brunello whole genome library  (Addgene inc)

Journal: Cancer prevention research (Philadelphia, Pa.)

Article Title: Genome-wide CRISPR screening reveals a PKA-driven resistance mechanism to metformin for oral cancer prevention that can be exploited by combination with NSAIDs

doi: 10.1158/1940-6207.CAPR-25-0264

Figure Lengend Snippet: (A) Schema of genome-wide CRISPR screen experimental design. Cartoon created with BioRender. (B) Table of genes encoding for AMPK subunits. (C) Top 20 normalized enrichment scores from pre-ranked GSEA run on beta-score-ranked genes in the Reactome Pathways obtained from MSigDB (left). Reactome Pathways with gene sets related to PKA signaling are bolded for emphasis (left). Table of genes encoding the regulatory subunits of PKA (right). (D) Western blot for Cal27 cells treated with metformin for 24 hours at the indicated doses. Total and phosphorylated CREB are shown, in addition to the phosphorylation status of the PKA substrate motif RRXS*/T* (PKA substrates). HSP90 was used as a loading control (left). Representative immunoblots are shown from n = 3 independent experiments. Quantification of p-CREB signal normalized to total CREB levels and p-PKA substrates normalized to control signal (right). Data are shown as mean ± SEM, n = 3. Statistical analysis was performed using one-way ANOVA. (E) Cell viability after 72 h treatment with 3 mmol/L metformin or 3 mmol/L metformin + 10 μM forskolin (Fsk) in Cal27 cells. Statistical analysis was performed using a t-test.

Article Snippet: For whole-genome screening, the human Brunello whole-genome CRISPR pooled library (RRID:Addgene_73179; gifted by David Root and John Doench) was employed.

Techniques: Genome Wide, CRISPR, Western Blot, Phospho-proteomics, Control

Journal: bioRxiv

Article Title: THOC1 complexes with SIN3A to regulate R-loops and promote glioblastoma progression

doi: 10.1101/2024.09.24.614748

Figure Lengend Snippet: A) Schematic of the whole-genome CRISPR-Cas9 knockout screen in human H4 GBM cells using the Brunello library to identify genes critical for GBM progression. Sequencing at days 0, 14, and 28 highlights top depleted guides as potential oncogenic drivers. B) Identification of THOC1 as a significant hit from the CRISPR screen. C) Pathway enrichment analysis showing THOC1’s involvement in several key pathways identified in the screen. D) Elevated THOC1 RNA expression in GBM compared to non-tumor samples, based on data from the GlioVis portal. E) Increased THOC1 protein expression in GBM tissue compared to normal brain tissue, as shown by Protein Atlas data. F) Western blot analysis showing higher baseline THOC1 expression in GBM patient-derived xenograft (PDX) line (GBM43) compared to a non-cancerous line. G) cBioPortal data indicating a low mutation rate of THOC1 in GBM (approximately 5%). H) Single-cell RNA sequencing data from GBMSeq showing high THOC1 expression localized to the tumor core.

Article Snippet: We initiated the process by infecting H4 human GBM cells with the Brunello whole-genome knockout library (Addgene, Cambridge, MA, USA), which included around 19,000 genes, each with 4 sgRNAs, along with 10,000 sgRNA non-targeting controls.

Techniques: CRISPR, Knock-Out, Sequencing, RNA Expression, Expressing, Western Blot, Derivative Assay, Mutagenesis, RNA Sequencing

Journal: PLOS Pathogens

Article Title: Genome-wide CRISPR/Cas9 screen identifies SLC39A9 and PIK3C3 as crucial entry factors for Ebola virus infection

doi: 10.1371/journal.ppat.1012444

Figure Lengend Snippet: ( A ) Schematic representation of the genome-wide CRISPR KO screen. Huh7.5.1-VP30-Cas9 CRISPR KO cells were infected with EBOVΔVP30-EGFP at MOI = 1, and GFP-negative cells were sorted at 3 days post-infection (dpi). Three rounds of infection and sorting were performed, and sgRNA abundance was analyzed using next-generation sequencing in both uninfected and selected cell populations. ( B ) Significance of enriched genes from three rounds of CRISPR KO screen based on MAGeCK analysis. The x and y-axes represent the mean log2 fold change of sgRNA counts from the first and second rounds of selection, respectively. Red dots indicate top candidates in the third selection, with circle diameter representing the number of enriched sgRNAs. Non-targeting controls are indicated as blue dots. The top 300 ranked gene list of three selections is provided in . ( C ) Venn diagram illustrating the overlap of top hits (p value < 0.005, ) from each round of the screens. ( D ) Gene Ontology Biological Process (GO BP) analysis of the top enriched genes from ( C ). The complete list of GO terms is available in . GO analysis was performed using ToppGene.

Article Snippet: Human whole-genome sgRNA library Brunello in backbone lentiGuide-Puro targeting 19114 genes and containing 76441 unique sgRNAs along with 1000 non-targeting controls (Addgene, #73178) [ , ].

Techniques: Genome Wide, CRISPR, Infection, Next-Generation Sequencing, Selection