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Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions

Abstract

Despite the importance of DNA methylation in health and disease, technologies to readily manipulate methylation of specific sequences for functional analysis and therapeutic purposes are lacking. Here we adapt the previously described dCas9–SunTag for efficient, targeted demethylation of specific DNA loci. The original SunTag consists of ten copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, which induces demethylation, we changed the linker length to 22 amino acids. The system attains demethylation efficiencies >50% in seven out of nine loci tested. Four of these seven loci showed demethylation of >90%. We demonstrate targeted demethylation of CpGs in regulatory regions and demethylation-dependent 1.7- to 50-fold upregulation of associated genes both in cell culture (embryonic stem cells, cancer cell lines, primary neural precursor cells) and in vivo in mouse fetuses.

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Figure 1: Targeted demethylation in ESCs using CRISPR–Cas9 and a peptide-repeat-based amplification.
Figure 2: Targeted demethylation of the imprinted gene H19 in ESCs using CRISPR–Cas9 and a peptide-repeat-based amplification.
Figure 3: In vitro and in vivo targeted demethylation using CRISPR–Cas9 and a peptide-repeat-based amplification.

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Acknowledgements

This work was supported by the Basic Science and Platform Technology Program for Innovative Biological Medicine from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT); The Japan Agency for Medical Research and Development (AMED) to I.H. and AMED-CREST and AMED to K.N.; and a Grant-in-Aid for Challenging Exploratory Research (grant number 15K14452) to K.N. The authors plan to make the reagents widely available to the academic community through Addgene (http://www.addgene.org/?gclid=CKvf88_a2ccCFQNwvAodSbUGiQ). We appreciate the technical assistance provided by The Research Support Center, Research Center for Human Disease Modeling, Kyushu University Graduate School of Medical Sciences.

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Authors

Contributions

I.H. conceived the project. I.H., S.M., T.H., K.H., and K.N. designed the experiments. S.M., H.N., T.H., K.N., M.K., K.O., and A.S. performed the experiments and analyzed the data. I.H., S.M., H.N., and K.N. wrote the manuscript.

Corresponding author

Correspondence to Izuho Hatada.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Design and sequence of system 1 for targeted demethylation.

(a) Design of system 1 for targeted demethylation. TET1CD was fused to a catalytic inactive Cas9 nuclease (dCas9) and expressed using the CAG promoter (pCAG-dCas9TET1CD). Each gRNA vector was generated by incorporating the target sequence into the gRNA cloning vector with the U6 promoter (Addgene, 41824) using Gibson assembly (New England BioLabs). (b) Full sequence of pCAG-dCas9TET1CD.

Supplementary Figure 2 Design and sequence of system 2 for targeted demethylation.

(a) Design of system 2 for targeted demethylation. dCas9 with ten copies of the GCN4 peptide was expressed using the CAG promoter (pCAG-dCas9-10xGCN4_v4). The length of the linker separating each 19 aa GCN4 peptide unit of the array was 5 aa. An anti-GCN4 peptide antibody (scFv)-sfGFP-TET1CD fusion protein was expressed using the CAG promoter (pCAG-scFvGCN4sfGFPTET1CD). Each gRNA vector was generated by incorporating the target sequence into the gRNA cloning vector with the U6 promoter (Addgene, 41824) using Gibson assembly (New England BioLabs). (b) Full sequence of pCAG-dCas9-10xGCN4_v4. (c) Full sequence of scFvGCN4sfGFPTET1CD.

Supplementary Figure 3 Design and sequence of system 3 for targeted demethylation.

(a) Design of system 3 for targeted demethylation. dCas9 with five copies of the GCN4 peptide was expressed using the CAG promoter (pCAG-dCas9-5xPlat2AflD). The length of the linker separating each 19 aa GCN4 peptide unit of the array was 22 aa. An anti-GCN4 peptide antibody (scFv)-sfGFP-TET1CD fusion protein was expressed using the CAG promoter (pCAG-scFvGCN4sfGFPTET1CD). Each gRNA vector was generated by incorporating the target sequence into the gRNA cloning vector with the U6 promoter (Addgene: 41824) using Gibson assembly (New England BioLabs). (b) Full sequence of pCAG-dCas9-5xPlat2AflD.

Supplementary Figure 4 Design and sequence of system 4 for targeted demethylation.

(a) Design of system 4 for targeted demethylation. dCas9 with four copies of the GCN4 peptide was expressed using the CAG promoter (pCAG-dCas9-3.5xSuper). The length of the linker separating each 19 aa GCN4 peptide unit of the array was 43 aa. An anti-GCN4 peptide antibody (scFv)-sfGFP-TET1CD fusion protein was expressed using the CAG promoter (pCAG-scFvGCN4sfGFPTET1CD). Each gRNA vector was generated by incorporating the target sequence into the gRNA cloning vector with the U6 promoter (Addgene: 41824) using Gibson assembly (New England BioLabs). (b) Full sequence of pCAG-dCas9-3.5xSuper.

Supplementary Figure 5 Validation of linker length and specificity of demethylation.

(a) The length of the linker separating each GCN4 peptide unit of the array fused to dCas9 is important for maximization of the demethylation activity. There are two possibilities to explain this. If the linker is too short, there is only a small amount of space for antibody-TET1CD fusion proteins to approach and bind to the GCN4 peptide array, resulting in poor demethylation activity (possibility 1). Alternatively, the amount of space is too small for antibody-TET1CD fusion proteins to work properly, resulting in poor demethylation activity (possibility 2). (b) Co-IP of the Cas9-peptide array and antibody-sfGFP-TET1CD fusion protein (systems 2–4). The Cas9-peptide array (with a HA tag) was immunoprecipitated with anti-HA magnetic beads and subjected to western blot analysis. The amount of co-immunoprecipitated antibody-sfGFP-TET1CD fusion protein (vector lacking the HA tag was used in this case) was quantified by western blotting with an anti-GFP antibody and normalized by the amount of the Cas9-peptide array quantified by western blotting with an anti-HA antibody. Normalized values are shown in the bar graph. Data are shown as the mean ± s.e.m. Statistical analyses were performed using an ANOVA with Tukey’s post-hoc test (N.S., not significant (p > 0.05)). (c) Demethylation activities of system 3 (dCas9-GCN4 and scFV-GFP-TET1CD), that with catalytic inactive TET1 (H1671Y, D1673A), that without scFv-GFP-TET1CD, and that without dCas9-GCN4. Demethylation of the STAT3-binding site was analyzed by COBRA. The gRNA used was target 2 of Gfap. Demethylation was analyzed as in Figure 1c. Data are shown as the mean ± s.e.m. (n = 3 from two independent experiments). The two-sided Student’s t-test was performed. ***P < 0.005.

Supplementary Figure 6 Quantification of the viability of ESCs into which systems 2, 3, and 4 were introduced.

(a) ESCs were transfected with the Gfap_2 gRNA using systems 2, 3, and 4. Two days after transfection, ESCs were stained with PI, and cell viability was quantified using a FACSVerse flow cytometer (BD Biosciences) with a 488-nm blue laser. Cells were categorized into four groups based on dye uptake and GFP signals. Quadrant UL shows PI-negative/GFP-positive cells, quadrant UR shows PI/GFP-positive cells, quadrant LL shows PI/GFP-negative cells, and quadrant LR shows PI-positive/GFP-negative cells. (b) The population of PI-positive cells among GFP-positive cells was compared among the systems. Data are shown as the mean ± s.e.m (n = 3 from two independent experiments). Statistical analyses were performed using an ANOVA with Tukey’s post-hoc test (N.S., not significant (p > 0.05)).

Supplementary Figure 7 Design and sequence of the all-in-one system for targeted demethylation.

(a) Design of the all-in-one system for targeted demethylation. This vector included the gRNA under the control of the U6 promoter, dCas9 with the GCN4 array (system 3), and the antibody-TET1CD fusion protein. Cloning was performed by linearization of an Afl II site and Gibson assembly-mediated incorporation of the gRNA insert fragment. (b) Full sequence of pPlatTET-gRNA2.

Supplementary Figure 8 Methylation surrounding the target site.

ESCs transfected with Gfap gRNA (Gfap_2) or a control using systems 2, 3 (same as Figure 1f), and 4 were sorted to isolate GFP-expressing cells, and methylation in the surrounding area was analyzed using bisulfite sequencing. Methylation for active and catalytically-dead TET1 is shown. Black/white circles indicate the percentage of methylation in each CpG site. Black indicates the methylation percentage. Each number beneath the circles indicates the position. The red bar indicates the STAT3-binding site. The blue bar indicates the target site. A scale is provided at the bottom. For each group, at least 14 randomly selected clones were sequenced and analyzed. The statistical significance between the two groups of the entire set of CpG sites was evaluated with the Mann-Whitney U-test (also called the Wilcoxon rank-sum test).

Supplementary Figure 9 Off-target analysis and genome-wide analysis of DNA methylation and gene expression.

(a) Methylation surrounding the off-target sites of the Gfap_2 gRNA. ESCs were transfected with this gRNA or a control using system 3, sorted, and analyzed for methylation in the area surrounding off-target sites by bisulfite sequencing. Matched sequences are written in red and mismatched sequences are written in black. Red bars indicate the homologous sequence to the Gfap target sites. Black/white circles indicate the percentage of methylation in each CpG site. Black indicates the methylation percentage. Each number beneath the circles indicates the position. A scale is provided at the bottom. For each group, at least 14 randomly selected clones were sequenced and analyzed. The statistical significance between the two groups of the entire set of CpG sites was evaluated with the Mann-Whitney U-test (also called the Wilcoxon rank-sum test). (b) BS-seq analysis of ESCs that were transfected with the all-in-one vector targeting Gfap (samples 1 and 3) or control (samples 2 and 4). Vectors with active (samples 1 and 2) or catalytically-dead (samples 3 and 4) TET1 were used. Sample 1 and 2 was mixed and randomly divided in half to generate samples a and b. Scatter plots of 1 vs. 2, 3 vs. 4, 1 vs. 3, and a vs. b are shown, along with the correlation coefficients. Each red line indicates the regression line of each sample. (c) BS-seq methylation landscape of the Gfap gene in samples 1–4. The STAT3-binding site and corresponding methylation are indicated by a pink bar and a pink square, respectively. (d) RNA-seq analysis of the same samples as in Supplementary Figure 9b. Scatter plots of 1 vs. 2, 3 vs. 4, and 1 vs. 3 are shown, along with the correlation coefficients.

Supplementary Figure 10 Methylation surrounding the H19 DMR CTCF-binding sites (m1 and m2).

ESCs were transfected with H19 DMR_2 gRNA using system 3, sorted, and analyzed for methylation in the area surrounding target site 2 by bisulfite sequencing. Methylation for active and catalytically-dead TET1 is shown. Black/white circles indicate the percentage of methylation in each CpG site. Black indicates the methylation percentage. Each number beneath the circles indicates the position. Red bars indicate the CTCF-binding sites (m1 and m2). The blue bar indicates the position of target 2. A scale is provided at the bottom. For each group, at least 14 randomly selected clones were sequenced and analyzed. The statistical significance between the two groups of the entire set of CpG sites was evaluated with the Mann-Whitney U-test (also called the Wilcoxon rank-sum test).

Supplementary Figure 11 Methylation surrounding the off-target sites of the H19DMR 1–4 gRNAs.

ESCs were transfected with these gRNAs using system 3, sorted, and analyzed for methylation in the area surrounding off-target sites by bisulfite sequencing. Matched sequences are written in red and mismatched sequences are written in black. Red bars indicate the homologous sequence to the H19 target sites. Black/white circles indicate the percentage of methylation in each CpG site. Black indicates the methylation percentage. Each number beneath the circles indicates the position. A scale is provided at the bottom. For each group, at least 14 randomly selected clones were sequenced and analyzed. The statistical significance between the two groups of the entire set of CpG sites was evaluated with the Mann-Whitney U-test (also called the Wilcoxon rank-sum test).

Supplementary Figure 12 Expression and methylation analysis of methylation-edited cells.

(a) Expression analysis of methylation-edited cells. Cells were transfected with gRNAs for RHOXF2B, CARD9, SH3BP2, or CNKSR1 using system 3 with active or catalytically-dead TET1CD. The cells were sorted and analyzed for expression by quantitative PCR. Data are shown as the mean ± s.e.m. (n = 3 from two independent experiments). The two-sided Student’s t-test was performed. N.S., not significant; **P < 0.01; ***P < 0.005. (b) Demethylation of the RHOXF2B, CARD9, SH3BP2, and CNKSR1 genes in human cells using system 3 with sorting. Demethylation activities for active and catalytically-dead TET1 are shown. Demethylation was analyzed as in Figure 1c. Data are shown as the mean ± s.e.m. (n = 3 from two independent experiments). The two-sided Student’s t-test was performed. N.S., not significant; ***P < 0.005.

Supplementary Figure 13 E18 brain sections that were electroporated with the vector targeting Gfap or the control vector at E14.

(a) E18 brain sections that were electroporated with the vector targeting Gfap or the control vector at E14. Brain sections obtained using catalytically-dead TET1 are also shown. Green, red, and blue indicate GFP, GFAP, and Hoechst, respectively. Magnified images of the boxed areas indicated are also shown in 1, 2, 1’, and 2’, respectively. The population of GFAP-positive cells among GFP-positive cells was significantly increased compared to the control. A scale bar is provided in the image. (b) The percentage of GFAP-positive cells among GFP-positive cells. The results obtained using catalytically-dead TET1 are also shown. Cortical sections at the same anatomical level were analyzed, and confocal images were taken with a confocal microscope. To assess astrocyte differentiation, at least 300 GFP-positive cells per sample (n=4–6 brains per group) were counted. GFAP-positive cells among GFP-positive cells were counted in high-magnification images, and each GFAP-positive cell was identified by GFAP staining around the nucleus, as indicated by both GFP and Hoechst. Data are shown as the mean ± s.e.m. Statistical analyses were performed using an ANOVA with Tukey’s post-hoc test (*p < 0.05, **p < 0.01, ***p < 0.001). (c) DNA methylation of the Gfap locus in GFP-positive cells in fetal brains that were electroporated with the vector targeting Gfap (Gfap_2, Gfap_3, and Gfap_0) or the control vector. The results obtained with catalytically-dead TET1 are also shown. GFP-positive cells were electroporated with the vectors at E14. At 24 h after electroporation, GFP-positive cells were sorted by FACS and used for DNA methylation analysis. The average methylation of CpGs at the Gfap locus analyzed by bisulfite sequencing is presented. At least three samples were used for analysis. Data are shown as the mean ± s.e.m. Statistical analyses were performed using an ANOVA with Tukey's post-hoc test (*p < 0.05).

Supplementary Figure 14 Luciferase reporter assay of the Gfap promoter.

A neural progenitor cell line from adult rat hippocampus (HCN cells) was co-transfected with the all-in-one vector (expressing gRNA for the control or Gfap locus) and a Gfap promoter-reporter plasmid, which expresses firefly luciferase under the regulation of the 2.6 kb Gfap promoter. The reporter assay using catalytically-dead TET1 is also shown. As an internal control, the sea pansy luciferase-expressing vector under the control of the human elongation factor-1a promoter was also co-transfected. One day after transfection, cells were stimulated with LIF (50 ng/ml) for 8 h and used for luciferase analysis. Firefly luciferase activities were determined by three independent transfections and normalized by comparison with Renilla luciferase activities as the internal control. Data are shown as the mean ± s.e.m. Statistical analyses were performed using an ANOVA with Tukey’s post-hoc test (*p < 0.05). ns, not significant (p > 0.05).

Supplementary Figure 15 A flowchart of the selection of off-target sites for bisulfite sequencing analysis.

Off-targets sites were searched for using a web tool called CRISPR direct (http://crispr.dbcls.jp/). By using this tool, the 12 bases in the 3’ region of the target sequence adjacent to the PAM were searched against the genome because this region contains critical residues determining target specificity. Next, the sites unsuitable for analysis (sequences containing repeats and those giving no PCR primers by Meth Primer (http://www.urogene.org/methprimer/) in the default condition except for the product size) were removed. As for the off-target analysis of Gfap, all these sites were selected and subjected to off-target analysis. As for the off-targets of H19, at least all the off-targets in which more than 16 of 20 bases match were selected. If there were fewer than three selected sites, sites of lower homology were selected.

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Morita, S., Noguchi, H., Horii, T. et al. Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat Biotechnol 34, 1060–1065 (2016). https://doi.org/10.1038/nbt.3658

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