Journal: bioRxiv

Article Title: Multiplexed Perturbation Enables Scalable Pooled Screens

doi: 10.1101/2025.08.14.669942

Figure Lengend Snippet: A) Illustration of low and high multiplicity of infection (MOI) lentiviral sgRNA delivery in CRISPR applications. B) Quantification of lentiviral insertions relative to viral concentration using dPCR from genomic DNA. C) eGFP expression (MFI, mean fluorescent intensity) from Puro-T2A-eGFP marker cassette used in sgRNA expression vectors correlates with quantity of transduced lentivirus. D) Correlation between fluorescent marker expression and dPCR-inferred copy numbers of lentiviral insertions. E) CRISPRi knockdown levels in K-562 cells with a single sgRNAs targeting five surface markers individually, measured by antibody staining and flow cytometry. F) Knockdown efficiency using pooled sgRNAs against five surface markers at increasing MOI. In B-D dots represent the mean of three replicates and bars the standard deviation from the mean.

Article Snippet: To quantify sgRNA expression cassette copy numbers of transduced cells, genomic DNA is extracted with the Monarch Spin gDNA Extraction Kit (New England Biolabs).

Techniques: Infection, CRISPR, Concentration Assay, Expressing, Marker, Knockdown, Staining, Flow Cytometry, Standard Deviation

Journal: bioRxiv

Article Title: Multiplexed Perturbation Enables Scalable Pooled Screens

doi: 10.1101/2025.08.14.669942

Figure Lengend Snippet: A) Schematic workflow of CRISPRi screens for cell essentiality and drug tolerance assessment. B) Experimental conditions for evaluating lentiviral MOI effects on CRISPRi screen performance. C) A compact sgRNA library targeting epigenetic regulator genes was transduced into K-562 CRISPRi cells with varying MOI and lentiviral insertions are quantified by dPCR from genomic DNA. D) Correlation between mOrange2 fluorescence intensity, expressed from the sgRNA expression vector, and lentiviral copy numbers determined by dPCR. E) Gene essentiality scores calculated via MAGeCK RRA algorithm across the three experimental conditions described in (B) .

Article Snippet: To quantify sgRNA expression cassette copy numbers of transduced cells, genomic DNA is extracted with the Monarch Spin gDNA Extraction Kit (New England Biolabs).

Techniques: Fluorescence, Expressing, Plasmid Preparation