Journal: Nature Communications

Article Title: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing

doi: 10.1038/s41467-020-14362-5

Figure Lengend Snippet: a Schematic of enCRISPRa containing three components: a dCas9-p300 fusion protein, the sgRNA with two MS2 hairpins, and the MCP-VP64 fusion protein. b Expression of MYOD upon dCas9 alone, dCas9-VP64 (V), dCas9-p300 (P), dCas9-VP64 + MCP-p300 (enCRISPRa-VP) or dCas9-p300 + MCP-VP64 (enCRISPRa-PV)-mediated enhancer activation in HEK293T cells. mRNA expression relative to nontransduced cells (control) is shown as mean ± SEM ( n = 4 experiments). The differences between control and dCas9 activators were analyzed by a one-way ANOVA. # P < 0.05, ### P < 0.001. The difference between different dCas9 activators were analyzed by a one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s. not significant. c Expression of HBE1 , HBG1/2 and HBB upon dCas9 alone, dCas9-VP64 (V), dCas9-p300 (P), or enCRISPRa (VP and PV)-mediated activation of the HS2 enhancer in HEK293T cells. mRNA expression relative to nontransduced cells is shown as mean ± SEM ( n = 4 experiments) and analyzed by a one-way ANOVA. d Expression of MYOD upon dxCas9-VPR, SunTag, SAM or enCRISPRa-mediated enhancer activation in HEK293T cells. mRNA expression relative to nontransduced cells is shown as mean ± SEM ( n = 4 experiments). The differences between control and dCas9 activators were analyzed by a one-way ANOVA. # P < 0.05, ### P < 0.001. The difference between different dCas9 activators were analyzed by a one-way ANOVA. *** P < 0.001. e Expression of HBE1 , HBG1/2 and HBB upon dxCas9-VPR, SunTag, SAM or enCRISPRa-mediated activation of HS2 in HEK293T cells. mRNA expression relative to nontransduced cells is shown as mean ± SEM ( n = 4 experiments) and analyzed by a one-way ANOVA. f Genome-wide analysis of dCas9 binding in HEK293T cells expressing HS2-specific sgRNA (two replicates sgHS2-rep1 and sgHS2-rep2) or nontargeting sgGal4. Data points for the sgRNA target regions and the predicted off-targets are shown as green and red, respectively. The x - and y axis denote the normalized read counts (left) or mean normalized read counts from n = 2 experiments (right). Source data are provided as a Source Data file.

Article Snippet: To generate the inducible dCas9-p300 expression vector, the p300 HAT core domain (p300 core) was PCR amplified from the pcDNA-dCas9-p300-Core vector (Addgene, Plasmid #61357) and cloned into Mlu I/ Bst XI digested pHR-TRE3G-KRAB-dCas9-P2A-mCherry backbone, which was a gift from Luke A. Gilbert .

Techniques: Expressing, Activation Assay, Genome Wide, Binding Assay

Journal: Nature Communications

Article Title: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing

doi: 10.1038/s41467-020-14362-5

Figure Lengend Snippet: a Schematic of enCRISPRi containing a dCas9-LSD1 fusion protein, the sgRNA with two MS2 hairpins, and the MCP-KRAB fusion protein. b Expression of β-globin genes in K562 cells upon dCas9-KRAB (K), dCas9-LSD1 (L) or enCRISPRi (LK and KL)-mediated repression of the HS2 enhancer using four HS2-targeting sgRNAs individually (sgHS2-1 to sgHS2-4) or combined (sgHS2-all). The nontargeting sgGal4 was analyzed as the control. mRNA expression relative to nontransduced cells is shown as mean ± SEM ( n = 4 experiments) and analyzed by a two-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s. not significant. c RNA-seq profiles in K562 cells upon dCas9-KRAB, dCas9-LSD1 or enCRISPRi-mediated repression of the HS2 enhancer using four sgRNAs (sgHS2-all). Scatter plot is shown for each gene by the mean of log2 normalized RNA-seq signals as transcripts per million or TPM ( n = 2 experiments) ( x axis) and log2 fold changes of mean TPM in cells expressing sgHS2 and nontransduced cells ( y axis). β-globin genes are indicated by red arrowheads. d Genome-wide analysis of dCas9 binding in K562 cells expressing HS2-specific sgRNA (sgHS2-rep1 and sgHS2-rep2) or nontargeting sgGal4. Data points for the sgRNA target regions and the predicted off-targets are shown as green and red, respectively. e Density maps are shown for DHS, ChIP-seq of H3K27ac, H3K4me1, H3K4me2, CTCF, and RNA-seq at the β-globin cluster (chr11: 5,222,500–5,323,700; hg19). The zoom-in view of the HS2 proximity region is shown on the top. Dashed lines denote the positions of sgRNAs. f Expression of β-globin genes in K562 cells coexpressing enCRISPRi and target-specific sgRNAs at various positions within the β-globin cluster, control sgRNAs (sgCtrl, sgTAD1, sgTAD2, sgCTCF1 and sgCTCF2) or nontargeting sgGal4. mRNA expression relative to nontransduced cells is shown as mean ± SEM. The differences between control sgGal4 and other sgRNAs were analyzed by a one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s. not significant. The differences between sgHS2 and other sgRNAs were analyzed by a one-way ANOVA. ## P < 0.01, ### P < 0.001. Source data are provided as a Source Data file.

Article Snippet: To generate the inducible dCas9-p300 expression vector, the p300 HAT core domain (p300 core) was PCR amplified from the pcDNA-dCas9-p300-Core vector (Addgene, Plasmid #61357) and cloned into Mlu I/ Bst XI digested pHR-TRE3G-KRAB-dCas9-P2A-mCherry backbone, which was a gift from Luke A. Gilbert .

Techniques: Expressing, RNA Sequencing Assay, Genome Wide, Binding Assay, ChIP-sequencing

Journal: Nature Communications

Article Title: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing

doi: 10.1038/s41467-020-14362-5

Figure Lengend Snippet: a Density maps are shown for ATAC-seq and H3K27ac ChIP-seq at TAL1 (chr1:47,679,000–47,722,000; hg19) in Jurkat cells. The TAL1 oncogenic SE is shown as blue shaded lines. The positions of sgRNAs and PAM sequences are shown as colored lines and boxes, respectively. b Chromatin occupancy of dCas9-p300 by target-specific (sgMut1, sgMut2, sgWT1 and sgWT2) or nontargeting sgGal4 in Jurkat or K562 cells. ChIP signals (% of input) are shown as mean ± SEM ( n = 4 experiments). c Expression of TAL1 mRNA in Jurkat cells upon enCRISPRa-mediated enhancer activation. Results are mean ± SEM ( n = 4 experiments). d Expression of TAL1 protein upon enCRISPRa-mediated enhancer activation. The quantified TAL1 expression is shown. e Activation of TAL1 SE promoted T-ALL growth in vitro. Relative absorbance by cell viability assays ( y axis) at different days of culture ( x axis) is shown ( n = 3 experiments). f Expression of TAL1 upon enCRISPRi-mediated enhancer repression in Jurkat cells. Results are mean ± SEM ( n = 4 experiments). g Expression of TAL1 protein upon enCRISPRi-mediated enhancer repression. h Repression of TAL1 SE impaired T-ALL growth in vitro ( n = 3 experiments). i Activation of TAL1 SE promoted T-ALL growth in NSG mice xenografted with Jurkat cells transduced with sgGal4, sgMut2 or sgWT2, respectively. Bioluminescence intensity is shown at 4 h, 2 and 4 weeks post transplantation. j Quantification of bioluminescence intensity is shown. Results are mean ± SEM ( n = 5 recipients per group). k Frequencies of leukemia cells in BM and PB of xenografted NSG mice 4 weeks post transplantation. Results are mean ± SEM ( n = 5, 5, and 4 recipients for sgGal4, sgMut2 and sgWT2, respectively). l Representative bloodsmear images of NSG mice 4 weeks post transplantation. The inset images indicate the zoom-in view. Representative leukemia cells are indicated by arrowheads. Scale bars, 200 and 20 µm for full and insert images, respectively. Results are mean ± SEM and analyzed by a one-way or two-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, ### P < 0.001, n.s. not significant. Source data are provided as a Source Data file.

Article Snippet: To generate the inducible dCas9-p300 expression vector, the p300 HAT core domain (p300 core) was PCR amplified from the pcDNA-dCas9-p300-Core vector (Addgene, Plasmid #61357) and cloned into Mlu I/ Bst XI digested pHR-TRE3G-KRAB-dCas9-P2A-mCherry backbone, which was a gift from Luke A. Gilbert .

Techniques: ChIP-sequencing, Expressing, Activation Assay, In Vitro, Transduction, Transplantation Assay

Journal: Nature Communications

Article Title: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing

doi: 10.1038/s41467-020-14362-5

Figure Lengend Snippet: a Schematic of site-specific KI of tetracycline-inducible dCas9-KRAB into the Col1a1 locus. Coexpression of dCas9-KRAB, sgRNA-MS2 and MCP-LSD1 leads to assembly of enCRISPRi complex in vivo. b Validation of dCas9-KRAB and rtTA KI or WT alleles by genotyping PCR. C57BL/6 WT mouse and targeted KH2-ESC were used as controls. Two independent heterozygous (Het) and homozygous (Hom) KI mice were analyzed. c Dox-inducible expression of dCas9-KRAB fusion protein was confirmed by Western blot in the targeted KH2-ESC and two independent dCas9-KRAB KI mice. β-tubulin was analyzed as the loading control. d Schematic of in vivo perturbation of lineage-specific enhancers in dCas9-KRAB KI mice. e In vivo enCRISPRi perturbation of Cebpa CREs revealed lineage-specific requirement of Cebpa enhancers during hematopoiesis. Waterfall plots are shown for target-specific sgRNAs (green and red dots) and nontargeting control sgRNAs (gray dots) by the mean normalized log2 fold changes in HSPCs, myeloid, T or B cells 16 weeks post BMT (T2) relative to pooled sgRNA-transduced HSPCs (T1) from two independent replicate screens ( n = 3 recipient mice per screen). Density maps are shown for ATAC-seq and H3K27ac ChIP-seq at the Cebpa locus (chr7:35,877,000–35,951,000; mm9) in bone marrow HSC, granulocytes (GN), monocytes (Mono), B, CD4+ and CD8+ T cells, respectively. The annotated Cebpa promoter (P) and enhancers (E1 to E4) are indicated by green and blue shaded lines. Results from independent replicate screens and statistical analyses are shown in Supplementary Fig. . f In vivo enCRISPRi perturbation of Spi1 CREs during hematopoiesis. Density maps are shown for ATAC-seq and H3K27ac ChIP-seq at the Spi1 locus (chr2:90,911,000–90,957,000; mm9) in bone marrow HSC, GN, Mono, B, CD4+ and CD8+ T cells, respectively. The annotated Spi1 promoter (P) and enhancer (E) are indicated by green and blue shaded lines. Results from independent replicate screens and statistical analyses are shown in Supplementary Fig. . Source data are provided as a Source Data file.

Article Snippet: To generate the inducible dCas9-p300 expression vector, the p300 HAT core domain (p300 core) was PCR amplified from the pcDNA-dCas9-p300-Core vector (Addgene, Plasmid #61357) and cloned into Mlu I/ Bst XI digested pHR-TRE3G-KRAB-dCas9-P2A-mCherry backbone, which was a gift from Luke A. Gilbert .

Techniques: In Vivo, Expressing, Western Blot, ChIP-sequencing

Journal: Nature Communications

Article Title: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing

doi: 10.1038/s41467-020-14362-5

Figure Lengend Snippet: a Schematic of in vivo pooled sgRNA-based multiloci perturbations of developmentally regulated enhancers in dCas9-KRAB KI mice. b In vivo perturbation of annotated CREs for five key hematopoietic TFs revealed the functional requirement of lineage-specific enhancers for HSC differentiation to one or multiple hematopoietic lineages. Waterfall plots are shown for target-specific sgRNAs (green and red dots) and nontargeting control sgRNAs (gray dots) by the mean normalized log2 fold changes in HSPCs, myeloid, T or B cells 16 weeks post BMT (T2) relative to pooled sgRNA-transduced HSPCs (T1) from two independent replicate screens ( n = 15 recipient mice per replicate screen). Results from independent replicate screens and statistical analyses are shown in Supplementary Fig. . c In vivo enCRISPRi of Runx1 CREs during hematopoiesis by single locus-based perturbation. Density maps are shown for ATAC-seq and H3K27ac ChIP-seq at the Runx1 locus (chr16:92,579,000–93,050,000; mm9) in bone marrow HSC, GN, Mono, B, CD4+ and CD8+ T cells, respectively. The annotated Runx1 promoters (P1 and P2) and enhancers (E1–E3) are indicated by green and blue shaded lines. Results from independent replicate screens and statistical analyses are shown in Supplementary Fig. . d In vivo enCRISPRi of Runx1 CREs during hematopoiesis by multiplexed perturbation. Results from independent replicate screens and statistical analyses are shown in Supplementary Fig. .

Article Snippet: To generate the inducible dCas9-p300 expression vector, the p300 HAT core domain (p300 core) was PCR amplified from the pcDNA-dCas9-p300-Core vector (Addgene, Plasmid #61357) and cloned into Mlu I/ Bst XI digested pHR-TRE3G-KRAB-dCas9-P2A-mCherry backbone, which was a gift from Luke A. Gilbert .

Techniques: In Vivo, Functional Assay, ChIP-sequencing

Journal: Nature Communications

Article Title: Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing

doi: 10.1038/s41467-020-14362-5

Figure Lengend Snippet: Density maps are shown for ChIP-seq of dCas9, active histone marks (H3K4me1, H3K4me2, and H3K27ac), repressive H3K9me3 and H3K27me3, GATA1, TAL1, and CTCF at the β-globin cluster (chr11: 5,222,500−5,323,700; hg19) in K562 cells coexpressing nontargeting sgGal4 (control or C) or sgHS2 with dCas9-KRAB (K), dCas9-LSD1 (L) or enCRISPRi (LK and KL). Regions showing increased or decreased ChIP-seq signals in enCRISPRi (LK) relative to control, dCas9-KRAB, dCas9-LSD1 or enCRISPRi (KL) (enCRISPRi—C, enCRISPRi—K, enCRISPRi—L, or enCRISPRi—KL) are depicted in green and red, respectively. Blue bars denote the sgRNA-targeted HS2 enhancer. Green bars denote the β-globin genes. Independent replicate experiments are shown as rep1 and rep2 in Supplementary Figs. and , respectively.

Article Snippet: To generate the inducible dCas9-p300 expression vector, the p300 HAT core domain (p300 core) was PCR amplified from the pcDNA-dCas9-p300-Core vector (Addgene, Plasmid #61357) and cloned into Mlu I/ Bst XI digested pHR-TRE3G-KRAB-dCas9-P2A-mCherry backbone, which was a gift from Luke A. Gilbert .

Techniques: ChIP-sequencing

Journal: Nature Communications

Article Title: Analysis of chromatin organization and gene expression in T cells identifies functional genes for rheumatoid arthritis

doi: 10.1038/s41467-020-18180-7

Figure Lengend Snippet: The region around an associated RA variant (rs9603616) on chromosome 13 intronic of the COG6 gene was targeted in the HEK2937T cell line, using p300 as the activator. a Fold change (qPCR) effect on FOXO1 gene expression compared to negative control. b Fold change (qPCR) effect on COG6 gene expression compared to negative control.

Article Snippet: Generation of the dCas9-p300 cell line and delivery of guides : HEK293T cells were first transduced lentivirally with the pLV-dCas9-p300-p2A-PuroR expression vector (Addgene #83889) and were selected with 2 µg mL −1 of puromycin and cultured for a week before being banked as a cell line.

Techniques: Variant Assay, Expressing, Negative Control

Journal: Genome Biology

Article Title: MICMIC: identification of DNA methylation of distal regulatory regions with causal effects on tumorigenesis

doi: 10.1186/s13059-018-1442-0

Figure Lengend Snippet: Validation of causal DNA methylation events in gastric cancer. a Representative results showing the negative regulation of WNT5B by methylation of its distal DRE (cg02935351, 22,595 bp from WNT5B TSS). Box plot shows the high, middle, and low expression groups of WNT5B, plotted against the methylation of the distal DRE in each group. MRA analysis was implemented by the gene set enrichment analysis (GSEA) method. GSEA graphs show tumor-suppressive signatures of WNT5B by MRA. Correlation analysis and MRA for other genes are shown in Additional file : Figure S5. b Confirmation that methylation of the distal DRE is the causal event for WNT5B regulation and cellular malignancy. qPCR results showing increased WNT5B expression in AGS cells treated with 5-AZA or dCas9-TET1, and decreased expression in cells transfected with dCas9-DNMT3A-3 L relative to controls. Cell migration assays showed that dCas9-DNMT3A-3 L targeting increased cell migration, while overexpression (OE) of WNT5B suppressed tumor cell migration. Significance was determined by t-test and error bars represent ± SD. c Bisulfite sequencing validation of increased methylation of the CpGs surrounding the dCas9-DNMT3A-3 L targeted DRE of WNT5B. In the lollipop diagram , black circles stand for methylated Cs and white circles for unmethylated Cs. Each box below corresponds to one CpG position in the genomic sequence. The colored bars summarize the methylation states of all sequences at that position with yellow for methylated Cs and blue for unmethylated Cs. d qPCR results for eight gastric cancer genes after dCas9-DNMT3A-3 L/TET1 epigenetic editing with dCas9-only as control, labelled as ctr1 and ctr2. Three independent replicates were conducted for each experiment. All DRE-target pairs tested here showed strong anti-correlation between expression and methylation, and the qPCR results showed dCas9-TET1 targeting increased gene expression, while dCas9-DNMT3A-3 L targeting inhibited gene expression ( p value < 0.01, student t-test). e Summary of cell migration assay results for eight gastric cancer genes, showing the causal effects of distal DRE methylation on cancer cell malignancy. See photos in Additional file : Figure S9. f The distal DRE region and promoter of each gene of interest were cloned into the pGL3 reporter vector and assayed for luciferase activity. The reporter constructs were also co-transfected with dCas9-DNMT3A-3 L (pro_enh + targeted_DNMT3A-3 L), resulting in decreased luciferase activity

Article Snippet: S3 for sgRNA sequences). pAC1405-pCR8-4xNLS_PUFa_2xNLS and pAC1371-pX-sgRNA-5xPBSa were gifts from Albert Cheng (Addgene plasmid #71888, Addgene #71903). dCas9 expression plasmid was generated by replacement of the cas9 with dCas9 cassette in px330 vector (px330, Addgene plasmid #42230, a gift from Feng Zhang; 3xFLAG-dCas9/pMXs-neo Addgene plasmid #51260, a gift from Hodaka Fujii).

Techniques: Biomarker Discovery, DNA Methylation Assay, Methylation, Expressing, Transfection, Migration, Over Expression, Methylation Sequencing, Sequencing, Control, Gene Expression, Cell Migration Assay, Clone Assay, Plasmid Preparation, Luciferase, Activity Assay, Construct

Journal: Genome Biology

Article Title: MICMIC: identification of DNA methylation of distal regulatory regions with causal effects on tumorigenesis

doi: 10.1186/s13059-018-1442-0

Figure Lengend Snippet: Validation of causal DNA methylation events in liver cancer. a Representative results showing the negative regulation of HDAC11 by methylation of its distal DRE (cg03190578, 3817 bp from HDAC11 TSS). Box plot shows the high, middle, and low expression groups of HDAC11, plotted against the methylation of the distal DRE in each group. GSEA graphs show oncogenic signatures of HDAC11 by MRA. Correlation analysis and MRA for other genes are shown in Additional file : Figure S10. b Confirmation that methylation of the distal DRE is the causal event for HDAC11 regulation and cellular malignancy. qPCR results showing increased HDAC11 expression in PLC8024 cells treated with 5-AZA or dCas9-TET1, and decreased expression in cells transfected with dCas9-DNMT3A-3 L relative to controls. Cell migration assays showed that dCas9-DNMT3A-3 L targeting suppressed cell migration, while overexpression (OE) of HDAC11 increased tumor cell migration. Significance was determined by t-test and error bars represent ± SD. c qPCR results for 11 liver cancer genes after dCas9-DNMT3A-3 L/TET1 epigenetic editing with dCas9-only as control, labelled as ctr1 and ctr2. Three independent replicates were conducted for each experiment. Ten out of 11 DRE-target pairs were predicted to be negatively regulated by DRE methylation, CBFA2T3 was predicted to be positively regulated by methylation of the distal DRE (cg20283771).The qPCR results ( p value < 0.01 by Student’s t-test) were consistent with the predictions (Additional file : Figure S10). d Summary of results for cell migration and proliferation assays for liver cancer genes, showing the causal effects of distal DRE methylation on cancer cell malignancy. See photos in Additional file : Figure S11. e The distal DRE region and promoter of each gene of interest were cloned into the pGL3 reporter vector and assayed for luciferase activity. The reporter constructs were also co-transfected with dCas9-DNMT3A-3 L (pro_enh + targeted_DNMT3A-3 L), resulting in decreased luciferase activity except CBFA2T3 positively correlated with its DRE methylation

Article Snippet: S3 for sgRNA sequences). pAC1405-pCR8-4xNLS_PUFa_2xNLS and pAC1371-pX-sgRNA-5xPBSa were gifts from Albert Cheng (Addgene plasmid #71888, Addgene #71903). dCas9 expression plasmid was generated by replacement of the cas9 with dCas9 cassette in px330 vector (px330, Addgene plasmid #42230, a gift from Feng Zhang; 3xFLAG-dCas9/pMXs-neo Addgene plasmid #51260, a gift from Hodaka Fujii).

Techniques: Biomarker Discovery, DNA Methylation Assay, Methylation, Expressing, Transfection, Migration, Over Expression, Control, Clone Assay, Plasmid Preparation, Luciferase, Activity Assay, Construct