Journal: bioRxiv

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

doi: 10.1101/2024.03.03.583177

Figure Lengend Snippet: (A) Schematic of the PWS locus on chr15 with common PWS deletions and the PWS gRNA library. Each thin vertical line represents a single gRNA. Genes colored blue are maternally imprinted, those that are pink are paternally imprinted, and those that are grey are not imprinted. (B) Summary of the PWS gRNA library. (C) Schematic of experimental protocol for CRISPRa/CRISPRi screens. (D) CRISPR screen results (zoomed in, see Supp. Fig. S1C) displayed as -log 10 (p ad j), where p ad j is the multiple-hypothesis-corrected p-value from DESeq2. Notable regions are highlighted in beige. (E) Summary of the PWS gRNA sub-library. (F) Flow cytometry of SNRPN-GFP MFI for validations of individual gRNAs of the pat SNRPN-2A-GFP CRISPRi dCas9 KRAB screen. MFI values normalized to Empty vector. One-way ANOVA followed by Dunnett’s multiple comparisons test vs. Empty. **p < 0.01, ***p < 0.001, ****p<0.0001. (G) qPCR of SNRPN-GFP from individual gRNA validations of each of the gRNAs in the mat1 and mat2 pools shown in . (H) Plot of -log 10 (p adj ) values of each gRNA in the VP64 dCas9 VP64 full library screen vs. Tet1c dCas9 sublibrary screen, plotting only the gRNAs present in both screens. (Significant p adj < 0.05.) (I) qPCR of SNRPN-GFP for validations of individual gRNAs of the mat SNRPN-2A-GFP CRISPRa Tet1c dCas9 screen. For qPCR in (G) and (I), fold change values are plotted mean +/-SD, but statistics were calculated on ddCt values (normalized to GAPDH and empty or NT vector sample); one-way ANOVA followed by Dunnett’s test vs. empty vector. ***p<0.001, ****p<0.0001. Unmarked comparisons are not significant.

Article Snippet: The Tet1c Cas9-T2A-Thy1.1 plasmid for transfection was generated by replacing BFP in Addgene #167983 with mouse Thy1.1 ( CD90 ).

Techniques: CRISPR, Flow Cytometry, Plasmid Preparation

Journal: bioRxiv

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

doi: 10.1101/2024.03.03.583177

Figure Lengend Snippet: (A) qPCR of SNRPN-GFP from total mRNA in patSNRPN-2A-GFP dCas9 KRAB lines. (B) qPCR of SNRPN-GFP from polyadenylated (poly-A) mRNA in patSNRPN-2A-GFP dCas9 KRAB lines. (C) qPCR of the indicated genes from total mRNA in patSNRPN-2A-GFP dCas9 KRAB lines. (D) Schematic of 3’ RACE-seq of SNRPN transcript. (E,F) Comparison of SNRPN 3’ UTR sequence variants in control cells with dCas9 KRAB and an empty gRNA vector and cells treated with either (E) a pat6 gRNA or (F) a pat8 gRNA (G) Sequences of the four most predominant 3’ UTR variants detected in all conditions. Colors in (E) and (F) match the correspondingly colored sequences in (G). (H) Plot of -log 10 (padj) values of each gRNA in the VP64 dCas9 VP64 full library screen vs. dCas9 KRAB screen. (Significant p adj < 0.05.) (I) qPCR of SNRPN-GFP in patSNRPN-2A-GFP dCas9 KRAB iPSCs with gRNAs from the pat4, mat1, and mat2 regions. (J) qPCR of SNRPN-GFP in matSNRPN-2A-GFP VP64 dCas9 VP64 iPSCs with gRNAs from the CRISPRi screen hits. (K) Plot of -log 10 (p adj ) values of each gRNA in the VP64 dCas9 VP64 full library screen vs. sublibrary screen. (Significant p adj < 0.05.) (L) Plot of -log 10 (p adj ) values of each gRNA in the Tet1c dCas9 sublibrary screen vs. dCas9 KRAB full library screen. Significant p adj < 0.05.

Article Snippet: The Tet1c Cas9-T2A-Thy1.1 plasmid for transfection was generated by replacing BFP in Addgene #167983 with mouse Thy1.1 ( CD90 ).

Techniques: Comparison, Sequencing, Control, Plasmid Preparation

Journal: bioRxiv

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

doi: 10.1101/2024.03.03.583177

Figure Lengend Snippet: (A) Schematic of chr15 in isogenic wildtype (WT) and PWS Type II deletion (ΔPWS) iPSCs. (B) qPCR of SNRPN in WT or ΔPWS iPSCs with VP64 dCas9 VP64 14 days after transduction with the indicated gRNA or gRNA pool. (C) qPCR of SNRPN in WT or ΔPWS iPSCs with Tet1c dCas9 14 days after transduction with the indicated gRNA or gRNA pool. For both qPCR plots, fold change values are plotted mean +/-SD, but statistics were calculated on ddCt values (normalized to GAPDH and WT ctrl sample); one-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 Tet1c dCas9 ΔPWS iPSCs, comparing mat3 g5 to NT gRNA. (F) HCR FlowFISH of VP64 dCas9 VP64 iPSCs (WT or ΔPWS) with the indicated gRNA. SNHG14 signal on X axis, with TBP as a control for cell size and staining. (G) HCR FlowFISH of Tet1c 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.

Article Snippet: The Tet1c Cas9-T2A-Thy1.1 plasmid for transfection was generated by replacing BFP in Addgene #167983 with mouse Thy1.1 ( CD90 ).

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

Journal: bioRxiv

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

doi: 10.1101/2024.03.03.583177

Figure Lengend Snippet: (A,C): qPCR of WT or ΔPWS VP64 dCas9 VP64 iPSCs with NT or mat1 g3 gRNA for either (A) SNORD116 or (C) sets of SNRPN transcript variants. (B,D) qPCR of WT or ΔPWS Tet1c dCas9 iPSCs with NT or mat3 g5 gRNA for either (B) SNORD116 or (D) sets of SNRPN transcript variants. Fold change values (relative to GAPDH ) are plotted mean +/-SD, but statistics were calculated on dCt values (normalized to GAPDH ); one-way ANOVA followed by Sidak’s multiple comparisons test for select groups WT + targeting gRNA vs. NT gRNA or ΔPWS + targeting gRNA vs. NT gRNA. *p<0.05, ***p<0.001,****p<0.0001. (E,F) Two replicates of HCR FlowFISH (Rep. 1 of each was shown in , respectively) of WT or ΔPWS iPSCs with either (E) VP64 dCas9 VP64 and NT or mat1/2 gRNAs, or (F) Tet1c dCas9 and NT or mat3 gRNAs. (G) Browser tracks of ATAC sequencing (rpkm-normalized BigWig) of ΔPWS or WT iPSCs with VP64 dCas9 VP64 and NT or mat1 g3 gRNA downstream of SNRPN, at the PWAR1 gene.

Article Snippet: The Tet1c Cas9-T2A-Thy1.1 plasmid for transfection was generated by replacing BFP in Addgene #167983 with mouse Thy1.1 ( CD90 ).

Techniques: Sequencing

Journal: bioRxiv

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

doi: 10.1101/2024.03.03.583177

Figure Lengend Snippet: (A) Genome browser visualization of RNA sequencing (rpkm-normalized BigWig) of VP64 dCas9 VP64 WT or ΔPWS iPSCs with NT or mat1 g3 gRNA, zoomed in on SNRPN upstream exons. (B) Targeted bisulphite sequencing of ΔPWS iPSCs with VP64 dCas9 VP64 or Tet1c dCas9 covering 24 CpG sites within the PWS locus (hg19 chr15: 25200353-25200693), 15 days post-transduction. Data are shown as the range of the data, with the plotted point being the median, n = 3 replicates. (C) qPCR of SNRPN in WT or ΔPWS iPSCs with VP64 dCas9 VP64 or Tet1c dCas9 38 days after transduction with the indicated gRNA, same cell samples as those collected for bisulphite sequencing depicted in . For both qPCR plots, fold change values are plotted mean +/-SD, but statistics were calculated on ddCt values (normalized to GAPDH and WT ctrl sample); one-way ANOVA followed by Dunnett’s test vs. ΔPWS NT gRNA ****p<0.0001. (D) Browser tracks of ATAC sequencing (rpkm-normalized BigWig) of ΔPWS or WT iPSCs with Tet1c dCas9 and NT or mat3 g5 gRNA. (E) Quantification of ATAC-seq reads (counts per million) at each of the two peaks at the PWS-IC (mat3 g5 is located within the first of the two peaks, see S4D). ΔPWS + NT vs. mat3 gRNA not significant, Tukey’s test following one-way ANOVA. (F) qPCR of SNRPN expression in ΔPWS iPSCs with NT or mat3 g5 gRNA, comparing 3 different Tet1c dCas9 constructs, all delivered by lentivirus.

Article Snippet: The Tet1c Cas9-T2A-Thy1.1 plasmid for transfection was generated by replacing BFP in Addgene #167983 with mouse Thy1.1 ( CD90 ).

Techniques: RNA Sequencing, Bisulfite Sequencing, Transduction, Sequencing, Expressing, Construct

Journal: bioRxiv

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

doi: 10.1101/2024.03.03.583177

Figure Lengend Snippet: (A) Targeted bisulphite sequencing of ΔPWS iPSCs with VP64 dCas9 VP64 covering 24 CpG sites within the PWS locus (hg19 chr15: 25200353-25200693), 38 days post-transduction. (B) Targeted bisulphite sequencing of ΔPWS iPSCs with Tet1c dCas9 covering 24 CpG sites within the PWS locus (hg19 chr15: 25200353-25200693), 38 days post-transduction. Data for (A) and (B) are shown as the range of the data, with the plotted point being the median, n = 3 replicates. (C) Browser tracks of ATAC sequencing (rpkm-normalized BigWig) of ΔPWS or WT iPSCs with VP64 dCas9 VP64 and NT or mat1 g3 gRNA. (D) Quantification of ATAC-seq reads (counts per million) at the peak at the mat1 g3 guide binding site (dashed line in (A)). ***p < 0.001, Tukey’s test following one-way ANOVA.

Article Snippet: The Tet1c Cas9-T2A-Thy1.1 plasmid for transfection was generated by replacing BFP in Addgene #167983 with mouse Thy1.1 ( CD90 ).

Techniques: Bisulfite Sequencing, Transduction, Sequencing, 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: 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: The Tet1c dCas9 plasmid (Addgene #232178) was generated by amplifying Tet1c from Addgene #108245, followed by Gibson assembly of Tet1c, dCas9, and BSD into a lentiviral expression backbone.

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: The Tet1c dCas9 plasmid (Addgene #232178) was generated by amplifying Tet1c from Addgene #108245, followed by Gibson assembly of Tet1c, dCas9, and BSD into a lentiviral expression backbone.

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: The Tet1c dCas9 plasmid (Addgene #232178) was generated by amplifying Tet1c from Addgene #108245, followed by Gibson assembly of Tet1c, dCas9, and BSD into a lentiviral expression backbone.

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: Transient expression of Tet1v4 dCas9 in ΔPWS iPSCs stably activates maternal PWS genes (A) Schematic of experimental protocol for transient delivery of Tet1v4 dCas9 plasmid and PWS gene expression analysis. (B) qPCR of dCas9 and SNRPN in WT and ΔPWS iPSCs after transient delivery of Tet1v4 dCas9 on day 0. Two-way ANOVA on ΔCt values (normalized to GAPDH ), followed by Dunnett’s test, compared to ΔPWS + NT gRNA; ∗ p < 0.05; ∗∗ p < 0.0001; ns, not significant. Data shown as mean ± SD. (C) qPCR of PWS genes in iPSC-derived neurons. Data plotted as mean fold change ± SD, but statistics computed on ΔΔCt (normalized to GAPDH and WT + NT). Two-way ANOVA followed by Dunnett’s test, compared to ΔPWS + NT gRNA; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, not significant. Data shown as mean ± SD. (D) Targeted bisulfite sequencing of ΔPWS iPSC-derived neurons ∼21 days post-differentiation, covering 24 CpG sites within the PWS locus (hg19 chr15: 25200353–25200693). Data shown as median ± range; n = 3 replicates. (E) 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 iNs expressing Tet1v4 dCas9. For all data shown in the figure, n = 2 replicates for WT NT; all other conditions, n = 3.

Article Snippet: The Tet1c dCas9 plasmid (Addgene #232178) was generated by amplifying Tet1c from Addgene #108245, followed by Gibson assembly of Tet1c, dCas9, and BSD into a lentiviral expression backbone.

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Gene Expression, Derivative Assay, Methylation Sequencing, Methylation

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: The Tet1c dCas9 plasmid (Addgene #232178) was generated by amplifying Tet1c from Addgene #108245, followed by Gibson assembly of Tet1c, dCas9, and BSD into a lentiviral expression backbone.

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

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