottom, Cell Signaling Technology cat.# 14C10) then α-Rabbit HRP secondary antibody (Sigma-Aldrich cat.# A6154). Membrane was imaged for the indicated durations after careful re-alignment of trimmed pieces.

  2. Supplementary Figure 2: Target gene activation is unaffected by overexpression of synthetic dCas9 fusion proteins. (72 KB)

    dCas9 fusion proteins were transiently co-transfected with an empty gRNA vector backbone and mRNA expression of IL1RN, MYOD, and OCT4 was assayed as in the main text. Red dashed line indicates background expression level from No DNA-transfected cells. n=2 independent experiments, error bars: s.e.m., no significant activation was observed for any target gene assayed.

  3. Supplementary Figure 3: Comparison of Sp. dCas9 and Nm. dCas9 gene induction from the HS2 enhancer with individual and pooled gRNAs. (133 KB)

    (a) Schematic display of the human β-globin locus including Streptococcus pyogenes dCas9 (Sp. dCas9) and Neisseria meningitidis dCas9 (Nm. dCas9) gRNA locations at the HS2 enhancer. Layered transcription profiles scaled to a vertical viewing range of 8 from nine ENCODE cell lines (GM12878, H1-hESC, HeLa-S3, HepG2, HSMM, HUVEC, K562, NHEK, and NHLF) is shown in addition to ENCODE p300 binding peaks in K562, A549 (EtOH.02), HeLA-S3, and SKN_SH_RA cell lines. An ENCODE HEK293T DNase hypersensitive site (HEK293T DHS) is shown in the HS2 Enhancer inset. (b–e) Relative transcriptional induction of HBE, HBG, HBD, and HBD transcripts from single and pooled Sp. dCas9 gRNAs (A–D) or single and pooled Nm. dCas9 gRNAs (A–E) in response to co-transfection with Sp. dCas9p300 Core or Nm. dCas9p300 Core respectively. gRNAs are tiled for each dCas9 ortholog corresponding to their location in GRCh37/hg19. Gray dashed line indicates background expression level in transiently co-transfected HEK293T cells. Note shared logarithmic scale among panels b–e. Numbers above bars in b–e indicate mean expression (n = at least 3 independent experiments, error bars: s.e.m.).

  4. Supplementary Figure 4: dCas9VP64 and dCas9p300 Core induce H3K27ac enrichment at IL1RN gRNA-targeted chromatin. (97 KB)

    The IL1RN locus on GRCh37/hg19 is shown along with IL1RN gRNA target sites. In addition layered ENCODE H3K27ac enrichment from seven cell lines (GM12878, H1-hESC, HSMM, HUVEC, K562, NHEK, and NHLF) is indicated with the vertical range setting set to 50. Tiled IL1RN ChIP qPCR amplicons (1–13) are also shown in corresponding locations on GRCh37/hg19. H3K27ac enrichment for dCas9VP64 and dCas9p300 Core co-transfected with four IL1RN-targeted gRNAs and normalized to dCas9 co-transfected with four IL1RN gRNAs is indicated for each ChIP qPCR locus assayed. 5ng of ChIP-prepared DNA was used for each reacton (n = 3 independent experiments, error bars: s.e.m.)

  5. Supplementary Figure 5: Direct comparison of VP64 and p300 Core effector domains between TALE and dCas9 programmable DNA binding proteins. (66 KB)

    (a) The GRCh37/hg19 region encompassing the IL1RN transcription start site is shown schematically along with IL1RN TALE binding sites and dCas9 IL1RN gRNA target sites. (b) Direct comparison of IL1RN activation in HEK293T cells when transfected with individual or pooled (A–D) IL1RN TALEVP64 fusion proteins or when co-transfected with dCas9VP64 and individual or pooled (A–D) IL1RN-targeting gRNAs. (c) Direct comparison of IL1RN activation in HEK293T cells when transfected with individual or pooled (A–D) IL1RN TALEp300 Core fusion proteins or when co-transfected with dCas9p300 Core and individual or pooled (A–D) IL1RN-targeting gRNAs. Note shared logarithmic scale between panels b and c. Numbers above bars in panels b and c indicate mean values. Tukey test, *P-value <0.05, n = at least 3 independent experiments, error bars: s.e.m.

  6. Supplementary Figure 6: TALE and ZF fusion protein expression. (50 KB)

    (a) Western blotting was carried out on cells transiently transfected with individual or pooled IL1RN TALE proteins. Nitrocellulose membranes were cut and probed with α-HA primary antibody (1:1000 dilution in TBST + 5% Milk, top, Covance cat.# MMS-101P) or α-GAPDH (bottom, Cell Signaling Technology cat.# 14C10) then α-Mouse HRP (Santa Cruz, sc-2005) or α-Rabbit HRP (Sigma-Aldrich cat.# A6154) secondary antibody, respectively. (b) Western blotting was carried out on cells transiently transfected with ICAM1 ZF-effector proteins and nitrocellulose membranes were cut and probed with α-FLAG primary antibody (top, Sigma-Aldrich cat.# F7425) or α-GAPDH (bottom, Cell Signaling Technology cat.# 14C10) then α-Rabbit HRP secondary antibody (Sigma-Aldrich cat.# A6154). Red asterisk indicates non-specific band.

  7. Supplementary Figure 7: dCas9p300 Core and dCas9VP64 do not display synergy in transactivation. (58 KB)

    (a) dCas9p300 Core was co-transfected at a 1:1 mass ratio to PL-SIN-EF1α-EGFP3 (GFP), dCas9, or dCas9VP64 with four IL1RN promoter gRNAs as indicated (n = 2 independent experiments, error bars: s.e.m.). (b) dCas9p300 Core was co-transfected at a 1:1 mass ratio to GFP, dCas9, or dCas9VP64 with four MYOD promoter gRNAs as indicated (n = 2 independent experiments, error bars: s.e.m.). No significant differences were observed using Tukey’s test (n.s).

  8. Supplementary Figure 8: Underlying chromatin context of dCas9p300 Core target loci. (126 KB)

    (a–d) Indicated loci are shown along with associated Streptococcus pyogenes gRNAs used in this study at corresponding genomic locations in GRCh37/hg19. ENCODE HEK293T DNase hypersensitivity enrichment is shown (note changes in scale) along with regions of significant DNase hypersensitivity in HEK293T cells (“DHS”). In addition ENCODE master DNase clusters across 125 cell types are shown. Layered ENCODE H3K27ac and H3K4me3 enrichment across seven cell lines (GM12878, H1-hESC, HSMM, HUVEC, K562, NHEK, and NHLF) is also displayed and scaled to a vertical viewing range of 50 and 150 respectively. Endogenous p300 binding profiles are also indicated for each locus and respective cell line. (e) An overview of the information provided in a–d.

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  1. Supplementary Text and Figures (1,291 KB)

    Supplementary Figures 1–8, Supplementary Tables 1–5 and Supplementary Notes 1 and 2

Additional data