lentiviruses expressing fuw dcas9 tet1cd  (Addgene inc)

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