Journal: bioRxiv

Article Title: Genome wide CRISPR screen for Pasteurella multocida toxin (PMT) binding proteins reveals LDL Receptor Related Protein1 (LRP1) as crucial cellular receptor

doi: 10.1101/2022.08.04.502755

Figure Lengend Snippet: Mouse embryonic fibroblasts (MEF) stably expressing Flag-Cas9-EGFP were transduced with a lentiviral CRISPR-library. Cells were treated three times with PMT(C1165S)DTa and surviving cells grown. Genomic DNA was extracted, inserted sgRNA amplified and sequenced.

Article Snippet: We utilized a well-functioning and validate genome-wide mouse lentiviral CRISPR guide RNA library v2 (Addgene).

Techniques: Stable Transfection, Expressing, Transduction, CRISPR, Amplification

Journal: bioRxiv

Article Title: Genome wide CRISPR screen for Pasteurella multocida toxin (PMT) binding proteins reveals LDL Receptor Related Protein1 (LRP1) as crucial cellular receptor

doi: 10.1101/2022.08.04.502755

Figure Lengend Snippet: The volcano plot shows gene expression changes between cells treated with lethal PMT-DTa chimera and those only transduced with the CRISPR library. All reads were mapped locally using BWA-MEM [ ,6], then quantified with featureCounts [4], and finally fold changes between the condition were calculated by DESeq2 [7] (see galaxy history). The volcano plot was drawn with the bioinfokit toolkit [8]. Significantly enriched genes, having a positive fold change above 0.584 and a p-value lower or equal 5%, are shown in blue. The significance thresholds are marked by gray-dotted lines. The ten most significant genes are highlighted with their name. Non-significant genes are colored in gray.

Article Snippet: We utilized a well-functioning and validate genome-wide mouse lentiviral CRISPR guide RNA library v2 (Addgene).

Techniques: Gene Expression, Transduction, CRISPR

Journal: bioRxiv

Article Title: Genome scale CRISPR screens identify actin capping proteins as key modulators of therapeutic responses to radiation and immunotherapy

doi: 10.1101/2024.01.14.575614

Figure Lengend Snippet: a, Schematic of the B16F10 genome-scale CRISPR KO screen is shown on the left. Validation experiment of WT B16F10 and Capza3 KO B16F10 cell survival following co-culture with OT-I CD8+ T cells is shown on the right. b, Screen analysis plot (left) and top 6 gRNAs enriched (right) in surviving B16F10 cells after low-dose radiation (1 Gy) treatment and co-culture with CD8 cytotoxic T cells. Results shown are from 2 biologic repeats of the screen. c, Screen analysis plot (left) and top 6 gRNAs enriched (right) in surviving B16F10 cells after low-dose radiation (1 Gy) treatment and anti-PD1 antibody treatment during co-culture with CD8 cytotoxic T cells. Results shown are from 2 biologic repeats of the screen. d, Screen analysis plot (left) and top 6 gRNAs enriched (right) in surviving B16F10 cells after anti-PD1 antibody treatment during co-culture with CD8 cytotoxic T cells. Results shown are from 2 biologic repeats of the screen. e, Validation experiment of WT B16F10 and Capza3 KO B16F10 cell survival. Capza3 KO B16F10 cells were transduced with lentivirus containing the Capza3 targeting gRNA and a GFP reporter. Prior to transduction of B16F10 cells, some cells were isolated to be used as a WT control. Flow analysis of % GFP positive cells (B16F10 Cas9 cells transduced with lentivirus that contains the Capza3 -targeting gRNA) following either: 1) co-culture with CD8 cytotoxic T cells or 2) treatment with low-dose radiation (1 Gy) and anti-PD1 antibody during co-culture with CD8 T cells. Representative plots are shown on the left and quantification is shown on the right. Significance testing was performed with two-way ANOVA. Validation experiments were performed using three biological replicates for each treatment condition. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Brie genome-scale CRISPR library , Addgene , 73633 , CRISPR lentiviral library.

Techniques: CRISPR, Biomarker Discovery, Co-Culture Assay, Transduction, Isolation, Control

Journal: bioRxiv

Article Title: Genome scale CRISPR screens identify actin capping proteins as key modulators of therapeutic responses to radiation and immunotherapy

doi: 10.1101/2024.01.14.575614

Figure Lengend Snippet:

Article Snippet: Brie genome-scale CRISPR library , Addgene , 73633 , CRISPR lentiviral library.

Techniques: CRISPR, Luciferase, Transfection, Control, Plasmid Preparation

Journal: Cell

Article Title: Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B

doi: 10.1016/j.cell.2019.04.035

Figure Lengend Snippet:

Article Snippet: Model-based Analysis of Genomewide CRISPR/Cas9 Knockout (MAGeCK) , , https://sourceforge.net/p/mageck/wiki/Home.

Techniques: Virus, Isolation, Recombinant, Transfection, Membrane, Sequencing, Software, CRISPR, Knock-Out

Journal: Nature Structural & Molecular Biology

Article Title: A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens

doi: 10.1038/s41594-024-01211-y

Figure Lengend Snippet: a , Schematic outline of cell surface proteome CRISPR screens (2,973 sgRNAs) in Cas9-expressing cancer cell models. b , c , Gene rankings for cell surface proteome CRISPR screens in five individual cell models ( b ) and the combined analysis ( c ) as calculated by the MAGeCK algorithm. The rankings of ITGAV (red), positive controls (yellow), negative controls (green) and the total library (gray) are indicated. d , Western blot of ITGAV and β-actin in MDA231-Cas9 + cells transduced with sgCtrl ( n = 2 independent sgRNA sequences) and sgITGAV ( n = 3 independent sgRNA sequences) for 3 days. e , Growth competition assay of MDA231-Cas9 + and PANC1-Cas9 + cells transduced with RFP-labeled sgCtrl (gray lines; two independent sgRNA sequences) and sgITGAV (red lines; three independent sgRNA sequences). Asterisk indicates that all three sgITGAV groups were significantly different ( P < 0.01) from the two sgCtrl groups ( n = 3 for each group). f , g , Cellular apoptosis detected by Annexin V + /DAPI − ( f ) and cell cycle monitored by EdU incorporation ( g ) in MDA231-Cas9 + cells transduced with sgCtrl and sgITGAV for 3 days ( n = 3 for each group). h , Survival curves for cancer patients with high (top quartile; n = 927) versus low (bottom quartile; n = 927) ITGAV expression (data source: GEPIA). Data are presented as the mean ± s.e.m. P values were calculated by two-sided Student’s t -test. FC, fold change; GBM, glioblastoma multiforme.

Article Snippet: To elucidate the cytoskeletal signaling pathways affected by depletion of ITGAV, we analyzed the DepMap genome-wide CRISPR screen consortium database ( https://depmap.org/portal/ ; BROAD Institute) and found higher correlations of CERES scores (CERES is a computational method to estimate gene-dependency levels from CRISPR–Cas9 essentiality screens) between ITGAV and RAC1 in the 769 tested cell models (Fig. , purple, and Extended Data Fig. ; Pearson coefficient = 0.482; rank 6 of 17,709 genes) than between ITGAV and CDC42 or RHOA (green).

Techniques: CRISPR, Expressing, Western Blot, Transduction, Competitive Binding Assay, Labeling

Journal: Nature Structural & Molecular Biology

Article Title: A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens

doi: 10.1038/s41594-024-01211-y

Figure Lengend Snippet: ( a ) Map of the ipUSEPR vector expressing a sgRNA together with a puromycin-resistant gene (PuroR) and a TagRFP fluorescent protein. Primers for Sanger (hU6-F_seq) and Illumina (DCF01 and DCR03) sequencing are listed. ( b–d ) Design and distribution of individual sgRNA frequencies RPMR (reads per million reads) in the CRISPR libraries targeting (B) cell surface proteome genes (n = 2,973 sgRNAs), (C) integrin family genes (n = 714 sgRNAs), and (D) coding regions of ITGAV (n = 412 sgRNAs). (B) 90.1%, (C) 97.7%, and (D) 96.3% of sgRNA in these libraries passed the QC by exhibiting RPMR ≥ 10. Data are represented as median ± interquartile range.

Article Snippet: To elucidate the cytoskeletal signaling pathways affected by depletion of ITGAV, we analyzed the DepMap genome-wide CRISPR screen consortium database ( https://depmap.org/portal/ ; BROAD Institute) and found higher correlations of CERES scores (CERES is a computational method to estimate gene-dependency levels from CRISPR–Cas9 essentiality screens) between ITGAV and RAC1 in the 769 tested cell models (Fig. , purple, and Extended Data Fig. ; Pearson coefficient = 0.482; rank 6 of 17,709 genes) than between ITGAV and CDC42 or RHOA (green).

Techniques: Plasmid Preparation, Expressing, Sequencing, CRISPR

Journal: Nature Structural & Molecular Biology

Article Title: A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens

doi: 10.1038/s41594-024-01211-y

Figure Lengend Snippet: ( a ) Combined gene ranking of the cell surface proteome CRISPR screens was calculated by the MAGeCK algorithm. The ranking of ITGAV (red), positive controls (yellow; target common essential genes), negative controls (green; target non-essential sequences), and total library (grey) are indicated. The pink box highlights the leading-edge essential genes with a combined Log2FC below -1.0. ( b ) Distribution of the positive (n = 12 genes) and negative (n = 5 genes) controls in the screen. Data are represented as median ± interquartile range. P value was calculated by two-sided Student’s t-test. ( c ) Three surface protein genes (ITGAV, ATP6AP2, and TFRC) were identified as the leading-edge essential genes. ( d ) Correlation of the CERES scores (computational method to estimate gene-dependency levels from CRISPR-Cas9 essentiality screens) and gene expression of ITGAV (left panel), ATP6AP2 (middle panel), and TFRC (right panel)(source: https://depmap.org/portal/ ; BROAD Institute). The cancer cell dependency on ITGAV is correlated with its expression. ( e ) Top ten candidate hits and ( f ) an overlap plot of the surface proteome CRISPR screens in five cell models. Red (ITGAV and ATP6AP2) indicates the common essential surface proteins in all screened cell types. Other colors (blue, orange, green, cyan, and pink) highlight the cell type-specific candidate genes.

Article Snippet: To elucidate the cytoskeletal signaling pathways affected by depletion of ITGAV, we analyzed the DepMap genome-wide CRISPR screen consortium database ( https://depmap.org/portal/ ; BROAD Institute) and found higher correlations of CERES scores (CERES is a computational method to estimate gene-dependency levels from CRISPR–Cas9 essentiality screens) between ITGAV and RAC1 in the 769 tested cell models (Fig. , purple, and Extended Data Fig. ; Pearson coefficient = 0.482; rank 6 of 17,709 genes) than between ITGAV and CDC42 or RHOA (green).

Techniques: CRISPR, Gene Expression, Expressing

Journal: Nature Structural & Molecular Biology

Article Title: A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens

doi: 10.1038/s41594-024-01211-y

Figure Lengend Snippet: CERES score is a computational method to estimate gene-dependency levels from CRISPR-Cas9 essentiality screens. The CERES scores of ITGAV (x-axis) and ( a ) Rho small GTPase genes RAC1, CDC42, and RHOA (y-axes; left, middle, and right respectively) and ( b ) ITGB1/3/5/6/8 (y-axes; top-left, top-middle, top-right, bottom-middle, bottom-right, respectively) in 769 cell models (dots) were obtained from the DepMap CRISPR screen consortium database (source: https://depmap.org/portal/ ; BROAD Institute). A higher Pearson coefficient ( r ) of the CERES scores between two genes indicates a higher likelihood the two genes are co-regulated in the tested cell models. The gene rank number is based on the Pearson coefficient ( r ) of the CERES scores between ITGAV and a total of 17,709 genes tested in the genome-wide CRISPR library screens.

Article Snippet: To elucidate the cytoskeletal signaling pathways affected by depletion of ITGAV, we analyzed the DepMap genome-wide CRISPR screen consortium database ( https://depmap.org/portal/ ; BROAD Institute) and found higher correlations of CERES scores (CERES is a computational method to estimate gene-dependency levels from CRISPR–Cas9 essentiality screens) between ITGAV and RAC1 in the 769 tested cell models (Fig. , purple, and Extended Data Fig. ; Pearson coefficient = 0.482; rank 6 of 17,709 genes) than between ITGAV and CDC42 or RHOA (green).

Techniques: CRISPR, Genome Wide

Journal: Nature Structural & Molecular Biology

Article Title: A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens

doi: 10.1038/s41594-024-01211-y

Figure Lengend Snippet: a , Model of integrin α (red) and β (blue) subunits and domain structures. The binding site of the extracellular ligand (yellow) is assembled upon heterodimerization of the α/β subunits. b , Schematic outline of integrin family CRISPR screens (712 sgRNAs) in Cas9-expressing MDA231 and PANC1 cells. c , Fold change of each sgRNA from day 0 to day 24 in MDA231-Cas9 + ( x axis) and PANC1-Cas9 + ( y axis) cells. The sgRNAs targeting ITGAV (red dots), ITGB5 (blue dots), positive controls (yellow triangles), negative controls (green triangles) and the total library (gray dots) are indicated. d , Heatmap showing CRISPR impact scores (median log 10 fold change of 25 sgRNAs) of each integrin subunit in the integrin network consisting of 24 distinct integrin α/β heterodimers. The solid lines indicate the integrin α/β pairs forming the RGD receptors (yellow), collagen receptors (pink), laminin receptors (brown) and leukocyte-specific receptors (green). The red dotted circle highlights αVβ5 as the top essential integrin heterodimer in cancer cells. e , Growth competition assay of MDA231-Cas9 + cells transduced with RFP-labeled sgCtrl (gray lines; two independent sgRNA sequences) and sgITGB1/3/5/6/8 (blue lines; three independent sgRNA sequences for each gene). Asterisk indicates that all three sgRNAs for each ITGB gene group were significantly different ( P < 0.01) from the two sgCtrl groups ( n = 3 for each group). f , Western blot of ITGB5 and β-actin in MDA231-Cas9 + cells transduced with sgCtrl ( n = 2 independent sgRNA sequences) and sgITGB5 ( n = 3 independent sgRNA sequences) for 3 days. g , h , Cellular apoptosis detected by Annexin V + /DAPI − ( g ) and cell cycle monitored by EdU incorporation ( h ) in MDA231-Cas9 + cells transduced with sgCtrl and sgITGB5 for 3 days ( n = 3 for each group). i , Gene ranking based on the Pearson coefficient ( r ) of CERES scores between ITGAV and ITGB5 (blue) compared with other ITGAV partner β subunit genes ITGB1 / 3 / 6 / 8 (yellow) in the 769 tested cell models (Extended Data Fig. ). Data are presented as the mean ± s.e.m. P values were calculated by two-sided Student’s t -test.

Article Snippet: To elucidate the cytoskeletal signaling pathways affected by depletion of ITGAV, we analyzed the DepMap genome-wide CRISPR screen consortium database ( https://depmap.org/portal/ ; BROAD Institute) and found higher correlations of CERES scores (CERES is a computational method to estimate gene-dependency levels from CRISPR–Cas9 essentiality screens) between ITGAV and RAC1 in the 769 tested cell models (Fig. , purple, and Extended Data Fig. ; Pearson coefficient = 0.482; rank 6 of 17,709 genes) than between ITGAV and CDC42 or RHOA (green).

Techniques: Binding Assay, CRISPR, Expressing, Competitive Binding Assay, Transduction, Labeling, Western Blot

Journal: Nature Structural & Molecular Biology

Article Title: A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens

doi: 10.1038/s41594-024-01211-y

Figure Lengend Snippet: a , Schematic outline of the ITGAV high-density CRISPR-tiling scan (412 sgRNAs) in MDA231-Cas9 + cells. b , 2D annotation of ITGAV CRISPR scan. The red line indicates the smoothened model of NCS derived from 348 sgRNAs (dots) targeting the coding exons of ITGAV . The median NCS of the positive control (gray dotted line; defined as −1.0) and negative control (defined as 0) sgRNAs are highlighted. The brown dashed box contains the β-propeller domain. The numbers 1–7 pinpoint the CRISPR-hypersensitive regions within the β-propeller domain. c , 3D annotation of ITGAV CRISPR scan NCS relative to AlphaFold structural modeling of ITGAV (AlphaFold ID: P06756). d , Enlarged view of the β-propeller domain showing the CRISPR-hypersensitive regions (numbers 1–7 as indicated in b ) pointing to the center cavity of the β-propeller HIP. The residues contributing to this aromatic-enriched pocket are highlighted. e , Schematic outline of the NanoBRET reporter system for detecting the ITGAV–ITGB5 interaction in living cells. f , Effect of alanine substitution of the ITGAV β-propeller HIP residues (brown; n = 3 for each group) on the NanoBRET signal compared with the wild-type ITGAV (gray; n = 3 for each group). Data are represented as mean ± s.e.m. P values were calculated by two-sided Student’s t -test. Ex, exon; TSS, transcription start site. TM, transmembrane.

Article Snippet: To elucidate the cytoskeletal signaling pathways affected by depletion of ITGAV, we analyzed the DepMap genome-wide CRISPR screen consortium database ( https://depmap.org/portal/ ; BROAD Institute) and found higher correlations of CERES scores (CERES is a computational method to estimate gene-dependency levels from CRISPR–Cas9 essentiality screens) between ITGAV and RAC1 in the 769 tested cell models (Fig. , purple, and Extended Data Fig. ; Pearson coefficient = 0.482; rank 6 of 17,709 genes) than between ITGAV and CDC42 or RHOA (green).

Techniques: CRISPR, Derivative Assay, Positive Control, Negative Control

Journal: Nature Structural & Molecular Biology

Article Title: A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens

doi: 10.1038/s41594-024-01211-y

Figure Lengend Snippet: a , 3D ‘docking box’ (cube) defined by the CRISPR-hypersensitive regions (numbers 1–7) within the ITGAV β-propeller domain. b , Compound (Cpd) ranking based on free binding energy (ΔG°) to the ‘docking box’ within the β-propeller domain predicted by AutoDock Vina. c , d , Heatmap showing relative CellTiter Glo (left) and CCK8 (right) signals (percentage of the signal for dimethyl sulfoxide; DMSO) in MDA231 cells incubated with 10 µM of 500 selected compounds ( c ) and the top nine effective compounds ( d ) for 3 days. Effective cell killing was defined as less than 10% relative signals for both CellTiter Glo and CCK8 assays. e , Schematic outline of flow cytometric measurement of cell surface integrin αVβ5 using a monoclonal antibody against integrin αVβ5 heterodimers. f , Effects of the top nine candidate compounds on cell surface integrin αVβ5 levels upon 1 h compound treatments ( n = 4 for each condition). g , h , Cellular apoptosis detected by Annexin V + /DAPI − ( g ) and cell cycle monitored by EdU incorporation ( h ) in MDA231 cells treated with Cpd_AV2 (40 µM) for 0 to 3 h ( n = 3 for each time point). i , Representative fluorescence images of F-actin (FITC, green) and nucleus (DAPI, blue) staining in MDA231 cells treated with control (DMSO) and Cpd_AV2 (40 µM) for 10 min. Scale bars, 20 µm. j , Violin plot showing the distribution of cell size (µm 2 ) in MDA231 cells treated with control (DMSO) and Cpd_AV2 (40 µM) for 10 min. Data are presented as the mean ± s.e.m. P values were calculated by two-sided Student’s t -test.

Article Snippet: To elucidate the cytoskeletal signaling pathways affected by depletion of ITGAV, we analyzed the DepMap genome-wide CRISPR screen consortium database ( https://depmap.org/portal/ ; BROAD Institute) and found higher correlations of CERES scores (CERES is a computational method to estimate gene-dependency levels from CRISPR–Cas9 essentiality screens) between ITGAV and RAC1 in the 769 tested cell models (Fig. , purple, and Extended Data Fig. ; Pearson coefficient = 0.482; rank 6 of 17,709 genes) than between ITGAV and CDC42 or RHOA (green).

Techniques: CRISPR, Binding Assay, Incubation, Fluorescence, Staining, Control

Journal: Nature Structural & Molecular Biology

Article Title: A novel class of inhibitors that disrupts the stability of integrin heterodimers identified by CRISPR-tiling-instructed genetic screens

doi: 10.1038/s41594-024-01211-y

Figure Lengend Snippet: The smoothened CRISPR tiling data (left panel; blue lines) of ( a ) BRD4, ( b ) AURKB, ( c ) CDK1, and ( d ) WEE1 were obtained from Munoz et al. On the right panels, the green boxes highlight the CRISPR-TICA region of interest based on the CRISPR sensitivity. The yellow arrows indicate the previously reported inhibitors for these proteins.

Article Snippet: To elucidate the cytoskeletal signaling pathways affected by depletion of ITGAV, we analyzed the DepMap genome-wide CRISPR screen consortium database ( https://depmap.org/portal/ ; BROAD Institute) and found higher correlations of CERES scores (CERES is a computational method to estimate gene-dependency levels from CRISPR–Cas9 essentiality screens) between ITGAV and RAC1 in the 769 tested cell models (Fig. , purple, and Extended Data Fig. ; Pearson coefficient = 0.482; rank 6 of 17,709 genes) than between ITGAV and CDC42 or RHOA (green).

Techniques: CRISPR