dcas9 vpr plasmid  (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: Genome-wide arrayed CRISPR activation screen for prion protein modulators

doi: 10.64898/2026.03.01.707423

Figure Lengend Snippet: Overview of the arrayed genome-wide CRISPRa screen and qgRNA library design. (A) Schematic of the arrayed CRISPR activation (CRISPRa) screen performed in U-251 MG cells stably expressing dCas9-VPR. Cells were transduced with the T.gonfio quadruple-guide RNA (qgRNA) lentiviral library, targeting human protein-coding genes at single-gene resolution. PrP C abundance was quantified four days post-transduction using a solution-based time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. (B) Schematic of the qgRNA-pYJA5 construct and cloning strategy underlying the T.gonfio CRISPRa library (adapted from Yin et al., Nat. Biomed. Eng., 2025 ). The ampicillin resistance gene (AmpR) was removed from the parental pYJA5 vector. sgRNA1–4 and the trimethoprim resistance gene (TmpR) were generated as three distinct PCR amplicons and assembled by Gibson cloning to generate the qgRNA-pYJA5 plasmid. Transformants were selected using trimethoprim. The full plasmid structure and detailed organization of the qgRNA cassette are shown. LTR, long terminal repeat; Ψ, packaging signal; PB, piggyBac transposon element; PuroR, puromycin resistance gene; hU6, mU6, hH1, and h7SK, RNA polymerase III promoters; sg, single-guide RNA. F and R arrows indicate primer positions used for single-colony PCR, Sanger sequencing, and next-generation sequencing validation.

Article Snippet: Human glioblastoma U-251 MG cells (Kerafast, Inc., Boston, MA, USA; Accession ID: CVCL_0021) stably expressing dCas9-VPR (pXPR_120, Addgene #96917) were maintained in T150 tissue culture flasks (TPP, Trasadingen, Switzerland).

Techniques: Genome Wide, CRISPR, Activation Assay, Stable Transfection, Expressing, Transduction, Fluorescence, Förster Resonance Energy Transfer, Construct, Cloning, Plasmid Preparation, Generated, Sequencing, Next-Generation Sequencing, Biomarker Discovery

Journal: bioRxiv

Article Title: Genome-wide arrayed CRISPR activation screen for prion protein modulators

doi: 10.64898/2026.03.01.707423

Figure Lengend Snippet: Establishment and optimization of the CRISPRa screening platform for PrP C quantification. (A) Western blot analysis of PrP C expression in lysates from the indicated human cell lines using the POM2 antibody. The cell lines tested include U-251 MG, SH-SY5Y wild type (SH WT), SH-SY5Y PRNP knockout (SH KO), SK-N-SH, LN229, HEK293, HeLa, HepG2, and HT-29. Actin was used as a loading control. (B) TR-FRET–based quantification of PrP C levels in the same panel of cell lines shown in (A). Data represent mean ± SEM from four independent measurements. U-251 MG cells exhibit intermediate PrP C expression, enabling detection of both positive and negative regulators in CRISPRa screens. (C) Western blot analysis demonstrating CRISPRa-mediated overexpression of PrP C in U-251 MG dCas9-VPR cells transduced with qgRNAs targeting PRNP or non-targeting (NT) controls. PrP C was detected using the POM2 antibody, and actin served as a loading control. PrP C induction was monitored over time post-transduction. (D) TR-FRET–based assay optimization testing different cell seeding densities at a multiplicity of infection (MOI) of 3. qgRNAs targeting PRNP served as positive controls and NT qgRNAs as negative controls. Z′-factor analysis was used to determine optimal screening conditions. Data are shown as sextuplicate measurements.

Article Snippet: Human glioblastoma U-251 MG cells (Kerafast, Inc., Boston, MA, USA; Accession ID: CVCL_0021) stably expressing dCas9-VPR (pXPR_120, Addgene #96917) were maintained in T150 tissue culture flasks (TPP, Trasadingen, Switzerland).

Techniques: Western Blot, Expressing, Knock-Out, Control, Over Expression, Transduction, Infection