cells bassik lab human crispr deletion library28  (Addgene inc)

Journal: Nature Structural & Molecular Biology

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

doi: 10.1038/s41594-026-01759-x

Figure Lengend Snippet: a , Schematic of CRISPR–Cas9 knock-in strategy. A C-terminal GFP-P2A-BFP tag was inserted into the FSP1 genomic locus before the stop codon. Cells expressing both GFP and BFP were enriched through multiple rounds of FACS. b , Fluorescence histograms of WT control and genomic FSP1 GFP-P2A-BFP knock-in cells. c , Immunoblot of lysates from control and FSP1 GFP-P2A-BFP cells from b . d , Fluorescence histograms of FSP1 GFP-P2A-BFP cells incubated with siRNAs against FSP1 for 72 h, including siRNAs from scramble control. e , Immunoblot of control and FSP1 GFP-P2A-BFP cells from d . f , FSP1 GFP-P2A-BFP subcellular distribution by live-cell microscopy. Cells were treated with 200 µM oleate for 24 h to induce lipid droplet formation, 500 nM LipiBlue to label lipid droplets and 5 µg ml −1 CellMask deep red to label the plasma membrane. g , Dose response of RSL3-induced cell death of WT and FSP1 GFP-P2A-BFP reporter cells in the presence or absence of 2 µM FSEN1 for 24 h. Data are shown as the mean ± s.e.m. of three wells of a 96-well plate from three independent experiments. h , Fluorescence histograms of FSP1 GFP-P2A-BFP cells treated with proteolysis inhibitors (5 mM 3-MA, 250 nM Baf-A1, 5 µM CB5083, 10 µM MLN7243 and 10 µM MG132) for 6 h. i , Quantification of MFI change in GFP:BFP ratio from h . Data are shown as the median ± s.e.m. of four independent experiments. NS, not significant.

Article Snippet: Genome-wide CRISPR–Cas9 screens were performed using the Bassik human CRISPR KO library (Addgene, pooled libraries 101926–101934).

Techniques: CRISPR, Knock-In, Expressing, Fluorescence, Control, Western Blot, Incubation, Microscopy, Clinical Proteomics, Membrane

Journal: Nature Structural & Molecular Biology

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

doi: 10.1038/s41594-026-01759-x

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 , Top, cloud plot indicating count numbers corresponding to FSP1 (color scale) and control (gray scale) sgRNAs. Bottom, distribution for the relative enrichment of FSP1 sgRNAs (blue lines). 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 KO library (Addgene, pooled libraries 101926–101934).

Techniques: Genome Wide, CRISPR, Control, Functional Assay

Journal: Nature Structural & Molecular Biology

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

doi: 10.1038/s41594-026-01759-x

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 , Heat maps 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 and NFE2L2 signaling, selenocysteine synthesis, CoQ synthesis and glycolysis. Genes are annotated with modes corresponding to gene effects and scores from batch retest of untreated and 100 nM RSL3-treated expression (BFP) screens. Red asterisks indicate genes reported to be regulated by NFE2L2. f , Heat maps 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 KO library (Addgene, pooled libraries 101926–101934).

Techniques: CRISPR, Expressing

Journal: Nature Structural & Molecular Biology

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

doi: 10.1038/s41594-026-01759-x

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 MFI change in GFP from d . Data are shown as the median ± s.e.m. of four independent experiments. f , Immunoblot of lysates from d . g , h , Ubiquitinated FSP1–GFP conjugates affinity-captured from lysates treated with 1 µM MG132 for 24 h ( g ) or expressing exogenous RNF8 variants ( h ). i , Kinetics of GFP fluorescence decay of FSP1 KO cells expressing FSP1–GFP in Cas9 control or RFK KO cells with or without the loss of RNF8 following Dox washout. j , 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). k , Model illustrating the mechanism by which vitamin B2 metabolism and FAD synthesis impact FSP1 activity and stability to prevent ferroptosis.

Article Snippet: Genome-wide CRISPR–Cas9 screens were performed using the Bassik human CRISPR KO library (Addgene, pooled libraries 101926–101934).

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

Journal: bioRxiv

Article Title: Antibody-Dependent Heterotypic Syncytia Drive COVID-19 Inflammation and Disease Progression

doi: 10.64898/2026.02.11.705426

Figure Lengend Snippet: A , Schematic of the CRISPR-Cas9 screen. B , Volcano plot of genes from the screen, ranked by fold change and adjusted P value. C , Representative flow cytometry dot plots of GW01-induced syncytia between A549-spike and FCER1G -KO (FcRγ-KO) THP-1 clones versus non-targeting control (NT), and quantification of syncytia(right inset)(mean ± SEM, n=3, * P <0.05, **** P <0.0001). D , Representative plots A549-spike fusion with ADAM10-KO THP-1 clones versus control and quantification of syncytia. E , Rescue of FcRγ: representative plots for FcRγ-KO-LVX (empty lentiviral vector), FcRγ-KO-FcRγ (FcRγ cDNA), and NT-LVX; with quantification of syncythia. F , Rescue of ADAM10: representative plots for ADAM10-KO-LVX (empty vector), ADAM10-KO-ADAM10 (ADAM10 cDNA), and NT-LVX, along with quantification of syncytia. G , Effect of CD64 or CD32 blocking antibodies on GW01-dependent syncytium formation measured by NanoLuc assay. H , Effect of the ADAM10 inhibitor GI254023X on GW01-dependent syncytium formation. I , Representative fluorescence images of syncytia (yellow merge) between THP-1-mCherry (red) and A549-spike (green; CFSE) at 3 hour post co-culture, with or without GI254023X. Scale bar, 100 μm. Data are presented as mean ± SEM, n=3, * P <0.05, *** P <0.001, **** P <0.0001 by one-way ANOVA.

Article Snippet: The genome-wide CRISPR sgRNA library (Addgene, 101926-101934) was packaged into lentiviruses.

Techniques: CRISPR, Flow Cytometry, Clone Assay, Control, Plasmid Preparation, Blocking Assay, Fluorescence, Co-Culture Assay

Journal: Nature Cell Biology

Article Title: DOT1L provides transcriptional memory through PRC1.1 antagonism

doi: 10.1038/s41556-025-01859-8

Figure Lengend Snippet: a-c . Bubble plots of -Log10(p-value) for: ( a ) MV4;11 SGC0946 genome-wide CRISPR screen at day 12, ( b ) murine MLLAF9 SGC0946 epigenetic-focused screen and ( c ) murine MLLAF9 VTP50469 epigenetic-focused screen at day 13. P-values calculated using the MAGECK algorithm and adjusted for multiple testing. d . Competition assay in MOLM13 control, RING1B, or KDM2B sgRNA cells treated with SGC0946 or VTP50469 . Data represents mean ± SD from n = 4 independent experiments. e . Competition assay in Murine MLLAF9 control, BCOR, or PCGF1 sgRNA cells treated with SGC0946 or VTP50469 . Data represents mean ± SD from n = 3 independent experiments. f-g . Competition assay in MOLM13 control, KMT2D sgRNA1/2 cells ( f , data represents mean ± SD from n = 3 independent experiments), or UTX sgRNA1/2 ( g , data from 2 independent experiments) treated with SGC0946 or VTP50469 for 7 days. h . Proliferation of MOLM13 control or PCGF1 sgRNA1/2 cells treated with SGC0946 or VTP50469 . Data represents mean ± SD from n = 3 independent experiments. i . Proliferation of control or PCGF1 KO MOLM13 with empty vector (EV) or wildtype PCGF1 (WT) treated with SGC0946 or VTP50469 . Data represents mean ± SD from n = 3 independent replicates. j . Proliferation of MOLM13 control or BCOR sgRNA1/2 cells treated with VTP50469 . Data represents mean ± SD from n = 3 independent experiments. k . Ezh2 and Eed sgRNA counts at day 2 and 13 from (c). l . DepMap scores for Ezh2 and Eed . Data represents mean ± SD from n = 6 MLL-rearranged cell lines. m . DepMap gene effect scores for MLL1, PCGF1, and BCOR. Data represents mean ± SD from n = 6 MLL-rearranged cell lines. n . Western blot of H3K79me2 and histone H3 in MOLM13 control, PCGF1 sgRNA1/2, and BCOR sgRNA1/2 cells treated with SGC0946 for 4 days. o . Dose response assay in murine MLLAF9 control or PCGF1 sgRNA1/2 cells treated with SGC0946 or VTP50469 . p . Proliferation assay in control or PCGF1 KO murine MLLAF9 cells treated with VTP50469 . Data represents mean ± SD from n = 3 independent experiments. q . Representative Annexin V gating strategy. Competition assay used BFP+ sgRNA cells with data as BFP + % normalised to DMSO. Source data available for d - f , h - o , and p .

Article Snippet: The whole genome Bassik Human CRISPR Knockout Library was obtained from Addgene.

Techniques: Genome Wide, CRISPR, Competitive Binding Assay, Control, Plasmid Preparation, Western Blot, Proliferation Assay

Journal: Nature Cell Biology

Article Title: DOT1L provides transcriptional memory through PRC1.1 antagonism

doi: 10.1038/s41556-025-01859-8

Figure Lengend Snippet: a , A schematic of the CRISPR survival screens. MV4;11 Cas9 or murine MLLAF9 Cas9 cells were infected with either a genome-wide or epigenetics-focused (1,134 genes) sgRNA CRISPR library. Cells were treated with DMSO, the Menin inhibitor VTP50469 (MENi, 100 nM) or the DOT1L inhibitor SGC0946 (DOT1Li, 5 μM). Samples were collected at days 12 and 24 (MV4;11 cells) and day 14 (murine MLLAF9 cells). b , A bubble plot showing the genes required for the efficacy of SGC0946 (day 24) in MV4;11 cells from the whole-genome CRISPR screen. P values were calculated using the MAGECK algorithm and adjusted for multiple testing. c , A schematic overview of the complexes of interest identified in the CRISPR screen: PRC1.1 and MLL3/4 with the enriched components highlighted in colour. d , A Venn diagram of the top 20 most significant hits from the murine MLLAF9 SGC0946 and VTP50469 survival screens performed using an epigenetics-focused CRISPR library. e , sgRNA negative selection competition assay in MV4;11 Cas9 cells transduced with non-silencing sgRNA (control) or two independent sgRNAs against BCOR or PCGF1. The percentage of sgRNA positive cells remaining over time. Data represent the mean ± s.d. from n = 3 independent experiments. f , A proliferation assay in control (ctrl) or two independent PCGF1 (left) or BCOR (right) sgRNAs in MV4;11 Cas9 cells treated with DMSO or SGC0946 5 μM as indicated. Data represent the mean ± s.d. from n = 3 independent replicates. g , A schematic of the layered DOT1L sgRNA negative selection competition assay. Two independent DOT1L sgRNAs were layered on top of cells already expressing BFP-control, PCGF1 or BCOR sgRNAs. These double KOs were then mixed with single KOs of each and the percentage of GFP + was measured by flow cytometry. h , sgRNA negative competition assay using MOLM13 Cas9 control, PCGF1 or BCOR KO-BFP cells transduced with two independent sgRNA against DOT1L or non-silencing control sgRNA linked with GFP. The percentage of sgRNA positive cells remaining over time are shown. Data represent the mean ± s.d. from n = 3 independent experiments. i , Bar plots of the percentage of Annexin V positive (apoptotic) control or PCGF1 KO murine MLLAF9 cells treated with either DMSO, SGC0946 (3 μM) or VTP50469 (300 nM) for 4 or 6 days. Data represent mean ± s.d. from n = 3 independent experiments. j , Proliferation assays with control or PCGF1/BCOR KO MOLM13 cells treated with ABT-199 (100 nM) or I-BET151 (1 μM). Data represent mean ± s.d. from n = 3 independent experiments. k , A proliferation assay in control or PCGF1/BCOR KO MOLM13 cells treated with DMSO or combined treatment with SGC0946 (5 μM) and VTP50469 (500 nM) as indicated. Data represent n = 3 independent experiments.

Article Snippet: The whole genome Bassik Human CRISPR Knockout Library was obtained from Addgene.

Techniques: CRISPR, Infection, Genome Wide, Selection, Competitive Binding Assay, Transduction, Control, Proliferation Assay, Expressing, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Genes driving three-dimensional growth of immortalized cells and cancer

doi: 10.1038/s41419-025-07719-5

Figure Lengend Snippet: a Representative images of 3D growth of HBLAK cells in ultra-low attachment plates. The scale bar represents 100 µm. b Plots illustrating the area of spheroids and CyQuant fluorescence readings over time. c Images showing that HBLAK cells do not grow but survive in the soft agar clonogenic assay while J82 cells are able to grow and form viable colonies. d The growth kinetics of HBLAK xenografts in FOXN−/− mice when injected at different cell numbers, showing a gradual decrease and eventually disappearance of the xenografts. M: million. e Workflow of the CRISPR screening in HBLAK cells. The cells were first transduced with spCas9 containing virus, which were re-infected with the DTKP Bassik library. The positively transfected cells, selected via antibiotic, were allowed to grow in 2D and 3D growth conditions. The surviving cells were sequenced to identify the sgRNAs that dropped out over time. f Workflow depicting how the FASTQ files of sequencing data were processed using the MAGeCK method to produce a phenotype score for each gene. g Scatter plots showing the diverse phenotype patterns in the 2D—day 14 and 3D—day 14 normalized against day 0 at respective growth setting.

Article Snippet: CRISPR-Cas9 depletion screen was conducted with Bassik Human CRISPR deletion library-Drug targets, kinases, and phosphatases (DTKP, Addgene, #101927) [ ] using HBLAK cells.

Techniques: CyQUANT Assay, Fluorescence, Clonogenic Assay, Injection, CRISPR, Transduction, Virus, Infection, Transfection, Sequencing