vpr  (Addgene inc)

Journal: ACS Omega

Article Title: Magneto-Photonic Gene Circuit for Minimally Invasive Control of Gene Expression in Mammalian Cells

doi: 10.1021/acsomega.5c13335

Figure Lengend Snippet: Characterization of EL222 constructs, light intensity and duration response. EL222 variants were obtained by replacing the VP16 transcription activation domain fused to EL222 with either VP64 or VPR activation domains. (A) Variable brightness LED matrix utilized for light stimulation and testing of EL222 variants. (B–F) Effect of light intensity on EL222-mediated transcription of the Firefly luciferase reporter gene. Cells expressing VP16-EL222 (B, D), VP64-EL222 (C, E), or VPR-EL222 (F) for 15 min, 30 min, or 2 h. Quantification of a Firefly luciferase reporter 24 h poststimulation showed levels of reporter expression that increase with LED strength. Regression analysis, represented by solid lines, shows the effect of LED strength on reporter expression can be approximated to a linear pattern at short exposure time or a sigmoidal pattern at longer time exposures. EL222-mediated Firefly luciferase expression at 60% LED intensity increased with stimulation time for cells expressing VP16-EL222 (G), VP64-EL222 (H), or VPR-EL222 (I).

Article Snippet: Thus, we engineered two new EL222 variants by replacing the existing VP16 TAD with either VP64 or VPR (transcription activation domains were cloned from Addgene plasmid #63798).

Techniques: Construct, Activation Assay, Luciferase, Expressing

Journal: ACS Omega

Article Title: Magneto-Photonic Gene Circuit for Minimally Invasive Control of Gene Expression in Mammalian Cells

doi: 10.1021/acsomega.5c13335

Figure Lengend Snippet: Optimizing Luminescent Activation of EL222 with NanoLuc. (A) Effect of substrate concentration, number of additions, and stimulation time on the EL222-mediated production of a SEAP reporter. Cells were provided with hCTZ ranging from 0 to 50 uM, with some groups receiving subsequent additions of substrate in intervals of 30 min, up to a maximum of 3 additions, as denoted by the number following the concentration. The substrate was left for a period of 3 h, and SEAP activity was measured the next morning. (B) Effect of substrate concentration, number of additions, and stimulation time on cell viability. Cell viability was determined via a cell titer blue assay; higher fluorescence denotes higher number of live cells. (C) Performance comparison of existing EL222 variants 24 h post LED stimulation. (D) Performance of VP64-EL222 and VPR-EL222 using NanoLuc luciferase for activation over an array of hCTZ concentrations. Statistical significance was calculated at a 5% significance level using one-way analysis of variance (ANOVA) followed by Dunnett’s test. (*) = P < 0.05, (**) = P < 0.01, (***) = P < 0.001, (****) = P = < 0.0001.

Article Snippet: Thus, we engineered two new EL222 variants by replacing the existing VP16 TAD with either VP64 or VPR (transcription activation domains were cloned from Addgene plasmid #63798).

Techniques: Activation Assay, Concentration Assay, Activity Assay, Fluorescence, Comparison, Luciferase

Journal: ACS Omega

Article Title: Magneto-Photonic Gene Circuit for Minimally Invasive Control of Gene Expression in Mammalian Cells

doi: 10.1021/acsomega.5c13335

Figure Lengend Snippet: Control of the VPR-EL222 circuit using the magneto receptive protein EPG. (A–J) Cells expressing one EPG-NanoLuc construct, VPR-EL222, and 5 × C120 SEAP were treated with 25 μM hCTZ and placed in a dark incubator. One plate received three rounds of EMF pulses following a 15 s ON 5 min OFF pattern, each round separated by 2 h. SEAP expression was measured 24 h after stimulation. No light (A) group and NanoLuc (B) act as negative controls for EMF response. RF114 (C) and fRR114 (D) showed a significant increase in SEAP production following magnetic stimulus. Results shown represent an average of three independent experiments; each separate experiment contains information collected from three individual wells. Statistical significance was calculated at a 5% significance level using two-way analysis of variance (ANOVA) followed by Sidak’s test. (*) = P < 0.05, (**) = P < 0.01, (***) = P < 0.001, (****) = P < 0.0001.

Article Snippet: Thus, we engineered two new EL222 variants by replacing the existing VP16 TAD with either VP64 or VPR (transcription activation domains were cloned from Addgene plasmid #63798).

Techniques: Control, Expressing, Construct

Journal: bioRxiv

Article Title: X-CODE: a dual RNA barcoding system for multi-platform clonal tracking and spatial phenotyping

doi: 10.64898/2026.02.10.705126

Figure Lengend Snippet: A ) Schematic of the X-CODE probe-based detection workflow (created with https://BioRender.com ). Individual barcode units (BUs) are detected using complementary fluorescent probes following rolling circle amplification (RCA), enabling single- or multiple-barcode identification within individual cells. B ) Flow cytometry contour plots of three barcoded TRAMP-C1 cell lines expressing BU-1, BU-2, or BU-1+BU-2. BU-1 and BU-2 were detected using Atto-550– and Cy5-conjugated probes, respectively. No RCA control was processed omitting the RCA step. WT sample are TRAMP-C1 cells without barcode. C ) Fluorescence confocal imaging of three barcoded clones, each carrying a unique combination of two out of four barcode units (Clone A: BU-1+BU-2; Clone B: BU-2+BU-3; Clone C: BU-3+BU-4). Top left: equal mix sample containing all three clones processed with the X-CODE detection workflow. In yellow, BU-1; in red, BU-2; in magenta, BU-3; in cyan, BU-4. Top right: detection of β-actin using the X-CODE probe protocol. The mix no-RCA negative control demonstrates loss of signal in the absence of RCA, and WT cells lack barcodes. Fluorophores used for detection: BU-1–Alexa 488, BU-2–Atto 550, BU-3–Cy5, BU-4–AF750, β-actin–Alexa 488. Scale bars, 20 µM. D ) Schematic representation of the combinatorial strategy developed for the assembly of the 15K and 2M barcode libraries (created with https://BioRender.com ). E ) NGS analysis of barcode representation in RM-1 cells transduced at low MOI with X-CODE libraries. Top: Scatter plot of NGS amplicon sequencing of short barcodes recovered from RM-1 cells transduced at low MOI with the 15K X-CODE library. Barcodes were PCR-amplified, gel-purified, and sequenced. The x axis shows individual barcode IDs, and the y-axis shows read counts. A total of 2,375 unique barcodes were detected; only barcodes with counts > 1 are plotted. Bottom: Equivalent analysis performed on RM-1 cells transduced at low MOI with the 2M X-CODE library. A total of 1,802 unique barcodes were identified; barcodes plotted are filtered for counts > 8,000. F ) Clonal spike-in retrieval sensitivity assay using X-CODE recall plasmid activation. Top schematic: Experimental workflow (created with https://BioRender.com ). A population of barcoded cells carrying a defined barcode was spiked into a bulk X-CODE–tagged cell population at defined ratios. Cells were transfected with dCas9-VPR and a barcode-specific recall plasmid, inducing GFP expression only in cells harbouring the cognate barcode. After 48 h, GFP⁺ cells were quantified by flow cytometry, sorted, and sequenced for validation. Bottom panels: Flow cytometry contour plots showing GFP activation across six spike-in dilution conditions (0%, 100%, 50%, 10%, 1%, 0.1%). The percentage refers to the fraction of spike-in cells in the initial mixture (red values). GFP⁺ enrichment scales with spike-in frequency, demonstrating barcode-specific recall sensitivity across >3 orders of magnitude, with minimal background at 0% and 0.1% spike-in control conditions.

Article Snippet: For functional recall, cells were transfected with the assembled recall plasmid and dCas9-VPR plasmid (Addgene; #63798) using Lipofectamine 3000 following the manufacturer’s instructions.

Techniques: Amplification, Flow Cytometry, Expressing, Control, Fluorescence, Imaging, Clone Assay, Negative Control, Sequencing, Purification, Sensitive Assay, Plasmid Preparation, Activation Assay, Transfection, Biomarker Discovery

Journal: iScience

Article Title: Recruitment of transcriptional effectors by Cas9 creates cis -regulatory elements and demonstrates distance-dependent transcriptional regulation

doi: 10.1016/j.isci.2025.114040

Figure Lengend Snippet: Recruitment of activation domain upregulates gene expression in a distance-dependent manner from multiple genomic sites. Activation maps shown as multi-track diagrams of six cardiomyocyte-specific gene loci spanning 140 Kbp each (A–C) Mybpc3 , Tnni1 , and Rcan1 , activated by sgRNA combination I. (D–F) Myh7 , Cox6a2 , and Myl3 , activated by sgRNA combination II. The TSS of each index gene is shown in red arrows. Tracks showing (from top to bottom): the genomic track with exons and introns in blue; activation track showing index gene fold change activation following dCas9-VPR targeting to the genomic site versus non-targeting gRNA control, as measured by RT-qPCR normalized to Gapdh in FIB (scale of activation for the track is shown in square brackets, average fold activation from each gRNA site in black bars, red dots over bars indicate SEM, all black bars have two-tailed t-test p < 0.05 vs. control, and sites with non-significant p > 0.05 activation are shown as pink bars on the negative scale for visibility, n = 3 for all sites); ATAC-seq and H3K27ac ChIP-seq tracks are shown for FIB in greens and CM in blue. HiC maps for each locus are shown above, with aqua colored lines indicating insulation locus boundaries. See also and .

Article Snippet: Plasmids expressing dCas9-VPR (SP-dCas9-VPR was a gift from George Church (Addgene plasmid # 63798; http://n2t.net/addgene:63798 ; RRID:Addgene_63798)), dCas9-KRAB domain (pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro was a gift from Charles Gersbach (Addgene plasmid # 71236; http://n2t.net/addgene:71236 ; RRID:Addgene_71236)), dCas9-p300 Core (pcDNA-dCas9-p300 Core was a gift from Charles Gersbach (Addgene plasmid # 61357; http://n2t.net/addgene:61357 ; RRID:Addgene_61357)) were acquired from Addgene.

Techniques: Activation Assay, Gene Expression, Control, Quantitative RT-PCR, Two Tailed Test, ChIP-sequencing, Insulation

Journal: iScience

Article Title: Recruitment of transcriptional effectors by Cas9 creates cis -regulatory elements and demonstrates distance-dependent transcriptional regulation

doi: 10.1016/j.isci.2025.114040

Figure Lengend Snippet: Activation from distal sites confers epigenetic marks at the activated site and at the gene promoter (A) A diagram of the Cox6a2 and Mybpc3 loci with a genomic track showing exons and introns in blue and ATAC-seq and H3K27ac ChIP-seq track in CM and FIB. A distal locus, marked by a black arrow and cross-hatched lines, was activated by dCas9-VPR in FIB. We then assessed Cox6a2 and Mybpc3 activation and the epigenetic marks at the distal site of activation and at the promoter of these genes (red arrow). As shown, prior to activation the distal sites lacked ATAC and H3K27ac marks in both FIB and CM, and the promoter lacked such marks in FIB. (B) qRT-PCR results show that dCas9-VPR targeting the 20 Kbp distal site induces strong gene activation. Data are shown as fold activation versus non-targeting gRNA control normalized to Gapdh, data are represented as mean ± SEM ( n = 3, ∗two-tailed t-test p < 0.01). (C) ChIP-qPCR analysis of H3K27ac (left) and H3K4mm (right) histone marks shows increased chromatin modification of both the distal activation site and the gene proximal promoter following activation of the distal site. For each promoter and distal site two separate primer pairs were used, amplifying regions ∼300 bp apart. Data are represented as mean ± SEM ( n = 5–8 for each primer pair) in N = 2–3 independent experiments (∗two-tailed t-test p < 0.05).

Article Snippet: Plasmids expressing dCas9-VPR (SP-dCas9-VPR was a gift from George Church (Addgene plasmid # 63798; http://n2t.net/addgene:63798 ; RRID:Addgene_63798)), dCas9-KRAB domain (pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro was a gift from Charles Gersbach (Addgene plasmid # 71236; http://n2t.net/addgene:71236 ; RRID:Addgene_71236)), dCas9-p300 Core (pcDNA-dCas9-p300 Core was a gift from Charles Gersbach (Addgene plasmid # 61357; http://n2t.net/addgene:61357 ; RRID:Addgene_61357)) were acquired from Addgene.

Techniques: Activation Assay, ChIP-sequencing, Quantitative RT-PCR, Control, ChIP-qPCR, Modification

Journal: iScience

Article Title: Recruitment of transcriptional effectors by Cas9 creates cis -regulatory elements and demonstrates distance-dependent transcriptional regulation

doi: 10.1016/j.isci.2025.114040

Figure Lengend Snippet: Activation with endogenous activation domains has similar features to activation with viral-derived activation domains (A) Diagram of CRISPR activators composed of dCas tethered to the activation domains of VP64, p65, and Rta (dCas9-VPR, top) or dCas tethered to the activation domains of the endogenous cardiac transcription factors GATA4, NKX2-5, and TBX5 (dCas9-GNT, bottom). (B) Activation maps as multi-track diagrams of two cardiomyocyte-specific gene loci ( Tnni1 and Cox6a2 ) are shown in a 70 Kbp window around the TSS. Tracks showing (from top to bottom): HiC maps for each locus, with aqua-colored lines indicating insulation locus boundaries; the genomic track with exons and introns in blue; activation tracks showing target gene average fold activation following dCas9-GNT in blue bars or activation with dCas9-VPR in black bars targeted to the genomic site versus non-targeting gRNA control, as measured by RT-qPCR normalized to Gapdh in FIB (scale of activation for the track is shown in square brackets, red dots over bars indicate SEM, only bars with two-tailed t-test p < 0.05 vs. control are shown, n = 3); tracks of ATAC-seq and H3K27ac ChIP-seq are shown for FIB in green and CM in blue. Diagram shows similar activation by dCas9-GNT and dCas9-VPR from distal sites lacking open chromatin or H3K27ac marks. (C) A diagram of the Myh7 locus with a genomic track showing exons and introns in blue and ATAC-seq and H3K27ac ChIP-seq track in CM and FIB. A distal site, 15 Kbp from the TSS, marked by a red dot and cross-hatched lines, was activated by dCas9-GNT in FIB. We then assessed the H3K27ac marks at the distal site of activation and at the promoter of these genes (red arrow). (D) qRT-PCR results show that dCas9-GNT targeting the 15 Kbp distal sites induce strong activation of Myh7 in FIB. Data are shown as fold activation vs. non-targeting gRNA control normalized to Gapdh; data are represented as mean ± SEM ( n = 3, ∗two-tailed t-test p < 0.005). (E) ChIP-qPCR analysis of H3K27ac activation marks shows increased chromatin modification of both the distal activation site and the gene proximal promoter following activation of the distal site by dCas9-GNT. For each promoter and distal site, two separate primer pairs were used, amplifying regions ∼300 bp apart. Data are represented as mean ± SEM ( n = 5–8 for each primer pair in N = 2–3 independent experiments, ∗two-tailed t-test p < 0.05). See also .

Article Snippet: Plasmids expressing dCas9-VPR (SP-dCas9-VPR was a gift from George Church (Addgene plasmid # 63798; http://n2t.net/addgene:63798 ; RRID:Addgene_63798)), dCas9-KRAB domain (pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro was a gift from Charles Gersbach (Addgene plasmid # 71236; http://n2t.net/addgene:71236 ; RRID:Addgene_71236)), dCas9-p300 Core (pcDNA-dCas9-p300 Core was a gift from Charles Gersbach (Addgene plasmid # 61357; http://n2t.net/addgene:61357 ; RRID:Addgene_61357)) were acquired from Addgene.

Techniques: Activation Assay, Derivative Assay, CRISPR, Insulation, Control, Quantitative RT-PCR, Two Tailed Test, ChIP-sequencing, ChIP-qPCR, Modification

Journal: iScience

Article Title: Recruitment of transcriptional effectors by Cas9 creates cis -regulatory elements and demonstrates distance-dependent transcriptional regulation

doi: 10.1016/j.isci.2025.114040

Figure Lengend Snippet: Recruitment of repression domain represses gene expression in a distance-dependent manner from multiple genomic loci (A) Multi-track diagrams of two cardiomyocyte specific gene loci ( Mybpc3 and Myh7 ) spanning 140 Kbp each. TSS of each gene is shown in red arrows. HiC maps for each locus are shown, with aqua-colored lines indicating insulation boundaries. Tracks showing (from top to bottom): the genomic track with exons and introns in blue; repression track showing index gene % repression in CM following dCas9-KRAB targeting to the genomic site with targeting versus non-targeting gRNA control, as measured by RT-qPCR normalized to Gapdh (scale of repression 0–100% in square brackets, average % repression from each gRNA site in black bars, red dots over bars indicate SEM, only bars with two-tailed t-test p < 0.05 vs. control are shown, n = 3). Activation track showing the same index gene activation in FIB following dCas9-VPR targeting to the genomic site versus non-targeting gRNA control, as measured by RT-qPCR normalized to Gapdh (scale of activation for the track is shown in square brackets, average fold activation from each gRNA site in black bars, red dots over bars indicate SEM, only bars with two-tailed t-test p < 0.05 vs. control are shown, n = 3). ATAC-seq and H3K27ac ChIP-seq tracks are shown for FIB in green and CM in blue. Diagram shows that like activation, repression can be achieved at a distance, by targeting non-regulatory chromatin, and can cross insulation boundaries. (B) Scatterplot of gene fold activation in FIB by dCas9-VPR as a function of % repression by dCas9-KRAB in CM as measured by RT-qPCR for multiple gRNA targeting sites in the Mybpc3 and Myh7 loci. Each dot represents the average of n = 3 measurements in CM and FIB. Plot shows activation and repression from these sites are generally correlated. A linear regression line is shown (Spearman’s Rho 0.34, n = 17). See also and ; .

Article Snippet: Plasmids expressing dCas9-VPR (SP-dCas9-VPR was a gift from George Church (Addgene plasmid # 63798; http://n2t.net/addgene:63798 ; RRID:Addgene_63798)), dCas9-KRAB domain (pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro was a gift from Charles Gersbach (Addgene plasmid # 71236; http://n2t.net/addgene:71236 ; RRID:Addgene_71236)), dCas9-p300 Core (pcDNA-dCas9-p300 Core was a gift from Charles Gersbach (Addgene plasmid # 61357; http://n2t.net/addgene:61357 ; RRID:Addgene_61357)) were acquired from Addgene.

Techniques: Gene Expression, Insulation, Control, Quantitative RT-PCR, Two Tailed Test, Activation Assay, ChIP-sequencing

Journal: iScience

Article Title: Recruitment of transcriptional effectors by Cas9 creates cis -regulatory elements and demonstrates distance-dependent transcriptional regulation

doi: 10.1016/j.isci.2025.114040

Figure Lengend Snippet: Recruitment of activation and repression domains controls gene expression in a distance-dependent manner from multiple genomic sites in human cells (A) Repression maps shown as multi-track diagrams in two cardiomyocyte-specific gene loci (MYBPC3 and MYH7), spanning 150 Kbp each. The TSS of each gene is shown in red arrows. Tracks showing (from top to bottom): the genomic track with exons and introns in blue; repression showing index gene % repression in iPSC-CM following Ad-dCas9-KRAB targeting versus non-targeting sgRNA control, measured by RT-qPCR normalized to GAPDH. ENCODE ATAC-seq and H3K27ac-seq tracks in hiPSC are shown in blue and red, respectively. Scale of repression 0%–100% in square brackets, average % repression from each sgRNA site in black bars, red dots over black bars represent SEM, only bards with two-tailed t-test p < 0.05 versus control are shown, n = 3 biological replicates. (B) Activation maps in human HEK293 fibroblasts shown as multi-track diagram of 3 CM-specific genes (TNNI1, COX6A2, and RCAN1) spanning 150 Kbp each. The TSS of each gene is shown in red arrows. Tracks showing (from top to bottom): the genomic track with exons and introns in blue, fold change activation following dCas9-VPR targeting versus non-targeting sgRNA control, measured by RT-qPCR normalized to GAPDH. ENCODE DNAse and H3K27ac-seq tracks in fibroblast cells are shown in blue and red, respectively. The scale for activation for the track is shown in square brackets, average fold activation for each sgRNA site in black bars, and red dots over black bars indicate SEM. All black bars have two-tailed t-test p < 0.05 versus control, n = 3 biological replicates.

Article Snippet: Plasmids expressing dCas9-VPR (SP-dCas9-VPR was a gift from George Church (Addgene plasmid # 63798; http://n2t.net/addgene:63798 ; RRID:Addgene_63798)), dCas9-KRAB domain (pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro was a gift from Charles Gersbach (Addgene plasmid # 71236; http://n2t.net/addgene:71236 ; RRID:Addgene_71236)), dCas9-p300 Core (pcDNA-dCas9-p300 Core was a gift from Charles Gersbach (Addgene plasmid # 61357; http://n2t.net/addgene:61357 ; RRID:Addgene_61357)) were acquired from Addgene.

Techniques: Activation Assay, Gene Expression, Control, Quantitative RT-PCR, Two Tailed Test