human crispr knockout library h3  (Addgene inc)

Journal: Cell Death & Disease

Article Title: Loss of ELF2 drives topotecan resistance in retinoblastoma revealed by genome-wide CRISPR-Cas9 screening

doi: 10.1038/s41419-025-08335-z

Figure Lengend Snippet: A The schematic diagram illustrates the workflow of genome-wide CRISPR-Cas9 knockout library screening (CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats). B The scatter plot depicts the results for topotecan positively selected hits in the CRISPR-Cas9 screening, with the top 20 hits shown in red. C KEGG analysis of the top 50 topotecan positively selected hits identified through genome-wide CRISPR-Cas9 knockout screening. D Relative cell viability of WERI-Rb1 and Y79 cells following treatment with topotecan or vehicle for 96 hours ( n = 3). E , F ELF2 protein expression in WERI-Rb1 and Y79 cells under topotecan treatment ( n = 3). Data are presented as means ± SD. Statistical analysis was performed using two-tailed Student’s t test ( D ) or one-way ANOVA and Tukey’s multiple comparison test ( F ); ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Article Snippet: The Human CRISPR Knockout Pooled Library (hGeCKO v2; Addgene #1000000048) was utilized to identify the genes responsible for topotecan resistance in retinoblastoma cells.

Techniques: Genome Wide, CRISPR, Knock-Out, Library Screening, Expressing, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: Vitamin B2 metabolism promotes FSP1 stability to prevent ferroptosis

doi: 10.1101/2025.08.05.668752

Figure Lengend Snippet: a , Schematic of CRISPR-Cas9 strategy used to generate the FSP1 GFP-P2A-BFP reporter system. b , Fluorescence histograms of wild-type control and genomic FSP1 GFP-P2A-BFP knock-in cells. c , Immunoblot of lysates from FSP1 GFP-P2A-BFP cells. endo., endogenous FSP1. d , Immunoblot of control and FSP1 GFP-P2A-BFP cells incubated with siRNAs against FSP1 for 72 h. e , Fluorescence histograms of FSP1 GFP-P2A-BFP cells from ( d ) including siRNAs from scramble control. f , Buoyant fractions, enriched with lipid droplets from FSP1 GFP-P2A-BFP cells, were isolated by sucrose gradient fractionation and analyzed by western blot. BF, buoyant fraction . g , FSP1 GFP-P2A-BFP subcellular distribution by live-cell microscopy. Cells were incubated with 200 µM oleate to induce lipid droplet formation, 500 nM LipiBlue to label lipid droplets, and 5 µg/mL CellMask Deep Red to label the plasma membrane. Line intensity plots showing colocalization between FSP1 GFP-P2A-BFP and organelle markers (right). h , Live-cell imaging of mCherry-expressing control and FSP1 GFP-P2A-BFP cells incubated with SYTOX Green and treated with 100 nM RSL3 for 24 h. Scale bar, 100 µm. i , Dose response of RSL3-induced cell death of wild-type and FSP1 GFP-P2A-BFP reporter cells in the presence of 2 µM FSEN1.

Article Snippet: Genome-wide CRISPR-Cas9 screens were performed using the Bassik Human CRISPR Knockout Library (Addgene pooled libraries nos.

Techniques: CRISPR, Fluorescence, Control, Knock-In, Western Blot, Incubation, Isolation, Fractionation, Microscopy, Clinical Proteomics, Membrane, Live Cell Imaging, Expressing

Journal: bioRxiv

Article Title: Vitamin B2 metabolism promotes FSP1 stability to prevent ferroptosis

doi: 10.1101/2025.08.05.668752

Figure Lengend Snippet: a , Schematic of the genome-wide CRISPR-Cas9 screening strategy. b , Gene effects and gene scores calculated for individual genes analyzed in the genome-wide CRISPR screen. c , Cloud plot indicating count numbers corresponding to FSP1 (color scale) and control (gray scale) sgRNAs (top). Frequency histogram indicating the distribution of the relative enrichment for FSP1 sgRNAs (blue lines, bottom). The gray line indicates the mean of the control sgRNA distribution. d , Bubble plot of the genes identified from the genome-wide screen with a 10% FDR cutoff, where each bubble color represents an enriched GO term functional cluster. e , Scatter plot of signed gene scores for individual genes from genome-wide screen (x-axis) versus batch retest screen (y-axis).

Article Snippet: Genome-wide CRISPR-Cas9 screens were performed using the Bassik Human CRISPR Knockout Library (Addgene pooled libraries nos.

Techniques: Genome Wide, CRISPR, Control, Functional Assay

Journal: bioRxiv

Article Title: Vitamin B2 metabolism promotes FSP1 stability to prevent ferroptosis

doi: 10.1101/2025.08.05.668752

Figure Lengend Snippet: a , Schematic of the CRISPR-Cas9 batch retest screening strategy. b-c , Gene effects and gene scores calculated for individual genes analyzed in the batch retest CRISPR screens for expression (BFP, b ) or stability (GFP:BFP, c ). d , Heatmaps of clustered genes based on the signed gene scores from GO enrichment analysis of the core transcriptional regulators. e , Schematic of cytosolic redox pathways including KEAP1/NFE2L2 signaling, selenocysteine synthesis, CoQ synthesis, glycolysis, etc. Genes are annotated with modes corresponding to gene effects and scores from batch retest basal and 100 nM RSL3-treated expression (BFP) screens. f , Heatmaps of clustered genes based on the signed gene scores from GO enrichment analysis of the core post-translational regulators.

Article Snippet: Genome-wide CRISPR-Cas9 screens were performed using the Bassik Human CRISPR Knockout Library (Addgene pooled libraries nos.

Techniques: CRISPR, Expressing

Journal: bioRxiv

Article Title: Vitamin B2 metabolism promotes FSP1 stability to prevent ferroptosis

doi: 10.1101/2025.08.05.668752

Figure Lengend Snippet: a , Schematic of sensitized UBAL degradation CRISPR-Cas9 screen. b , Gene effects and gene scores calculated for individual genes analyzed in the targeted degradation screen. c , STRING network map of the identified genes with a 10% FDR cutoff, where each bubble color represents an enriched GO term functional cluster. d , Fluorescence histograms of FSP1 KO cells expressing FSP1-GFP in the indicated cell lines. e , Quantification of median fluorescence intensity (MFI) change in GFP from ( d ). f , Immunoblot of lysates from ( d ). g-h , Kinetics of GFP fluorescence decay of FSP1 KO cells expressing FSP1-GFP in control or RFK KO cells with or without the loss of RNF8 ( g ) or RFK/RNF8 DKO (double knock-out) cells expressing the indicated RNF8 variants ( h ) following doxycycline washout. i , FSP1-GFP or RNF8 S-tag immunoprecipitation from FSP1 KO cells expressing FSP1-GFP in the indicated cell lines and blotting for RNF8 (top) or FSP1 (middle). j , Model illustrating the mechanism by which vitamin B2 metabolism and FAD synthesis impacts FSP1 activity and stability to prevent ferroptosis.

Article Snippet: Genome-wide CRISPR-Cas9 screens were performed using the Bassik Human CRISPR Knockout Library (Addgene pooled libraries nos.

Techniques: CRISPR, Functional Assay, Fluorescence, Expressing, Western Blot, Control, Knock-Out, Immunoprecipitation, Activity Assay

Journal: JCI Insight

Article Title: Imlunestrant a next-generation oral SERD overcomes ESR1 mutant resistance in estrogen receptor–positive breast cancer

doi: 10.1172/jci.insight.188051

Figure Lengend Snippet: ( A ) Cartoon schematic of the genome-wide CRISPR/Cas9-knockout (KO) screen; (step 1) T47D cells are infected with the H3 lentiviral library of gRNAs, (step 2) infected cells are selected using puromycin, (step 3) cells are collected for untreated baseline (Day 0), or treated with DMSO or 1 nM imlunestrant, (step 4) after treatment for 14 or 31 days cells are collected, gRNAs amplified, and sequenced, followed by (step 5) gRNA analysis using MAGeCK algorithm. ( B ) MAGeCK β score and ranking results for gRNAs enriched or depleted after treatment with DMSO versus Day 0 for 14 days or ( C ) 31 days. ( D ) MAGeCK β score and ranking results for gRNAs enriched or depleted after treatment with imlunestrant versus Day 0 for 14 days or ( E ) 31 days. Scatter plot with each dot represents a gene in the genome-wide gRNA library, red are enriched after treatment (β ≥ +1), blue are depleted after treatment (β ≤ –1), black are insignificant after treatment, genes in the ER pathway are orange, the CDK pathway are purple, and the MTORC1 pathway are green. ( F ) β scores for individual genes depleted after imlunestrant treatment in the ER pathway, ( G ) the CDK pathway, or ( H ) the MTORC1 pathway.

Article Snippet: For the CRISPR screen we used the H3 genome wide gRNA library (Addgene #133914) that targets 18,000 annotated genes in the human genome, with 6 gRNAs per gene on average for a total of 117,587 gRNAs and 3,842 control gRNAs targeting AAVS1, ROSA26, and CCR5.

Techniques: Genome Wide, CRISPR, Knock-Out, Infection, Amplification