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

Journal: bioRxiv

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

doi: 10.64898/2026.03.01.707423

Figure Lengend Snippet:

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, Significance Assay