2a gfp sequence  (Addgene inc)

Journal: bioRxiv

Article Title: The evolution of gene regulation in mammalian cerebellum development

doi: 10.1101/2025.03.14.643248

Figure Lengend Snippet: (A) Scheme on assignment of human CREs to different evolutionary clades using DeepCeREvo. (B) Human CREs inferred as preserved in different clades based on DeepCeREvo prediction scores on orthologous regions across 227 mammalian species for selected NMFs. (C) Proportions of CREs assigned to different clades, grouped by minimum age of the sequence. (D) Fraction of human CREs in each NMF across different evolutionary clades, with orthologous regions accessible in mouse (top) or marmoset (bottom). (E) Normalized fragment counts in mature granule cells (GC_defined) for regions that are orthologous to human CREs in NMF_14. Regions are grouped according to their predicted evolutionary histories. Boxes represent the interquartile range and whiskers extend to extreme values within 1.5 times the interquartile range from the box. (F, G) Luciferase reporter assays in mouse primary granule cells testing the enhancer activity of CREs predicted to be eutherian-shared (F) or having emerged in the last 43 million years (G). CREs were placed in front of the SV40 promoter in forward (left) or reverse (right) orientation. Bars and error bars display the mean normalized and scaled reporter activity and its range; points denote biological replicates. P -values relative to the constructs without an enhancer were estimated using linear mixed models, corrected for multiple testing using the Benjamini-Hochberg method, and are shown for each orientation only for bars with log 2 (fold change) ≥ 0.5. ***, P < 0.001; **, P < 0.01.

Article Snippet: Next, SV40 promoter sequence was amplified from pHRdSV40-scFv-GCN4-sfGFP-VP64-GB1-NLS vector , which was a gift from Ron Vale (Addgene plasmid #60904), using KAPA HiFi HotStart ReadyMix (Roche) and primers with overhanging homology arms (table S13).

Techniques: Sequencing, Luciferase, Activity Assay, Construct

Journal: bioRxiv

Article Title: The evolution of gene regulation in mammalian cerebellum development

doi: 10.1101/2025.03.14.643248

Figure Lengend Snippet: (A) Schematic illustration of isolation and culturing of mouse P7 granule cells. DIV, days in vitro ; SAG, Smoothened Agonist. (B) Phase contrast micrographs of granule cells at DIV3 and DIV6. (C, D) UMAPs of 1,167 DIV3 and 2,784 DIV6 granule cells, coloured by sample (C) or cell state (D). (E) Relative cell type abundances in the DIV3 and DIV6 granule cell datasets. (F) Fraction of highly variable CREs identified in the mouse ( in vivo ) snATAC-seq data that are also accessible in the mouse granule cells cultured in vitro . (G) Luciferase reporter assays in mouse primary granule cells testing the enhancer activity of human CREs predicted to have emerged in the last 43 million years. CREs were placed in front of the SV40 promoter in forward (left) or reverse (right) orientation. Bars and error bars display the mean normalized and scaled reporter activity and its range; points denote biological replicates. P -values relative to the constructs without an enhancer were estimated using linear mixed-effects models, corrected for multiple testing using the Benjamini-Hochberg method, and are shown only for bars with log 2 (fold change) ≥ 0.5. ***, P < 0.001.

Article Snippet: Next, SV40 promoter sequence was amplified from pHRdSV40-scFv-GCN4-sfGFP-VP64-GB1-NLS vector , which was a gift from Ron Vale (Addgene plasmid #60904), using KAPA HiFi HotStart ReadyMix (Roche) and primers with overhanging homology arms (table S13).

Techniques: Isolation, In Vitro, In Vivo, Cell Culture, Luciferase, Activity Assay, Construct

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 Tet1v4 dCas9 lentiviral expression plasmid (Addgene #232180) was generated by replacing the VP64 dCas9 VP64 sequence in our previously-generated VP64 dCas9 VP64 -2A-BSD plasmid with the Tet1c-XTEN80-dCas9 sequence in Addgene #167983 via Gibson assembly.

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 Tet1v4 dCas9 lentiviral expression plasmid (Addgene #232180) was generated by replacing the VP64 dCas9 VP64 sequence in our previously-generated VP64 dCas9 VP64 -2A-BSD plasmid with the Tet1c-XTEN80-dCas9 sequence in Addgene #167983 via Gibson assembly.

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 Tet1v4 dCas9 lentiviral expression plasmid (Addgene #232180) was generated by replacing the VP64 dCas9 VP64 sequence in our previously-generated VP64 dCas9 VP64 -2A-BSD plasmid with the Tet1c-XTEN80-dCas9 sequence in Addgene #167983 via Gibson assembly.

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 Tet1v4 dCas9 lentiviral expression plasmid (Addgene #232180) was generated by replacing the VP64 dCas9 VP64 sequence in our previously-generated VP64 dCas9 VP64 -2A-BSD plasmid with the Tet1c-XTEN80-dCas9 sequence in Addgene #167983 via Gibson assembly.

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 Tet1v4 dCas9 lentiviral expression plasmid (Addgene #232180) was generated by replacing the VP64 dCas9 VP64 sequence in our previously-generated VP64 dCas9 VP64 -2A-BSD plasmid with the Tet1c-XTEN80-dCas9 sequence in Addgene #167983 via Gibson assembly.

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

Journal: Nucleic Acids Research

Article Title: Efficient activation of hundreds of LTR12C elements reveals cis -regulatory function determined by distinct epigenetic mechanisms

doi: 10.1093/nar/gkae498

Figure Lengend Snippet: The dCas9-SunTag-based CRISPRa systems induce LTR12C expression from proximal promoter regions. ( A ) Percentage of upregulated LTR families in human cell lines following 5-aza-CdR treatment. The four cell lines were treated with PBS (control) or 300 nM 5-aza-CdR for 24 h and harvested at 5 days after the treatment. The upregulated LTR copies [fold change (FC) >2 by comparison with PBS-treated human cell lines] are categorized into each LTR family. The top 10 most highly abundant LTR families are highlighted with distinct colors. ( B ) Schematics of dCas9 constructs and fusion proteins. The constructs contain dCas9, SunTags separated by 22-amino-acid linkers (Multi SunTag), 2A self-cleaving peptide (2A), single-chain variable fragment (ScFv), green fluorescence protein (GFP), VP64/p300 and gRNA. The fusion proteins with single gRNA are inferred to transactivate multiple copies of LTR12C. ( C ) A sequence alignment of LTR12C elements deposited in the RepeatMasker database. Each row represents one LTR12C element. Heatmap indicates percent identity (0 to 90%); blank indicates gaps. Arrowhead indicates gRNA positions. Promoter and enhancer region is defined as 400 bp upstream region from TSS. ( D ) Relative expression of LTR12C based on reverse transcription qPCR (RT-qPCR) after transfection of the dCas9-SunTag-VP64 construct with represented gRNAs in HEK293T cells. Error bars represent standard error of the mean (SEM) from three independent biological replicates.

Article Snippet: The VP64 sequence was amplified from pcDNA-dCas9-VP64 plasmid (Addgene #47107) by polymerase chain reaction (PCR).

Techniques: Expressing, Control, Comparison, Construct, Fluorescence, Sequencing, Reverse Transcription, Quantitative RT-PCR, Transfection

Journal: Nucleic Acids Research

Article Title: Efficient activation of hundreds of LTR12C elements reveals cis -regulatory function determined by distinct epigenetic mechanisms

doi: 10.1093/nar/gkae498

Figure Lengend Snippet: The transactivation capacity of the dCas9-SunTag-VP64 system for LTR12C is higher than that of the dCas9-SunTag-p300 system. ( A ) Relative RNA expression of LTR12C and selected coding genes for RHOXF2B , IL1RN and OCT4 following represented CRISPRa as determined by RT-qPCR in HEK293T cells. Primers of LTR12C were designed at multiple loci. Error bars represent SEM from three independent biological replicates. P -values were calculated using the two-tailed Student’s t -test: *** P < 0.001. ( B ) The volcano plots show expression changes of LTR copies (not only LTR12C) after transfection of represented constructs with LTR12C-targeting gRNA in HEK293T cells. The log 2 FC values and −log 10 ( P -value) were calculated by comparison with expression of LTR copies in cells transfected with constructs expressing nontargeting gRNA. Dashed lines are the threshold of the two-tailed Wilcoxon signed-rank test P -value <0.05 (horizontal) or FC > 2 (vertical). The red (upper-right area) and blue (upper-left area) points represent upregulated and downregulated LTR copies with statistical significance, respectively. The pie charts show percentage of the upregulated LTR copies in each LTR family. The top five most highly abundant LTR families are highlighted with distinct colors. ( C ) The distribution of expression level (RPKM) of LTR12C in the represented groups in HEK293T cells. ‘All LTR12C’ refers to LTR12C copies that were deposited in RepeatMasker. Each box represents the data between the 25th and 75th quartiles. The whiskers are drawn down to the 10th percentile and up to the 90th percentile. White bars indicate median values. Adjusted P -values were calculated using Mann–Whitney U test and Holm’s method: *** P < 0.001. ( D ) LTR12C copies overlapping among upregulated LTR12C by dCas9-SunTag-VP64, dCas9-SunTag-p300 and 5-aza-CdR in HEK293T cells.

Article Snippet: The VP64 sequence was amplified from pcDNA-dCas9-VP64 plasmid (Addgene #47107) by polymerase chain reaction (PCR).

Techniques: RNA Expression, Quantitative RT-PCR, Two Tailed Test, Expressing, Transfection, Construct, Comparison, MANN-WHITNEY

Journal: Nucleic Acids Research

Article Title: Efficient activation of hundreds of LTR12C elements reveals cis -regulatory function determined by distinct epigenetic mechanisms

doi: 10.1093/nar/gkae498

Figure Lengend Snippet: The off-target events by single gRNA-based dCas9-SunTag-VP64 were limited. ( A ) Percentage of dCas9-HA binding LTR families represented among the LTRs upregulated after dCas9-SunTag-VP64-based CRISPRa in HEK293T cells, as detected in Figure , left panel. ( B ) Representative genomic regions showing LTR12C-specific transactivation in HEK293T cells. The track views represent RNA expression based on RNA-seq (upper) and dCas9-HA binding sites based on ChIP-seq (lower). Green (bottom) bars indicate genomic position of retrotransposons with their strand orientation. Arrowhead (blue) on the retrotransposons indicates gRNA targeting sites with one mismatch. Arrowhead (green) on the track view indicates poly(A) signal site. ( C ) Representative genomic region comprising LTR12C and other retrotransposons that gained robust expression in HEK293T cells.

Article Snippet: The VP64 sequence was amplified from pcDNA-dCas9-VP64 plasmid (Addgene #47107) by polymerase chain reaction (PCR).

Techniques: Binding Assay, RNA Expression, RNA Sequencing, ChIP-sequencing, Expressing