Fig. 2: CRISPR library design and screening in AD-bMSCs reveals TP53 and PTEN as key regulators of cell proliferation.
From: Pooled CRISPR screens identifies key regulators of bovine stem cell expansion for cultured meat
A Composition of the designed CRISPR sgRNA library. The library consists of 3000 sgRNAs targeting 603 genes, grouped into: Transcription Factors (TF, 2099 sgRNAs/484 genes), Top Gene Effect Score (TGES, 304 sgRNAs/73 genes), Bottom Gene Effect Score (BGES, 161 sgRNAs/46 genes), and controls (436 sgRNAs). B Mean Gene Effect Score across all DepMap datasets for each library subgroup, indicating predicted impact on cell fitness upon gene knockout. C, D CRISPR screen enrichment analysis. AD-bMSCs were transduced with Cas9-expressing vector followed by the designed sgRNA library (n = 3). AD-bMSCs were sampled on days 3, 7, 10, 16, 24, and 30 post-library transduction. C Minimum hypergeometric test (mHG) enrichment analysis. Log2 fold-change (y-axis) plotted against log2FC rank (x-axis) over time. Genes with top Gene Effect Scores (TGES) showed significant enrichment (red), while genes with bottom scores (BGES) showed significant depletion (blue). mHG p values are displayed for each day: TGES enrichment (top left, red) and BGES depletion (top right, blue). D Volcano plots of screen results by day. −log10 p value (x-axis) plotted against log2 fold-change (y-axis), calculated using MAGeCK. Red: significantly enriched gene-knockouts (leading with TP53 and PTEN); Blue: significantly depleted gene-knockouts; Gray: non-changing gene-knockouts. Dashed lines indicate significance thresholds (p < 0.05, |log2FC | > 1).
