Journal: Cell Genomics

Article Title: Activation of the imprinted Prader-Willi syndrome locus by CRISPR-based epigenome editing

doi: 10.1016/j.xgen.2025.100770

Figure Lengend Snippet: High-throughput screens reveal regulatory elements of maternal and paternal SNRPN alleles (A) Schematic of the PWS locus on chr15 with common PWS deletions and the PWS gRNA library. Each thin vertical line represents an sgRNA. Genes colored blue are maternally imprinted, those that are pink are paternally imprinted, and those that are gray are not imprinted. (B) Summary of the PWS gRNA library. (C) Schematic of experimental protocol for CRISPRa/CRISPRi screens. (D) CRISPR screen results (magnified, see Figure S1 E) displayed as −log 10 ( p adj ), where p adj is the multiple-hypothesis-corrected p value from DESeq2. Notable regions are highlighted in red. Note that genes SNORD107 and SNORD64 in the schematic are intended to help orient the reader, and due to the genes’ small size, locations are approximate and not drawn to scale. (E) qPCR of SNRPN-GFP for validations of individual gRNAs of the pat SNRPN-2A-GFP CRISPRi dCas9 KRAB screen with either dCas9 KRAB or dCas9 only (no effector) to control for steric hindrance. Fold-change values normalized to NT gRNA within either dCas9 KRAB - or dCas9-only conditions. (F) qPCR of SNRPN-GFP from individual or pooled gRNA validations of selected gRNAs in the mat1 and mat2 regions. (G) Summary of the PWS gRNA sub-library. (H) qPCR of SNRPN-GFP in mat SNRPN-GFP iPSCs with Tet1c dCas9 14 days after transduction with the indicated gRNA. For qPCR in (E), (F), and (H), fold change values are plotted as mean ± SD, but statistics were calculated on ΔΔCt values (normalized to GAPDH and empty or NT vector sample); for (E), two-way ANOVA followed by Tukey's multiple comprisons test vs. NT; for (F) and (G), one-way ANOVA, followed by Dunnett’s test vs. empty vector. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001 relative to NT/empty vector. Unmarked comparisons are not significant.

Article Snippet: Plasmid containing Tet1c coding sequence , Addgene , #108245.

Techniques: High Throughput Screening Assay, CRISPR, Control, Transduction, Plasmid Preparation

Journal: Cell Genomics

Article Title: Activation of the imprinted Prader-Willi syndrome locus by CRISPR-based epigenome editing

doi: 10.1016/j.xgen.2025.100770

Figure Lengend Snippet: Tet1c and VP64 activate maternally imprinted PWS genes in ΔPWS iPSCs (A) Schematic of chr15 in isogenic wild-type (WT) and PWS type II deletion (ΔPWS) iPSCs. (B–D) (B) qPCR of SNRPN in WT or ΔPWS iPSCs with (C) VP64 dCas9 VP64 and (D) Tet1v4 dCas9 14 days after transduction with the indicated gRNA. For both qPCR plots, fold change values are plotted as mean ± SD, but statistics were calculated on ΔΔCt values (normalized to GAPDH and WT ctrl sample); 1-way ANOVA, followed by Dunnett’s test vs. ΔPWS NT gRNA ∗∗∗∗ p < 0.0001. (D) Differential expression analysis of total RNA sequencing of VP64 dCas9 VP64 ΔPWS iPSCs, comparing mat1 g3 to NT gRNA. (E) Differential expression analysis of total RNA sequencing of Tet1v4 dCas9 ΔPWS iPSCs, comparing mat3 g5 to NT gRNA. (F and G) HCR FlowFISH assessing SNRPN (transcript variant 1) signal in (F) VP64 dCas9 VP64 and (G) Tet1v4 dCas9 iPSCs (WT or ΔPWS) with the indicated gRNA. SNRPN (transcript variant 1) signal on X axis, with TBP as a control for cell size and staining. (H) HCR FlowFISH assessing SNHG14 signal in VP64 dCas9 VP64 iPSCs (WT or ΔPWS) with the indicated gRNA. (I and J) Targeted bisulfite sequencing of WT and ΔPWS iPSCs with (I) VP64 dCas9 VP64 and (J) Tet1v4 dCas9 covering 24 CpG sites within the PWS locus (hg19 chr15: 25200353–25200693), 2 weeks post-transduction. Data for (I) and (J) are shown as the range of the data, with the plotted point being the median; n = 3 replicates. (K) Read-level methylation analysis showing number of methylated cytosines in a CpG context per read (containing a total of 24 CpGs) in each of the indicated conditions in WT or ΔPWS iPSCs expressing Tet1v4 dCas9.

Article Snippet: Plasmid containing Tet1c coding sequence , Addgene , #108245.

Techniques: Transduction, Quantitative Proteomics, RNA Sequencing, Variant Assay, Control, Staining, Methylation Sequencing, Methylation, Expressing

Journal: Cell Genomics

Article Title: Activation of the imprinted Prader-Willi syndrome locus by CRISPR-based epigenome editing

doi: 10.1016/j.xgen.2025.100770

Figure Lengend Snippet: Tet1c and VP64 alter chromatin accessibility and/or DNA methylation at the PWS locus (A) Browser tracks of ATAC-seq (reads per kilobase per million mapped reads [RPKM]-normalized BigWig) of WT and ΔPWS iPSCs with VP64 dCas9 VP64 and NT or mat1 g3 gRNA. (B) Quantification of ATAC-seq reads (counts per million [CPM]) at the peak at the mat1 g3 binding site (dashed line in A). ∗∗∗ p < 0.001, 1-way ANOVA followed by Tukey’s test. (C) H3K4me3 CUT&RUN (CPM-normalized BigWig) of WT and ΔPWS iPSCs with VP64 dCas9 VP64 and NT or mat1 g3 gRNA, magnified and shown in full in Figure S4 C. n = 2 replicates as shown. (D) Quantification of CUT&RUN reads (CPM) shown in (C) at the annotated peak adjacent to the mat1 g3 binding site. (E) Browser tracks of ATAC-seq (RPKM-normalized BigWig) of WT and ΔPWS iPSCs with Tet1v4 dCas9 and NT or mat3 g5 gRNA. n = 2 or 3 replicates as shown. (F) Quantification of ATAC-seq reads (CPM) at each of 2 peaks within the PWS-IC. ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; Tukey’s test following 2-way ANOVA. N = 2 or 3 as shown in Figure 3 E. (G) H3K4me3 CUT&RUN (CPM-normalized BigWig) of WT and ΔPWS iPSCs with Tet1v4 dCas9 and NT or mat3 g5 gRNA, magnified at PWS-IC and shown in full in Figure S4 E. n = 2 replicates as shown in figure. (H) Quantification of CUT&RUN reads (CPM) shown in (G) at the annotated peak at the PWS-IC.

Article Snippet: Plasmid containing Tet1c coding sequence , Addgene , #108245.

Techniques: DNA Methylation Assay, Binding Assay

Journal: Cell Genomics

Article Title: Activation of the imprinted Prader-Willi syndrome locus by CRISPR-based epigenome editing

doi: 10.1016/j.xgen.2025.100770

Figure Lengend Snippet:

Article Snippet: Plasmid containing Tet1c coding sequence , Addgene , #108245.

Techniques: DNA Methylation Assay, Sequencing, Recombinant, Plasmid Preparation, Expressing, Software

Journal: Nucleic Acids Research

Article Title: Artificial escape from XCI by DNA methylation editing of the CDKL5 gene

doi: 10.1093/nar/gkz1214

Figure Lengend Snippet: Programmable transcription of the CDKL5 gene. ( A ) UCSC genome browser snapshot of the target sites of the six sgRNAs directed against the CDKL5 promoter on Xp22.13. DNase hypersensitive sites and H3K4me3, often found near promoters are derived from ENCODE. Sense sgRNAs are shown in blue, antisense sgRNAs in red. ( B ) CDKL5 mRNA fold change relative to mock-treated cells in U87MG cells determined by RT-qPCR resulting from programmable transcription using a dCas9-no effector (dC) or dCas9-VP64 (dC-V) in combination with different pools of three to six sgRNAs targeted to the CDKL5 promoter 48 h after transient transfection. # Significantly different from dCas9 sgRNAs 1–3, n = 3 independent experiments, Tukey's HSD, P < 0.05. ( C ) CDKL5 mRNA fold change relative to mock-treated cells in BE2C determined by RT-qPCR resulting from programmable transcription using dCas9-no effector or dCas9-VP64 co-expressed with sgRNAs 1–3 48 h after transient transfection. ( D ) CDKL5 mRNA fold change relative to mock-treated cells in Lenti-X 293T determined by RT-qPCR resulting from programmable transcription using dCas9-no effector or dCas9-VP64 co-expressed with sgRNAs 1–3 48 h after transient transfection. # Significantly different from dCas9 sgRNAs 1–3, n = 3 independent experiments, Student's t -test P <0.05.

Article Snippet: Transfections were performed in 12-well plates using either a mock-treatment (diluted transfection reagent) or 700 ng dCas9 expression vector (Fuw-dCas9-Tet1CD-P2A-BFP, Addgene plasmid #108245; Fuw-dCas9-Tet1CD_IM, Addgene plasmid # 84479; pLV hUbC-dCas9-T2A-GFP, Addgene plasmid # 53191; pLV hUbC-dCas9 VP64-T2A-GFP, Addgene plasmid # 53192) and 300 ng of equimolar pooled sgRNA expression vectors.

Techniques: Derivative Assay, Quantitative RT-PCR, Transfection

Journal: Nucleic Acids Research

Article Title: Artificial escape from XCI by DNA methylation editing of the CDKL5 gene

doi: 10.1093/nar/gkz1214

Figure Lengend Snippet: Targeted reactivation of CDKL5 from the inactive X allele. ( A ) Allele specific read counts for the mRNA expression of the active (Xa) or inactive (Xi) CDKL5 allele of mock-treated SH-SY5Y or after constitutive expression of dCas9 effector domains dCas9 (dC), dCas9-VP64 (dC-V), dCas9-TET1CD (dC-T) or a combination of dCas9-VP64 and dCas9-TET1CD (dC-V+dC-T) and sgRNAs 1–3 after 21 days post-transduction. # Significantly different from mock-treated, ‡ significantly different from dCas9, n = 3 independent experiments, Tukey's HSD, P < 0.05. ( B ) Relative Xi CDKL5 mRNA expression of mock-treated or stably transduced SH-SY5Y relative to CDKL5 Xa mRNA expression of mock-treated cells as determined by allele-specific RT-qPCR after 21 days post-transduction. # Significantly different from dC, ‡ significantly different from dC-V, † significantly different from dC-T, n = 3 independent experiments, Tukey's HSD, P < 0.05 ( C ) Relative Xa CDKL5 mRNA expression in mock-treated and stably transduced SH-SY5Y cells determined by allele-specific RT-qPCR after 21 days post-transduction. # Significantly different from mock-treated, ‡ significantly different from dCas9, n = 3 independent experiments, Tukey's HSD, all P < 0.05.

Article Snippet: Transfections were performed in 12-well plates using either a mock-treatment (diluted transfection reagent) or 700 ng dCas9 expression vector (Fuw-dCas9-Tet1CD-P2A-BFP, Addgene plasmid #108245; Fuw-dCas9-Tet1CD_IM, Addgene plasmid # 84479; pLV hUbC-dCas9-T2A-GFP, Addgene plasmid # 53191; pLV hUbC-dCas9 VP64-T2A-GFP, Addgene plasmid # 53192) and 300 ng of equimolar pooled sgRNA expression vectors.

Techniques: Expressing, Transduction, Stable Transfection, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Artificial escape from XCI by DNA methylation editing of the CDKL5 gene

doi: 10.1093/nar/gkz1214

Figure Lengend Snippet: dCas9-TET1CD causes removal of DNA methylation from the CDKL5 CGI promoter. ( A ) UCSC genome browser snapshot of the target sites of sgRNAs 1–3 directed against the CDKL5 promoter on Xp22.13 and a large CpG Island (>1 kb) spanning the transcriptional start site of CDKL5 . The black box represents a >200 bp region assessed for targeted DNA methylation changes containing 24 individual CpG dinucleotides (drawn to scale). ( B ) 5-methylcytosine levels in a CpG context (5meCG) over total CpG context as assessed by targeted bisulfite sequencing across 11 CpG dinucleotides in mock-treated cells or cells transduced to constitutively express dCas9-no effector (dC) or dCas9 fused to either VP64 (dC-V) or TET1CD (dC-T), a combination thereof (dC-V+dC-T) or a catalytically inactive TET1CD (dC-dT). X-axis depicts the individual CpG position relative to the amplicon (not drawn to scale). ( C ) Mean 5-methylcytosine levels in a CpG context over all 11 CpG dinucleotides in all treatment groups. # Significantly different from mock-treated cells, ‡ significantly different from dCas9, † significantly different from dC-dT, ¥ significantly different from dC-T, n = 3 independent experiments, Tukey's HSD, all P < 0.05.

Article Snippet: Transfections were performed in 12-well plates using either a mock-treatment (diluted transfection reagent) or 700 ng dCas9 expression vector (Fuw-dCas9-Tet1CD-P2A-BFP, Addgene plasmid #108245; Fuw-dCas9-Tet1CD_IM, Addgene plasmid # 84479; pLV hUbC-dCas9-T2A-GFP, Addgene plasmid # 53191; pLV hUbC-dCas9 VP64-T2A-GFP, Addgene plasmid # 53192) and 300 ng of equimolar pooled sgRNA expression vectors.

Techniques: DNA Methylation Assay, Methylation Sequencing, Amplification

Journal: Nucleic Acids Research

Article Title: Artificial escape from XCI by DNA methylation editing of the CDKL5 gene

doi: 10.1093/nar/gkz1214

Figure Lengend Snippet: Depletion of the XCI hallmark histone modification H3K27me3. ( A ) UCSC genome browser snapshot of the target sites of sgRNAs 1–3 directed against the CDKL5 promoter on Xp22.13 and H3K27me3 peaks derived from ENCODE. Black boxes show the regions assessed by ChIP-qPCR. ( B ) Input normalized H3K27me enrichment levels determined by ChIP-qPCR in region A of the CDKL5 promoter in mock-treated cells or cells transduced to constitutively express dCas9-no effector (dC) or dCas9 fused to either VP64 (dC-V) or TET1CD (dC-T). ( C ) Input normalized H3K27me enrichment levels determined by ChIP-qPCR in region B of the CDKL5 promoter. ( D ) Input normalized H3K27me enrichment levels determined by ChIP-qPCR in region C of the CDKL5 promoter. ( E ) Input normalized H3K27me enrichment levels determined by ChIP-qPCR in the promoter of the nearest neighboring gene promoter, SCML2 . ( F ) Input normalized H3K27me enrichment levels determined by ChIP-qPCR in the promoter of a distal gene, MECP2 , that serves as a negative control. # Significantly different from mock-treated cells, n = 3 independent experiments, P < 0.05.

Article Snippet: Transfections were performed in 12-well plates using either a mock-treatment (diluted transfection reagent) or 700 ng dCas9 expression vector (Fuw-dCas9-Tet1CD-P2A-BFP, Addgene plasmid #108245; Fuw-dCas9-Tet1CD_IM, Addgene plasmid # 84479; pLV hUbC-dCas9-T2A-GFP, Addgene plasmid # 53191; pLV hUbC-dCas9 VP64-T2A-GFP, Addgene plasmid # 53192) and 300 ng of equimolar pooled sgRNA expression vectors.

Techniques: Modification, Derivative Assay, ChIP-qPCR, Negative Control

Journal: Nucleic Acids Research

Article Title: Artificial escape from XCI by DNA methylation editing of the CDKL5 gene

doi: 10.1093/nar/gkz1214

Figure Lengend Snippet: Global DNA hypomethylation due to constitutive dCas9-TET1CD expression. ( A ) Thirty-two CpG positions shown with their respective location on the X-chromosome (hg19) from the 850K MethylationEPIC array across the CDKL5 promoter were used to assess gene-wide changes in DNA methylation levels represented as changes in the beta value of the TSS200, TSS1500, 5′UTR and gene body of CDKL5 . After transduction with dCas9-no effector (dC), dCas9-TET1CD (dC-T) and a catalytically inactive TET1CD (dC-dT), we found reduced DNA methylation levels in the TSS1500 and TSS200 region of cells transduced with dCas9-TET1CD. The red line demonstrates the sgRNA binding sites in the CDKL5 promoter. *Significantly differentially methylated positions for further assessment. ( B ) Side-by-side assessment of significantly differentially methylated positions in the CDKL5 promoter with a mean difference in beta value of <0.05. # Significantly different from dC, † significantly different from dC-dT, n = 2 independent experiments, FDR < 5%. ( C ) Histogram of the number of genes by the number of significantly hypomethylated sites of dCas9-TET1CD transduced cells when compared to dCas9 or a catalytically inactive TET1 fused to dCas9 demonstrates that the majority of genes shows only a single probe falling within the respective promoter region. ( D ) Side-by-side assessment of significantly differentially methylated positions in the COL9A3 promoter with a mean difference in beta value of <0.05. # Significantly different from dC, † significantly different from dC-dT, n = 2 independent experiments, FDR < 5%. ( E ) Venn diagram of shared genes between dCas9-TET1CD comparisons with dCas9 or a catalytically inactive TET1CD mutant shows an overlap of 48 genes between the two groups. ( F ) A flow chart diagram representing the analysis pipeline for genome-wide methylation effects of dCas9-TET1CD, starting from a total number of probes, down to significantly differentially methylated sites and ultimately differentially methylated genes.

Article Snippet: Transfections were performed in 12-well plates using either a mock-treatment (diluted transfection reagent) or 700 ng dCas9 expression vector (Fuw-dCas9-Tet1CD-P2A-BFP, Addgene plasmid #108245; Fuw-dCas9-Tet1CD_IM, Addgene plasmid # 84479; pLV hUbC-dCas9-T2A-GFP, Addgene plasmid # 53191; pLV hUbC-dCas9 VP64-T2A-GFP, Addgene plasmid # 53192) and 300 ng of equimolar pooled sgRNA expression vectors.

Techniques: Expressing, DNA Methylation Assay, Transduction, Binding Assay, Methylation, Mutagenesis, Genome Wide

Journal: Nucleic Acids Research

Article Title: Artificial escape from XCI by DNA methylation editing of the CDKL5 gene

doi: 10.1093/nar/gkz1214

Figure Lengend Snippet: Off-target analysis of CRISPR/dCas9 effectors by RNA-seq. ( A ) Volcano plot of significance (FDR adjusted P value) versus fold change for differential DESeq2 expression analysis of mock-treated, dCas9-VP64 (dC-V), dCas9-TET1CD (dC-T) or dCas9-VP64 and dCas9-TET1CD (dC-V+dC-T) guided by sgRNAs 1–3 to the CDKL5 promoter compared to a dCas9-no effector control (dC). Differentially expressed genes are highlighted in red (FDR < 1%, log fold change >1), predicted CRISPR off-target sites are highlighted in blue and the CDKL5 target gene is highlighted in green. The number of downregulated genes is found in the upper left of each panel, the number of upregulated genes is found in the upper right of each panel. ( B ) Venn diagram showing the overlap of differentially expressed genes between all conditions and the putative off-target list. A single gene, CNTNAP2 is shared between all four groups as a putative off-target. ( C ) Venn diagram showing the overlap between differentially expressed genes and differentially methylated positions identified in a comparison between dCas9-TET1CD and dCas9 and potential CRISPR off-targets.

Article Snippet: Transfections were performed in 12-well plates using either a mock-treatment (diluted transfection reagent) or 700 ng dCas9 expression vector (Fuw-dCas9-Tet1CD-P2A-BFP, Addgene plasmid #108245; Fuw-dCas9-Tet1CD_IM, Addgene plasmid # 84479; pLV hUbC-dCas9-T2A-GFP, Addgene plasmid # 53191; pLV hUbC-dCas9 VP64-T2A-GFP, Addgene plasmid # 53192) and 300 ng of equimolar pooled sgRNA expression vectors.

Techniques: CRISPR, RNA Sequencing, Expressing, Control, Methylation, Comparison

Journal: Nature Communications

Article Title: Epigenetic interaction between UTX and DNMT1 regulates diet-induced myogenic remodeling in brown fat

doi: 10.1038/s41467-021-27141-7

Figure Lengend Snippet: A Pyrosequencing analysis of DNA methylation at Myod1 promoter in BAT1 adipocytes transfected with lentiviral vectors expressing dCas9-TET1CD along with lentiviral vectors expressing either Myod1 -targeting gRNA or scramble non-targeting gRNA ( n = 4/group). *indicates statistical significance between the two groups by two-tailed unpaired Student’s t -test. B Quantitative PCR analysis of Myod1 and BAT-specific gene expression in BAT1 adipocytes transfected with lentiviral vectors expressing dCas9-TET1CD along with lentiviral vectors expressing either Myod1 -targeting gRNA or scramble non-targeting gRNA ( n = 4/group). *indicates statistical significance between the two groups by two-tailed unpaired Student’s t -test. For ( A , B ), 4-day differentiated BAT brown adipocytes were transfected with lentiviral vectors FUW-dCas9-TET1CD along with lentiviral vectors pgRNA-mCherry encoding either scramble-gRNA or Myod1-targeting gRNA using Amaxa Nucleofector II Electroporator with an Amaxa cell line nucleofector kit L. Cells were harvested 2 days after for pyrosequencing ( A ) or gene expression ( B ) analysis. C , D Body weight ( C ) and Body composition ( D ) in mice with iBAT injection of lentiviruses expressing dCas9-TET1CD plus lentiviruses expressing either targeting Myod1-gRNA-mCherry or non-targeting scramble-gRNA-mCherry ( n = 4/group). *indicates statistical significance between the two groups by two-tailed unpaired Student’s t -test. E – G Methylation levels at Myod1 promoter ( E , n = 8/group), Myogenic marker gene expression (F, n = 7 for dCas9 + scramble, and 5 for dCas9 + Myod1 gRNA), and BAT-specific gene expression ( G , n = 7 for dCas9+scramble, and 6 for dCas9 + Myod1 gRNA) in iBAT of mice with iBAT injection of lentiviruses expressing dCas9-TET1CD plus lentiviruses expressing either targeting Myod1-gRNA-mCherry or non-targeting scramble-gRNA-mCherry. *Indicates statistical significance between the two groups by Mann–Whitney’s nonparametric U test in ( E ), ( F ) and ( G ). ( H ) Representative IHC staining of UCP1 (upper panel) and MyHC (lower panel) in iBAT of mice with iBAT injection of lentiviruses expressing dCas9-TET1CD plus lentiviruses expressing either targeting Myod1-gRNA-mCherry or non-targeting scramble-gRNA-mCherry ( n = 3 replicates). For ( C – H ), 3-month-old chow-fed male C57BL/6J mice were bilaterally injected with lentiviruses expressing dCas9-TET1CD plus lentiviruses expressing either targeting Myod1-gRNA-mCherry or non-targeting scramble-gRNA-mCherry into iBAT for up to 2 months. All data are expressed as mean ± SEM.

Article Snippet: For ( A , B ), 4-day differentiated BAT brown adipocytes were transfected with lentiviral vectors FUW-dCas9-TET1CD along with lentiviral vectors pgRNA-mCherry encoding either scramble-gRNA or Myod1-targeting gRNA using Amaxa Nucleofector II Electroporator with an Amaxa cell line nucleofector kit L. Cells were harvested 2 days after for pyrosequencing ( A ) or gene expression ( B ) analysis.

Techniques: DNA Methylation Assay, Transfection, Expressing, Two Tailed Test, Real-time Polymerase Chain Reaction, Injection, Methylation, Marker, Immunohistochemistry

Journal: Clinical Epigenetics

Article Title: Epigenetic reactivation of tumor suppressor genes with CRISPRa technologies as precision therapy for hepatocellular carcinoma

doi: 10.1186/s13148-023-01482-0

Figure Lengend Snippet: Upregulation of tumor suppressor genes by CRISPRa in Hep3B HCC cells. A Schematic representation of CRISPRa consisting of Sp dCas9 C-terminally fused to the tripartite transactivator VPR (VP64, p65, and Rta) and coupled with the gRNA-MS2-MCP system that recruits the bipartite transactivator p65-HSF1 for targeted epigenetic editing. As indicated for each tumor suppressor gene (TSG), gRNAs, designated as G1, G2, G3, and G4, direct CRISPRa to the forward (right arrow) or reverse (left arrow) DNA strand within the regulatory region and proximal promoter of the TSG. gRNA numbering (±) refers to the distance in base pairs from the transcription start site (TSS) of each targeted TSG. B – H Reactivation of TSGs by CRISPRa was evaluated by qRT-PCR 48 h after transient transfection. Fold change in TSG mRNA expression from transfected cells with CRISPRa and TSG-targeting gRNAs, and normal hepatocytes was normalized to control transfections with empty vector (EV) and compared to CRISPRa with no gRNA (NO G) for statistical analysis. From left to right: B HHIP : * P = 0.0390, **** P < 0.0001; C MT1M : *** P = 0.0004, **** P < 0.0001; D PZP : *** P = 0.0006, **** P < 0.0001, *** P = 0.0002, * P = 0.0359, ** P = 0.0013, **** P < 0.0001; E TTC36 : ** P = 0.0013, ** P = 0.0022, *** P = 0.0001, *** P = 0.0004, **** P < 0.0001; F MT1E : * P = 0.0349, **** P < 0.0001, *** P = 0.0002; G miR-122-5p : ** P = 0.0014, * P = 0.0119, **** P < 0.0001; H PTGR1 : *** P = 0.0001, **** P < 0.0001. Data presented as means ± SEM ( n = 3), and P values were determined by unpaired t- test. Sp dCas9 Streptococcus pyogenes deactivated Cas9 protein adopted for epigenome engineering, MS2 RNA aptamer, MCP MS2 coat protein, HSF1 heat shock factor 1, Chr chromosome, (+) forward DNA strand, (−) reverse DNA strand, and MIX 4G combination of all four gRNAs targeting a TSG

Article Snippet: The pLV hU6-sgRNA hUbC-dCas9-TET1-CD-T2A-Puro lentiviral vector was generated from the pLV hU6-sgRNA hUbC-dCas9-KRAB-T2A-Puro (Addgene plasmid # 71236, a gift from Charles Gersbach) by replacing the KRAB domain with the catalytic domain of TET-1 from Fuw-dCas9-Tet1CD (Addgene plasmid # 84475, a gift from Rudolf Jaenisch).

Techniques: Quantitative RT-PCR, Transfection, Expressing, Plasmid Preparation

Journal: Clinical Epigenetics

Article Title: Epigenetic reactivation of tumor suppressor genes with CRISPRa technologies as precision therapy for hepatocellular carcinoma

doi: 10.1186/s13148-023-01482-0

Figure Lengend Snippet: Maximizing reactivation of highly downregulated tumor suppressor genes in Hep3B and HuH-7 HCC cells. A Schematic representation depicting the CRISPRa toolbox developed for epigenetic editing. Sp dCas9 C-terminally fused to VPR, i.e., Sp dCas9-VPR ( dark blue ); gRNA-MS2-MCP-p65-HSF1 recruiting system ( light blue ); Sp dCas9 C-terminally fused to TET1 catalytic domain, i.e., Sp dCas9-TET1-CD ( dark pink ); gRNA-MS2-MCP-TET1-CD recruiting system ( light pink ); and Sp dCas9 ( yellow ). B – H Fold change in MT1M ( B ), HHIP ( C , D ), PZP ( E , F ), and TTC36 ( G , H ) mRNA expression evaluated by qRT-PCR 96 h after transient transfection in Hep3B and HuH-7 cells. Cells were transfected with combinations of CRISPRa along with the most potent tumor suppressor gene-targeting gRNA/s, or with no gRNA (NO G) as control. Relative gene expression was normalized and compared to cells transfected with empty vector control (EV) for statistical analysis. From left to right: B MT1M : **** P < 0.0001, *** P = 0.0002; C HHIP : **** P < 0.0001, *** P = 0.0008; D HHIP : **** P < 0.0001, *** P = 0.0002; E PZP : *** P = 0.0002, **** P < 0.0001, ** P = 0.0022, *** P = 0.0003, *** P = 0.0001; F PZP : **** P < 0.0001; G TTC36 : **** P < 0.0001, * P = 0.0190, ** P = 0.0030; H TTC36 : **** P < 0.0001, ** P = 0.0088, *** P = 0.0007. Data presented as means ± SEM ( n = 3), and P values were determined by unpaired t- test. Sp dCas9 Streptococcus pyogenes deactivated Cas9 protein adopted for epigenome engineering, VPR VP64, p65, Rta, MS2 RNA aptamer, MCP MS2-coat protein, HSF1 heat shock factor 1, TET1-CD Ten-Eleven Translocation methylcytosine dioxygenase 1-catalytic domain, MIX 4G combination of four gRNAs

Article Snippet: The pLV hU6-sgRNA hUbC-dCas9-TET1-CD-T2A-Puro lentiviral vector was generated from the pLV hU6-sgRNA hUbC-dCas9-KRAB-T2A-Puro (Addgene plasmid # 71236, a gift from Charles Gersbach) by replacing the KRAB domain with the catalytic domain of TET-1 from Fuw-dCas9-Tet1CD (Addgene plasmid # 84475, a gift from Rudolf Jaenisch).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Translocation Assay