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
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- 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.
- 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.
- 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.).
- 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.)
- 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.
- 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.
- 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).
- 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.
- Supplementary Text and Figures (1,291 KB)
Supplementary Figures 1–8, Supplementary Tables 1–5 and Supplementary Notes 1 and 2