Journal: Aging (Albany NY)

Article Title: Circular RNA RHOT1 promotes progression and inhibits ferroptosis via mir-106a-5p/STAT3 axis in breast cancer

doi: 10.18632/aging.202608

Figure Lengend Snippet: MiR-106a-5p induces ferroptosis by targeting STAT3 in breast cancer cells. ( A ) The interaction of miR-106a-5p and STAT3 3’ UTR was identified by bioinformatic analysis using Targetscan ( http://www.targetscan.org/vert_72/ ). ( B – D ) The MDA-MB-231 and T47D cells were treated with the miR-106a-5p mimic or control mimic. ( B ) The luciferase activities of wild type STAT3 (STAT3 WT) and STAT3 with the miR-106a-5p-binding site mutant (STAT3 MUT) were determined by luciferase reporter gene assays in the cell. ( C ) The mRNA expression of STAT3 was analyzed by qPCR in the cells. ( D ) The protein expression of STAT3 and β-actin was tested by Western blot analysis in the cells. ( E ) The MDA-MB-231 and T47D cells were treated control shRNA, circRHOT1 shRNA, or co-treated with circRHOT1 shRNA and miR-106a-5p inhibitor. The protein expression of STAT3 and β-actin was assessed by Western blot analysis in the cells. ( E , F ) The MDA-MB-231 and T47D cells were treated with 5 mmol/L erastin, co-treated with 5 mmol/L erastin and miR-106a-5p mimic, or o-treated with 5 mmol/L erastin, miR-106a-5p mimic, and pcDNA.1-STAT3. The cell growth was analyzed by MTT assays. ( G – I ) The MDA-MB-231 and T47D cells were treated control shRNA, miR-106a-5p mimic, or co-treated with miR-106a-5p mimic and pcDNA.1-STAT3. ( G ) The levels of iron were analyzed by Iron Assay Kit. ( H ) The levels of ROS were measure by flow cytometry analysis in the cells. ( I ) The expression of GPX4, SLC7A11, and β-actin was measured by Western blot analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circRHOT1 shRNA, the corresponding control shRNA, the pcDNA3.1-circRHOT1 overexpression vector, the pcDNA3.1-STAT3 overexpression vector, miR-106a-5p mimic, miR-106a-5p inhibitor, and corresponding control were synthesized and purchased (GenePharma, China) (GenScript, China).

Techniques: Luciferase, Binding Assay, Mutagenesis, Expressing, Western Blot, shRNA, Iron Assay, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: Circular RNA RHOT1 promotes progression and inhibits ferroptosis via mir-106a-5p/STAT3 axis in breast cancer

doi: 10.18632/aging.202608

Figure Lengend Snippet: CircRHOT1 contributes to breast cancer progression by miR-106a-5p/STAT3 axis. ( A – D ) The MDA-MB-231 and T47D cells were treated control shRNA, circRHOT1 shRNA, or co-treated with circRHOT1 shRNA and miR-106a-5p inhibitor or pcDNA.1-STAT3. ( A , B ) The cell viability was measured by MTT assays in the cells. ( C , D ) The cell apoptosis was measure by flow cytometry analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circRHOT1 shRNA, the corresponding control shRNA, the pcDNA3.1-circRHOT1 overexpression vector, the pcDNA3.1-STAT3 overexpression vector, miR-106a-5p mimic, miR-106a-5p inhibitor, and corresponding control were synthesized and purchased (GenePharma, China) (GenScript, China).

Techniques: shRNA, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: Circular RNA RHOT1 promotes progression and inhibits ferroptosis via mir-106a-5p/STAT3 axis in breast cancer

doi: 10.18632/aging.202608

Figure Lengend Snippet: CircRHOT1 promotes the tumor growth of breast cancer in vivo . ( A – E ) The effect of circRHOT1 on tumor growth of breast cancer cells in vivo was analyzed by nude mice tumorigenicity assay by injected with the MDA-MB-231 cells treated with control shRNA or circRHOT1 shRNA. ( A ) Representative images of dissected tumors from nude mice were presented. ( B ) The average tumor volume was calculated and shown. ( C ) The average tumor weight was calculated and shown. ( D ) The expression levels of miR-106a-5p were measured by qPCR in the tumor tissues of the mice. ( E ) The protein expression of STAT3 and β-actin was assessed by Western blot analysis in the tumor tissues of the mice. N = 5. Data are presented as mean ± SD. Statistic significant differences were indicated: ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circRHOT1 shRNA, the corresponding control shRNA, the pcDNA3.1-circRHOT1 overexpression vector, the pcDNA3.1-STAT3 overexpression vector, miR-106a-5p mimic, miR-106a-5p inhibitor, and corresponding control were synthesized and purchased (GenePharma, China) (GenScript, China).

Techniques: In Vivo, Tumorigenicity Assay, Injection, shRNA, Expressing, Western Blot

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH inhibits the proliferation and induces apoptosis of NP cells. ( A ) The expression levels of circITCH were measured by qPCR in the NP tissues of IDD patients (n=90) and normal cases (n=90). ( B – F ) The NP cells were infected with lentiviral plasmids carrying circITCH shRNA or corresponding control shRNA or transfected with the pcDNA3.1 or the pcDNA3.1-circITCH overexpression vector. ( B ) The expression levels of circITCH were examined by qPCR in the cells. ( C , D ) The cell proliferation was analyzed by CCK-8 assays in the cells. ( E ) Cell apoptosis was tested by flow cytometry analysis in the cells. ( F ) The expression of Bax, caspase3, cleaved caspase3 (c-caspase3), caspase9, and cleaved caspase9 (c-caspase9) was measured by Western blot analysis. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Expressing, Infection, shRNA, Transfection, Over Expression, Plasmid Preparation, CCK-8 Assay, Flow Cytometry, Western Blot

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH promotes ECM degradation of degenerative NP cells. ( A – E ) The NP cells were infected with lentiviral plasmids carrying circITCH shRNA or corresponding control shRNA or transfected with the pcDNA3.1 or the pcDNA3.1-circITCH overexpression vector. ( A – D ) The mRNA expression of collagen II ( A ), aggrecan ( B ), MMP13 ( C ), and ADAMTS4 ( D ) was measured by qPCR in the cells. ( E ) The protein expression of collagen II, aggrecan, MMP13, ADAMTS4, and β-actin was tested by Western blot analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Infection, shRNA, Transfection, Over Expression, Plasmid Preparation, Expressing, Western Blot

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH serves as a miR-17-5p sponge in NP cells. ( A ) The potential interaction between circITCH and miR-17-5p was identified by the bioinformatic analysis using ENCORI ( http://starbase.sysu.edu.cn/index.php ). ( B , C ) The NP cells were treated with the miR-17-5p mimic or control mimic. ( B ) The expression levels of miR-17-5p were measured by qPCR in the cells. ( C ) The luciferase activities of wild type circITCH (circITCH WT) and circITCH with the miR-17-5p-binding site mutant (circITCH MUT) were determined by luciferase reporter gene assays in the cells. ( D ) The NP cells were infected with lentiviral plasmids carrying circITCH shRNA or corresponding control shRNA or transfected with the pcDNA3.1 or the pcDNA3.1-circITCH overexpression vector. The expression of miR-17-5p was analyzed by qPCR in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Expressing, Luciferase, Binding Assay, Mutagenesis, Infection, shRNA, Transfection, Over Expression, Plasmid Preparation

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: MiR-17-5p targets SOX4 in NP cells. ( A ) The interaction of miR-17-5p and SOX4 3’ UTR was identified by bioinformatic analysis using Targetscan ( http://www.targetscan.org/vert_72/ ). ( B – E ) The NP cells were treated with the miR-17-5p mimic or control mimic. ( B ) The expression levels of miR-17-5p were measured by qPCR in the cells. ( C ) The luciferase activities of wild type SOX4 (SOX4 WT) and SOX4 with the miR-17-5p-binding site mutant (SOX4 MUT) were determined by luciferase reporter gene assays in the cell. ( D ) The mRNA expression of SOX4 was analyzed by qPCR in the cells. ( E ) The protein expression of SOX4 and β-actin was tested by Western blot analysis in the cells. ( F ) The protein expression of collagen II, aggrecan, MMP13, ADAMTS4, and β-actin was analyzed by Western blot analysis in the NP cells treated with control mimic, miR-17-5p mimic, or co-treated with miR-17-5p mimic and pcDNA3.1-SOX4 overexpression vector. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Expressing, Luciferase, Binding Assay, Mutagenesis, Western Blot, Over Expression, Plasmid Preparation

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH activates Wnt/β-catenin signaling by targeting miR-17-5p/SOX4 axis. ( A ) The NP cells were transfected with control shRNA, lentiviral plasmids carrying circITCH shRNA, or co-treated with lentiviral plasmids carrying circITCH shRNA and miR-17-5p inhibitor. The protein expression of SOX4 and β-actin was tested by Western blot analysis in the cells. ( B , C ) The NP cells were transfected with control shRNA, lentiviral plasmids carrying circITCH shRNA, or co-treated with lentiviral plasmids carrying circITCH shRNA and pcDNA3.1-SOX4 overexpression vector, lentiviral plasmids carrying circITCH shRNA, pcDNA3.1-SOX4 overexpression vector, and miR-17-5p mimic, or lentiviral plasmids carrying circITCH shRNA and LiCl. The expression of Wnt1, β-catenin, c-Myc, Cyclin D1, and β-actin was analyzed by Western blot analysis in the cells. The results of Western blot analysis were quantified by ImageJ software. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Transfection, shRNA, Expressing, Western Blot, Over Expression, Plasmid Preparation, Software

Journal: Aging (Albany NY)

Article Title: Circular RNA ITCH promotes extracellular matrix degradation via activating Wnt/β-catenin signaling in intervertebral disc degeneration

doi: 10.18632/aging.203036

Figure Lengend Snippet: CircITCH contributes to ECM degradation of degenerative NP cells by modulating miR-17-5p/SOX4/Wnt/β-catenin signaling. ( A – C ) The NP cells were transfected with control shRNA, lentiviral plasmids carrying circITCH shRNA, or co-treated with lentiviral plasmids carrying circITCH shRNA and pcDNA3.1-SOX4 overexpression vector, miR-17-5p inhibitor, or LiCl. ( A ) The cell proliferation was analyzed by CCK-8 assays in the cells. ( B ) Cell apoptosis was tested by flow cytometry analysis in the cells. ( C ) The protein expression of collagen II, aggrecan, MMP13, ADAMTS4, and β-actin was analyzed by Western blot analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: * P < 0.05, ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circITCH shRNA, the corresponding control shRNA, the pcDNA3.1-circITCH overexpression vector, the lentiviral plasmids carrying SOX4 shRNA, the corresponding control shRNA, the pcDNA3.1-SOX4 overexpression vector, miR-17-5p mimic and inhibitor were obtained (GenePharma, China) (GenScript, China).

Techniques: Transfection, shRNA, Over Expression, Plasmid Preparation, CCK-8 Assay, Flow Cytometry, Expressing, Western Blot

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