human dnmt3a catalytic domain  (Addgene inc)

Journal: bioRxiv

Article Title: Systematic comparison of dCas9-based DNA methylation epimodifiers over time indicates efficient on-target and widespread off-target effects

doi: 10.1101/2025.03.15.641804

Figure Lengend Snippet: Early methylation editing efficiency of the dCas9-epimodifiers. a. Schematic of the design of the dCas9-epimodifiers. b. Genomic localization of BACH2 promoter targeted by the specific BACH2 -targeting gRNA 8 (g8). c. Experimental design where HEK293T cells were transfected with plasmids encoding gRNA-dCas9-epimodifier-EGFP (3A, 3A3L, 3A3A, 3A-KRAB, mut3A, M.SssI, SunTag) or CRISPRoff (co-transfected with a gRNA-encoding plasmid). Successfully transfected cells were sorted 3 days post transfection (p.t.) by GFP-based fluorescent activated cell sorting (FACS). d. DNA methylation of CpGs in BACH2 promoter 3 days p.t. with epimodifiers expressing either BACH2 -targeting gRNA8 (g8) or non-targeting control gRNA (NTC). Methylation levels in the control cells including cells expressing dCas9-deactivated DNMT3A (d3A) or non-transfected cells (NT) are depicted by the black and grey lines, respectively. e. Tukey plot, comparison Δβ (gRNA-NT) of the methylation changes induced by the g8 versus NTC across all tools. Boxplots summarize the distribution, with positive Δβ indicating increased methylation. f . Example of methylation changes at one of the predicted off-target loci ( PAX5 : Chr. 9 36,92,2430-36,92,3069, hg38), with methylation change induced by g8 or NTC compared to NT control (upper graph). The basal methylation level at each CpG is represented by the histogram plot (lower graph). Methylation data, assessed by targeted methylation sequencing, are available in Supplementary Table 3.

Article Snippet: M3-dCAS9-DNMT3A-DNMT3A (dCas9-3A-3A, Addgene #218776) plasmid made by the amplification of the human DNMT3A catalytic domain from the pdCas9-DNMT3A-EGFP (Addgene #71666) plasmid using the primers containing the FseI restriction sites.

Techniques: Methylation, Transfection, Plasmid Preparation, FACS, DNA Methylation Assay, Expressing, Control, Comparison, Sequencing

Journal: bioRxiv

Article Title: Systematic comparison of dCas9-based DNA methylation epimodifiers over time indicates efficient on-target and widespread off-target effects

doi: 10.1101/2025.03.15.641804

Figure Lengend Snippet: Factors influencing on-target methylation editing. a. Schematic of the designed. b. Experimental design where HEK293T cells were transfected with dCas9-DNMT3A-EGFP plasmids encoding BACH2 -targeting gRNA8 ( BACH2 -dCas9-3A), non-targeting control gRNA (non-targeting-dCas9-3A), gRNA scaffold (scaffold-dCas9-3A) or no gRNA scaffold (empty-dCas9-3A). Successfully transfected cells were sorted 3 days post-transfection (p.t.) based on different GFP-intensity using fluorescent activated cell sorting (FACS). c. Impact of expression levels of dCas9-DNMT3A, based on sorting cells with varying levels of GFP intensity, in combination with strong (pCAG), moderate (EF1a), or weak (UBC) promoters, on methylation deposition induced by BACH2 gRNA, NTC gRNA, gRNA scaffold or no gRNA scaffold. d. The expression levels of dCas9-DNMT3A under promoters with varying strengths assessed across samples with different GFP mean fluorescent intensities. e. Methylation deposition by dCas9-epimodifier comprising the catalytic domain of DNMT3A harboring the R882H mutation (dCas9-mut3A). f. Methylation deposition following transfection with BACH2 -targeting gRNA8 ( BACH2 -dCas9-3A), non-targeting control gRNA (non-targeting-dCas9-3A) and gRNA scaffold (scaffold-dCas9-3A) 24, 48 and 72 hours (h) p.t..

Article Snippet: M3-dCAS9-DNMT3A-DNMT3A (dCas9-3A-3A, Addgene #218776) plasmid made by the amplification of the human DNMT3A catalytic domain from the pdCas9-DNMT3A-EGFP (Addgene #71666) plasmid using the primers containing the FseI restriction sites.

Techniques: Methylation, Transfection, Control, FACS, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Systematic comparison of dCas9-based DNA methylation epimodifiers over time indicates efficient on-target and widespread off-target effects

doi: 10.1101/2025.03.15.641804

Figure Lengend Snippet: Early transcriptomic changes induced by the dCas9-epimodifiers. a . Hierarchical clustering and principal component analysis of RNA-sequencing data 3 days post-transfection with the dCas9-epimodifiers (3A, 3A3L, 3A3A, 3A-KRAB, mut3A, M.SssI, SunTag) or CRISPRoff (co-transfected with a gRNA-encoding plasmid). b . Correlation between transcriptional changes driven by BACH2 -targeting (g8) and non-targeting control (NTC) gRNAs in comparison to the control deactivated DNMT3A (d3A) with the blue and pink colors reflecting downregulated and upregulated transcripts, respectively, between g8 and NTC. c . Total number of differentially expressed genes (DEGs, upper graph) and proportion in percentage (middle graph) of upregulated (pink color) and downregulated (blue color) DEGs between each dCas9-epimodifier and control d3A with |log 2 FC| > 1. The number of DEGs shared with at least one other epimodifier is detailed in the lower pairwise sharedness plot (the darker the color the highest number of genes being shared). d . Clustering of gene ontology terms, using GeneSetCluster tool, derived from the transcriptome of each epimodifier expressing either g8 or NTC compared to the control d3A, the Jaccard score depicted in grey gradient represents the degree of gene sharedness between sets (the darker the colors, the higher the number of genes being shared). The normalized enrichment scores (NES) illustrating upregulation and downregulation are indicated in orange and green colors, respectively for each cluster. e . STRING network of the core interconnected genes shared between epimodifiers identified using Density-Based Spatial Clustering of Applications with Noise algorithm. Pie charts summarize the dCas9-epimodifiers involved in the gene overlap for each sub-network, distinguishing the fraction shared between CRISPRoff and dCas9-mut3A from other shared genes. f . BACH2 promoter differential methylation region (DMR, mean of CpGs β values) and gene expression (in counts per million, CPM, upper graph) and correlation between differences at BACH2 promoter methylation (Δβ g8-NTC ) and gene expression (log 2 g8/NTC) between g8 and NTC gRNAs (lower graph).

Article Snippet: M3-dCAS9-DNMT3A-DNMT3A (dCas9-3A-3A, Addgene #218776) plasmid made by the amplification of the human DNMT3A catalytic domain from the pdCas9-DNMT3A-EGFP (Addgene #71666) plasmid using the primers containing the FseI restriction sites.

Techniques: RNA Sequencing, Transfection, Plasmid Preparation, Control, Comparison, Derivative Assay, Expressing, Methylation, Gene Expression

Journal: bioRxiv

Article Title: Systematic comparison of dCas9-based DNA methylation epimodifiers over time indicates efficient on-target and widespread off-target effects

doi: 10.1101/2025.03.15.641804

Figure Lengend Snippet: Stability of the on-target methylation editing over time. a. Experimental design where HEK293T cells were transfected with plasmids encoding gRNA-dCas9-epimodifier-EGFP (3A, 3A3L, 3A3A, 3A-KRAB, mut3A, M.SssI, SunTag) or CRISPRoff (co-transfected with a gRNA-encoding plasmid). Successfully transfected cells were sorted based on GFP or BFP signal 3 days post-transfection (p.t.) using fluorescent activated cell sorting (FACS), GFP/BFP positive cells were cultured and harvested at different time points for methylation and expression analyses. b. Methylation of CpGs at BACH2 promoter 3 (D3), 7 (D7), 14 (D14), 21 (D21) and 30 (D30) days p.t. with the epimodifiers expressing either BACH2 -targeting gRNA8 (g8) or non-targeting gRNA control (NTC). Yellow arrow indicated position of BACH2 -targeting gRNA (g8). Methylation levels in the control conditions including cells expressing dCas9-deactivated DNMT3A (d3A) or non-transfected cells (NT) are depicted by the black and grey lines, respectively. Comparison of g8-driven methylation levels for all constructs per time point is illustrated by different turquoise blue colors. The net effect of g8 in comparison to NTC is represented on the right panel with the methylation differences of the differentially methylated CpGs (circles) centered on median (line), 25th and 75th percentile bounds (darker color) and minimum and maximum extending to the lowest / highest values (light color). Methylation data, assessed by targeted methylation sequencing, are available in Supplementary Tables 3 & 4.

Article Snippet: M3-dCAS9-DNMT3A-DNMT3A (dCas9-3A-3A, Addgene #218776) plasmid made by the amplification of the human DNMT3A catalytic domain from the pdCas9-DNMT3A-EGFP (Addgene #71666) plasmid using the primers containing the FseI restriction sites.

Techniques: Methylation, Transfection, Plasmid Preparation, FACS, Cell Culture, Expressing, Control, Comparison, Construct, Sequencing

Journal: bioRxiv

Article Title: Systematic comparison of dCas9-based DNA methylation epimodifiers over time indicates efficient on-target and widespread off-target effects

doi: 10.1101/2025.03.15.641804

Figure Lengend Snippet: Genome-wide methylation induced by the four most potent dCas9-epimodifiers over time. a. Violin plot showing methylation level distributions across sample groups for day 3 (D3), 7 (D7) and 30 (D30) post transfection (p.t.). Overlayed boxplots indicate the interquartile range, and open circles mark the median values. Density plot is visualizing the overall distribution of methylation levels across all samples. b. Correlation between methylation changes at differentially methylated regions (DMRs) driven by BACH2 -targeting gRNA8 (g8) or non-targeting control gRNA (NTC) in comparison to the control deactivated DNMT3A (d3A) at each time point, with the blue and purple colors reflecting hypomethylated and hypermethylated loci, respectively, between g8 and NTC. c . Effect size (violin plot) and total number (horizontal bar plot) of the DMRs induced by each dCas9-epimodifier at each time point. The fraction (%) of the DMRs that overlap between two conditions is depicted by the pairwise sharedness plot (the darker the yellow color the higher percentage of genes being shared). d . Fraction of DMRs stably altered between time points (gradient of grey colors) or specifically found only at day 3, 7 or 30 p.t. (white color). e. Top ‘Biological Processes’ gene ontology terms obtained using overrepresentation analysis of the DMR-genes that are shared between dCas9-epimodifiers. The circle size and color reflect the FDR significance and number of genes, respectively. f. Distribution of the DMRs according to chromatin state segmentation by hidden Markov model (ChromHMM) in HEK293T cells obtained from International Human Epigenome Consortium. The methylation data, assessed by EPIC array, is available in Supplementary Table 8. The statistics accompanying ChromHMM enrichment are available in Supplementary Table 9. TSS, transcription start site, Heterochrom, heterochromatin. g. Weblogo representation of sequence preferences for CRISPRoff gRNA8, showing GC enrichment, particularly in GCGC motifs. ANOVA – Tukey–Kramer’s test (sig P-value p < 0.05).

Article Snippet: M3-dCAS9-DNMT3A-DNMT3A (dCas9-3A-3A, Addgene #218776) plasmid made by the amplification of the human DNMT3A catalytic domain from the pdCas9-DNMT3A-EGFP (Addgene #71666) plasmid using the primers containing the FseI restriction sites.

Techniques: Genome Wide, Methylation, Transfection, Control, Comparison, Stable Transfection, Sequencing