Journal: bioRxiv

Article Title: Parallel CRISPR screens reveal pathways controlling the cell surface levels of the attractant receptor FPR1

doi: 10.1101/2025.04.21.649864

Figure Lengend Snippet: Clonally selected β-arrestin double CRISPR knockout cells are compared to pooled double β-arrestin knockout cell lines using the FPR1 endocytosis assay. Cells were untreated or stimulated with 1 μM fMLF. (A) A representative plot showing fMLF-induced decrease in surface FPR1 in the control sgRNA expressing cells. (B) Pooled double β-arrestin knockout cells have a smaller shift after fMLF stimulation. (C) Representative plots obtained from twelve clonally selected β-arrestin double knockout cell lines. Phenotypes observed in clonally selected and pooled knockout cell lines resemble each other.

Article Snippet: Bassik Lab Human CRISPR Deletion Library , Addgene catalog #101926- 101934, was packaged into lentiviral particles using TransIT-2020 transfection reagent (VWR, catalog #10767-014).

Techniques: CRISPR, Knock-Out, Endocytosis Assay, Control, Expressing, Double Knockout

Journal: bioRxiv

Article Title: Parallel CRISPR screens reveal pathways controlling the cell surface levels of the attractant receptor FPR1

doi: 10.1101/2025.04.21.649864

Figure Lengend Snippet: (A) Schematic representation of a selection of hits found in the parallel whole-genome CRISPR screens. Genes shown are significant hits in at least one screen, and they were organized based on prior literature. The effect scores for each gene are represented as colored rectangles. The scores are only shown if the gene was a hit in the particular screen (pFDR< 0.05). Negative scores indicate a decrease in surface FPR1, while positive scores indicate an increase. For hits regulating FPR1 surface expression post-stimulation (shown in pink and green), the effect scores obtained from the integrated analysis of the two screens were presented. (B) Top hits from the surface FPR1 expression screen. FPR2 and FPR3 were shown for comparison and are not identified as hits in either of the screens. (C) Significant hits from the integrated analysis of FPR1 internalization, recycling, or exocytosis. Scatter plots in B and C represent the same dataset (log Pscore, with the basal surface expression score on the y-axis and post-stimulation score on the x-axis) with different genes highlighted.

Article Snippet: Bassik Lab Human CRISPR Deletion Library , Addgene catalog #101926- 101934, was packaged into lentiviral particles using TransIT-2020 transfection reagent (VWR, catalog #10767-014).

Techniques: Selection, CRISPR, Expressing, Comparison

Journal: International journal of molecular sciences

Article Title: Onvansertib and Navitoclax Combination as a New Therapeutic Option for Mucinous Ovarian Carcinoma.

doi: 10.3390/ijms26020472

Figure Lengend Snippet: Figure 2. Genes identified by CRISPR/Cas9 screening in EFO27/Cas9. (A) There were 12 genes with an FDR < 0.05 compared to the not treated groups T1 and T0. These are the genes that, if depleted, lead to cell death. (B) Comparing T1-treated with T1-untreated genes, the selection based on the FDR < 0.1 of individual sgRNAs allowed for the identification of three genes with potential synthetic lethality with onvansertib. For each indicated gene, blue lines represent the gRNAs that were downregulated, while the red lines are the gRNAs that were upregulated. CRISPR-seq data were processed using the MAGeCK pipeline [36] in the MAGeCKFlute R package [37].

Article Snippet: For screening, we used the Lentiviral Bassik Human CRISPR Deletion Library—Apoptosis and Cancer (Addgene #101926), composed of 31,324 sgRNAs, targeting 3015 genes, with about 10 sgRNAs targeting each gene.

Techniques: CRISPR, Selection

Journal: bioRxiv

Article Title: CLCC1 promotes hepatic neutral lipid flux and nuclear pore complex assembly

doi: 10.1101/2024.06.07.597858

Figure Lengend Snippet: A) Top : Screen conditions were optimized in Huh7 cells treated for 24 h with 1 µg/mL triacsin C or 100 µM oleate and analyzed by fluorescence microscopy of cells labeled with BODIPY 493/503 (LDs) and DAPI (nuclei). Bottom : Flow cytometry BODIPY 493/503 fluorescence histograms of untreated, 1 µg/mL triacsin C- and 100 µM oleate-treated Huh7 cells. B) Schematic of FACS-based CRISPR-Cas9 screen approach to identify genes that regulate neutral lipid abundance, using BODIPY 493/503 as a neutral lipid reporter. C) Volcano plot indicating the gene effects (i.e., phenotype) and gene scores (i.e., confidence) for individual genes from batch retest screens in Huh7 cells. Gene effects and scores are calculated from two biological replicates. Positive (red) and negative (blue) genes of interest are highlighted. D) 265 of the 285 credible hits mapped in STRING confidence (text mining, experiments, physical interactions, genetic interactions, functional pathways) grouped manually by GO functional annotations. E) Schematic of parallel CRISPR screens under eleven different metabolic stress conditions. F) Heatmap of clustered genes based on gene score across all conditions. Boxes 1-4 indicate clusters of core positive regulators and boxes 5-10 indicate clusters of core negative regulators.

Article Snippet: Guide sequences for CLCC1, TMEM41B, VMP1, and safe-targets (sgSAFE #5784) (see Table 1) were selected from the Bassik Human CRISPR Knockout Library (Addgene, 101926, 101927, 101928, 101929, 101930, 101931, 101932, 101933, 101934).

Techniques: Fluorescence, Microscopy, Labeling, Flow Cytometry, CRISPR, Functional Assay

Journal: bioRxiv

Article Title: CLCC1 promotes hepatic neutral lipid flux and nuclear pore complex assembly

doi: 10.1101/2024.06.07.597858

Figure Lengend Snippet: A) Quantification of the amount of neutral lipid from flow cytometry histograms (representative histograms shown in ). Data represent mean ± SD across three biological replicates. ****p<0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test . B) Breakdown of the custom lipid metabolism CRISPR-Cas9 library used in batch retest screens. C) Pairwise comparison of gene scores between the genome-wide and batch retest screens. Scores were adjusted as positive or negative based upon the direction of gene effect. D) Significant gene clusters from . Nodes are marked based on directionality of effect (red or blue), gene effect size, and confidence score.

Article Snippet: Guide sequences for CLCC1, TMEM41B, VMP1, and safe-targets (sgSAFE #5784) (see Table 1) were selected from the Bassik Human CRISPR Knockout Library (Addgene, 101926, 101927, 101928, 101929, 101930, 101931, 101932, 101933, 101934).

Techniques: Flow Cytometry, CRISPR, Comparison, Genome Wide

Journal: bioRxiv

Article Title: CLCC1 promotes hepatic neutral lipid flux and nuclear pore complex assembly

doi: 10.1101/2024.06.07.597858

Figure Lengend Snippet: A) Cluster of top negative regulators of lipid storage from metabolic state-dependent CRISPR- Cas9 screens. B) Representative confocal images of lipid droplets using BODIPY 493/503 in control (expressing safe targeting sgRNA) and CLCC1 KO cells under basal conditions or following treatment with 1 µg/mL triacsin C for 24 h, 100 µM oleate for 24 h, or serum starve for 48 h. C) Quantification of the number of LDs from (C). Data represent mean ± SD of > 100 cells across three biological replicates. ****p<0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test . D) Quantification of the area of LDs from (C). Data represent mean ± SD of > 100 cells across three biological replicates. ****p<0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test . E) Representative transmission EM images of negative stained Huh7 cells expressing a safe targeting sgRNA (control) or sgRNAs against CLCC1. F) TLC resolving of TAG, CE, and polar lipids in Huh7 control and CLCC1 KO cells. Quantification of TAG (left graph) and CE (right graph) bands normalized to phospholipids. Data represent mean ± SD of three biological replicates. ****p<0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test . G) Immunoblot analysis of three Clcc1 fl/fl mice injected with either AAV-GFP (control) or AAV- Cre (Clcc1 HepKO ). Samples were analyzed four weeks post-injection. H) Fold change in liver weight normalized to body weight for control and Clcc1 HepKO mice. n > 9 . I) Body weight of the indicated control and Clcc1 HepKO mice. n > 9 . J) Representative images of livers of control and CLCC1 HepKO m K,L) Quantification of TAG (K) and CE (L) normalized to PL using TLC. Data represent mean ± SD of six mice. ****p<0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test . M) Representative oil red O stained liver sections from control and Clcc1 HepKO mice. N) Representative transmission EM images of negative stained control and Clcc1 HepKO m. O) Quantification of AST, LDL and HDL from clinical analyzer. Data represent mean ± SD of > four mice. ****p<0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test .

Article Snippet: Guide sequences for CLCC1, TMEM41B, VMP1, and safe-targets (sgSAFE #5784) (see Table 1) were selected from the Bassik Human CRISPR Knockout Library (Addgene, 101926, 101927, 101928, 101929, 101930, 101931, 101932, 101933, 101934).

Techniques: CRISPR, Control, Expressing, Transmission Assay, Staining, Western Blot, Injection

Journal: bioRxiv

Article Title: CLCC1 promotes hepatic neutral lipid flux and nuclear pore complex assembly

doi: 10.1101/2024.06.07.597858

Figure Lengend Snippet: A) Confidence score for CLCC1, TMEM41B, and VMP1 from two genome wide CRISPR screens, the neutral lipid (i.e., BODIPY 493/503) screen in the current manuscript and a previous PLIN2-GFP screen . The sign, positive or negative, indicates the effect of gene depletion on neutral lipids or PLIN2-GFP levels. B) Immunoblot of the indicated proteins in control and CLCC1 KO Huh7 cells. C) Immunoblot of TMEM41B in control and TMEM41B KO Huh7 cells. D) Immunoblot of VMP1 in control and VMP1 KO Huh7 cells. E) Representative fluorescence microscopy images of PLIN2 (green) and LDs (blue) in control and TMEM41B KO and VMP1 KO cells. Scale bar represents 10 µm. F) Representative fluorescence microscopy images of PLIN2 (green) and LDs (blue) in control and TMEM41B KO cells treated with MTP inhibitor (MTPi) for 72 h. Zoom images of the boxed regions are shown on the right. Scale bar represents 10 µm. G) Quantification of LDs and LD PLIN2 staining of control and TMEM41B KO cells incubated in the presence and absence of MTP inhibitor (MTPi) as in panel F.

Article Snippet: Guide sequences for CLCC1, TMEM41B, VMP1, and safe-targets (sgSAFE #5784) (see Table 1) were selected from the Bassik Human CRISPR Knockout Library (Addgene, 101926, 101927, 101928, 101929, 101930, 101931, 101932, 101933, 101934).

Techniques: Genome Wide, CRISPR, Western Blot, Control, Fluorescence, Microscopy, Staining, Incubation