Journal: The Journal of Biological Chemistry

Article Title: Whole-genome CRISPR screening identifies PI3K/AKT as a downstream component of the oncogenic GNAQ–focal adhesion kinase signaling circuitry

doi: 10.1016/j.jbc.2022.102866

Figure Lengend Snippet: PI3K/AKT pathway activation drives resistance to FAKi in GNAQ-mutant UM. A , schematic of whole-genome CRISPR screen experimental design. Created with Biorender.com . B , cell viability represented by beta score where a positive beta score indicates positive selection (resistance) (beta >0.5, indicated by dotted line ), and a negative beta score indicates negative selection (sensitivity) (beta <−0.5, indicated by dotted line ) under FAKi treatment. C , over-representation analysis of top sgRNAs (FDR <0.015) with positive beta score using KEGG and Biocarta gene sets. Color intensity of bars fade by decreasing −Log 10 p value. D , overall survival analysis of UM The Cancer Genome Atlas patient cohort with high ( top 25%) or low ( bottom 25%) expression of PI3K/AKT/mTOR Hallmark gene signature. Dotted lines indicate 95% confidence interval. E , cell viability of 92.1 UM cells after siRNA knockdown of PTEN or TSC2 compared with control siRNA in response to VS-4718 (FAKi) treatment for 72 h, percent viability is normalized to vehicle treatment (mean ± SD, n = 3). F , phosphorylation of FAK, AKT, and S6 after siRNA-mediated knockdown of CRISPR top hits (PTEN and TSC2) in 92.1 UM cells. Representative immunoblots are shown from n = 3 independent experiments. FAKi, focal adhesion kinase inhibitor; FDR, false discovery rate; KEGG, Kyoto Encyclopedia of Genes and Genomes; mTOR, mammalian target of rapamycin; PTEN, phosphatase and tensin homolog; sgRNA, single-gide RNA; TSC2, tuberous sclerosis complex 2; UM, uveal melanoma.

Article Snippet: The human Brunello whole-genome CRISPR pooled library was a gift from David Root and John Doench (Addgene #73178).

Techniques: Activation Assay, Mutagenesis, CRISPR, Selection, Expressing, Knockdown, Control, Phospho-proteomics, Western Blot

Journal: bioRxiv

Article Title: ARF4-mediated Retrograde Trafficking Drives Chemoresistance in Glioblastoma

doi: 10.1101/2021.07.18.451328

Figure Lengend Snippet: A) CRISPR-Cas9 knockout screening was performed using the Brunello whole-genome library, covering 19,000+ guides, ∼2000 non-targeting controls, and 4 sgRNAs per gene. A DMSO-treated population was compared to a TMZ-treated population, and guides were sequenced at d0 and d14. B) Comparison of d14 versus day 0 revealed segments of guides that were enriched and depleted. C) TMZ-sensitivity and TMZ-resistance genes identified and pursued were filtered with multiple CRISPR-appropriate analysis algorithms D) Specific genes were then examined for enrichment and depletion at day 0 and day 14. Known TMZ-resistance genes were depleted at d14, reflecting the validity of our screen. Key TMZ-sensitivity genes were identified as enriched ( MLH1, MSH6, PMS2, MSH2 ), further reflecting validity of the screen. E) Enrichment mapping identified key pathways across identified TMZ-sensitivity and TMZ-resistance genes. Enrichment mapping was performed in the Enrichr application. F) Of the 5966 genes identified as TMZ-resistance genes, 150 were significant by DESeq2 and MAGeCK algorithms. 34 were filtered based on novelty, ability to study, and clinical significance. 4 were finally chosen for validation to reflect critical pathways from the screen. Analysis was performed in Prism 8, using ANOVA to compare row-means to determine significance or using log-rank tests to determine survival significance *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Article Snippet: H4 human GBM cells were infected with the whole-genome knockout Brunello library (Addgene, Cambridge, MA, USA), which included ∼19,000 genes with 4 sgRNAs per gene and 10,000 sgRNA non-targeting controls.

Techniques: CRISPR, Knock-Out, Comparison, Biomarker Discovery

Journal: Nature Communications

Article Title: dFLASH; dual FLuorescent transcription factor activity sensor for histone integrated live-cell reporting and high-content screening

doi: 10.1038/s41467-025-58488-w

Figure Lengend Snippet: a HEK293T (upper panel) or U2OS (lower panel) mcdFLASH-HIF/iCas9-BFP cells were used to validate CRISPR KO loss of function screening by short term (3 days) inducible (dox) knockout of HIF-1α or gain of function with knockout of VHL. Data is representative of n = 2 independent experiments. b Schematic of the whole genome KO screening strategy. Briefly, clonal dFLASH-HRE reporter lines were transduced with a dox inducible Cas9-TagBFP virus and selected with blasticidin (BlastS) and for homogeneous Cas9-BFP induction. The Brunello whole genome KO library, ~77,000 guide RNAs targeting 19,114 genes, was then introduced and cells selected with puromycin (Puro) prior to a short-term (5 days) Cas9 induction with dox and FACS isolation of low activity pools. Created in BioRender. Peet, D. (2025) https://BioRender.com/m13q226 c dFLASH-HIF benchmarking against another published pooled HIF CRISPR screen. Loss of function CRISPR screen performed with Hela 3xHRE-mCherry ODD (Ortmann et al. 2021) vs HEK293T or U2OS dFLASH-HIF CRISPR screens. The number of enriched MAGeck CRISPR hits ( p < 0.05, one-tailed) were quantified and plotted. d Volcano plots of dFLASH-HRE CRISPR screens in HEK293T (left panel) or U2OS (right panel) demonstrating the strong selection of obligate regulators HIF-1α and ARNT. Differentially regulated genes from DEseq2 (generalised linear model with negative binomial distribution) with a p -value < 0.001 (grey dotted horizontal line, two-sided Wald test) and >0.6 LFC (grey dotted vertical line) are marked as red dots. Data presented is from n = 3 independent CRISPR screens. Source data are provided as a Source Data file.

Article Snippet: The Brunello whole genome KO library (Addgene #92379) was propagated in electrocompetent NEBstables by 5 parallel electroporations of 100 ng plasmid and 25 μl of electrocompetent NEBstables (lab made) using a Biorad Gene pulser (1.8 Kv, 200 mA, 25 μF) using 0.1 cm cuvettes (Biorad #165-2089), resulting in ~500x coverage.

Techniques: CRISPR, Knock-Out, Transduction, Virus, Isolation, Activity Assay, One-tailed Test, Selection

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

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) Flow cytometry quantification of viral infection in WT and KO Huh7.5.1-VP30 cells. Top-enriched candidate genes, identified through statistical analysis , were selected. Huh7.5.1-VP30 cells were transduced with two individual sgRNAs targeting each hit, followed by EBOVΔVP30-EGFP infection at MOI = 1. At 48hpi, cells were fixed, and viral infection (GFP-positive rate) was analyzed by flow cytometry, normalized to infected WT Huh7.5.1-VP30 cells. ( B ) Validation of SLC39A9 and PIK3C3 KO efficiency through immunoblotting ( left ); Huh7.5.1-VP30 cells transduced with sgRNAs were infected with EBOVΔVP30-EGFP virus at MOI = 1. Huh7 WT or KO infected cells were analyzed by RT-qPCR to determine EBOV NP RNA abundance ( right ). (C) At 48hpi, supernatants were collected for titration, and focus-forming units per mL (FFU/mL) were calculated. Complementation of Huh7.5.1-VP30 KO cells with respective cDNAs of PIK3C3 ( D ) or SLC39A9 ( E ). SLC39A9 and PIK3C3 protein levels were detected by WB ( upper ), and virus-infected cells were quantified by flow cytometry at 2dpi ( lower ). ( F ) NTC or KO Huh7 cells were challenged with authentic EBOV (MOI = 0.1) for 3 days. RT-qPCR assay determined EBOV RNA abundance, normalized to GAPDH. Error bars represent SD of the mean from one representative experiment with triplicates. Significance assessed by one-way ANOVA, with asterisks indicating significant differences: NS, no significance (p ≥ 0.05); *, p < 0.05; **, p < 0.01; ***, p < 0.001. The experiments were repeated in three independent trials with similar results.

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: Flow Cytometry, Infection, Transduction, Biomarker Discovery, Western Blot, Virus, Quantitative RT-PCR, Titration

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 ) Huh7 WT or KO cells were inoculated with EBOVΔVP30-EGFP virus at MOI = 10 at 37°C for 3h; absence of transcription factor VP30 in Huh7 cells stuck the virus in the entry step ( lower ). Unbound viruses were washed three times with PBS and cellular RNA was extracted for RT-qPCR analysis. Quantification results are showing on the ( B ). (C) Schematic view of the EBOV replicon assay. Huh7-4P replicon cells, transduced with NTC, PIK3C3, or SLC39A9 targeting sgRNAs, were assessed for viral replication through supernatant Gluc activity or cellular viral RNA level. ( D ) KO efficiency of replicon cells detected by PIK3C3 and SLC39A9 immunoblotting. NTC or KO replicon cells were analyzed for Gluc activity (E) and RNA level (F) after 3 days. ( G ) Schematic view of the EBOV virus-like particle (VLP) production assay. NTC, PIK3C3, or SLC39A9 targeting sgRNAs were transduced into HEK293T cells, and KO efficiency was confirmed by WB ( H ). Co-transfection of EBOV VP40, NP, and GP expression vectors in NTC or KO 293T cells produced VLPs. Concentrated VLP supernatants were collected for WB analysis. ( I ) WB analysis and quantification of concentrated EBOV VLPs. All three viral proteins were detected, and intensity quantification was performed using ImageJ. Experiments were repeated at least three times with similar results; error bars represent the SD of the mean from one representative experiment with triplicates. Significance assessed by one-way ANOVA, with asterisks indicating significant differences: ns, no significance (p ≥ 0.05); *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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: Virus, Quantitative RT-PCR, Transduction, Activity Assay, Western Blot, Cotransfection, Expressing, Produced