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Addgene inc whole genome crispr knockout grna library

( a ) Schematic view of ex vivo <t>CRISPR/Cas9</t> screening in mouse primary CD8 + T-cells. ( b ) Volcano plot showing results of ex vivo CRISPR/Cas9 genome-wide screenings. The screenings were repeated independently once. The p-values were calculated using the α-robust rank aggregation (α-RRA) algorithm in MAGeCK. ( c ) Verification of candidate genes by individual single gRNAs. The relative expression levels of surface PD-1 protein and PD-1 mRNA were measured by FACS as mean fluorescent intensity (MFI) and RT-qPCR, respectively. The verification assays were biologically replicated twice. ( d ) GSEA of significantly enriched KEGG pathways in genome-wide screening. The enrichment score (ES) and statistical significance were calculated using the clusterProfiler (version 3.12.0) R package.

Whole Genome Crispr Knockout Grna Library, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions"

Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

Journal: eLife

doi: 10.7554/eLife.108724

( a ) Schematic view of ex vivo CRISPR/Cas9 screening in mouse primary CD8 + T-cells. ( b ) Volcano plot showing results of ex vivo CRISPR/Cas9 genome-wide screenings. The screenings were repeated independently once. The p-values were calculated using the α-robust rank aggregation (α-RRA) algorithm in MAGeCK. ( c ) Verification of candidate genes by individual single gRNAs. The relative expression levels of surface PD-1 protein and PD-1 mRNA were measured by FACS as mean fluorescent intensity (MFI) and RT-qPCR, respectively. The verification assays were biologically replicated twice. ( d ) GSEA of significantly enriched KEGG pathways in genome-wide screening. The enrichment score (ES) and statistical significance were calculated using the clusterProfiler (version 3.12.0) R package.

Figure Legend Snippet: ( a ) Schematic view of ex vivo CRISPR/Cas9 screening in mouse primary CD8 + T-cells. ( b ) Volcano plot showing results of ex vivo CRISPR/Cas9 genome-wide screenings. The screenings were repeated independently once. The p-values were calculated using the α-robust rank aggregation (α-RRA) algorithm in MAGeCK. ( c ) Verification of candidate genes by individual single gRNAs. The relative expression levels of surface PD-1 protein and PD-1 mRNA were measured by FACS as mean fluorescent intensity (MFI) and RT-qPCR, respectively. The verification assays were biologically replicated twice. ( d ) GSEA of significantly enriched KEGG pathways in genome-wide screening. The enrichment score (ES) and statistical significance were calculated using the clusterProfiler (version 3.12.0) R package.

Techniques Used: Ex Vivo, CRISPR, Genome Wide, Expressing, Quantitative RT-PCR

( a ) Schematic view of in vivo CRISPR/Cas9 screening in mouse primary CD8 + T-cells. ( b ) Volcano plot showing results of ex vivo CRISPR/Cas9 screening. The screenings were repeated independently once. The p-values were calculated using the α-RRA algorithm in MAGeCK. ( c ) Volcano plot showing results of in vivo CRISPR/Cas9 screenings. The screenings were repeated independently once. The p-values were calculated using the α-RRA algorithm in MAGeCK.

Figure Legend Snippet: ( a ) Schematic view of in vivo CRISPR/Cas9 screening in mouse primary CD8 + T-cells. ( b ) Volcano plot showing results of ex vivo CRISPR/Cas9 screening. The screenings were repeated independently once. The p-values were calculated using the α-RRA algorithm in MAGeCK. ( c ) Volcano plot showing results of in vivo CRISPR/Cas9 screenings. The screenings were repeated independently once. The p-values were calculated using the α-RRA algorithm in MAGeCK.

Techniques Used: In Vivo, CRISPR, Ex Vivo

( a ) CRISPR/Cas9 knockout of B4galt1 (sgB4galt1) (sg2) in CD8 + T-cells increases expression of PD-1 before and after co-culture with B16F10-OVA cells. The MFIs of PD-1 were measured by FACS (n=6). The relative mRNA levels of PD-1 were measured by quantitative RT-qPCR (n=6). The p-values were calculated using a two-tailed Student’s t -test. ( b ) The effect of B4galt1 knockout on PD-1 surface expression could be rescued by overexpression of either long- or short-isoform B4galt1 (n=3). The p-values were calculated using a two-tailed Student’s t -test. ( c ) CRISPR/Cas9 knockout of B4galt1 in CD8 + T-cells increases expression of TNFα and IFNγ after co-culture with B16F10-OVA cells. The relative mRNA levels were measured by quantitative RT-qPCR (n=3). The secreted TNFα and IFNγ in medium were measured by ELISA (n=6). The p-values were calculated using a two-tailed Student’s t -test. ( d ) CRISPR/Cas9 knockout of B4galt1 in OT-I CD8 + T-cells increases in vitro specific killing activities on B16F10-OVA cells (n=3). The p-values were calculated using a two-tailed Student’s t -test. ( e ) Schematic view of B4GALT1 knockdown in human NY-ESO-1 TCR-T-cells. ( f ) Knockdown of B4GALT1 in human NY-ESO-1 TCR-T-cells by shRNA increases in vitro killing activities on A375 cells (n=5). The p-values were calculated using a two-tailed Student’s t -test. ( g ) Knockdown of B4GALT1 in human NY-ESO-1 TCR-T-cells increases expression of TNFα and IFNγ after co-culture with A375 cells. The secreted TNFα and IFNγ in medium were measured by ELISA (n=3). The p-values were calculated using a two-tailed Student’s t -test. ( h ) Heatmap demonstrating differentially expressed genes (DEGs) between B4galt1 knockout and control mouse OT-I CD8 + T-cells after co-culture. The genes in TCR signaling pathway are labeled on the left side. ( i ) Volcano plot showing upregulated and downregulated genes (p-value <0.01) in B4galt1 knockout mouse OT-I CD8 + T-cells after co-culture. The genes in TCR signaling pathway are labeled with dark blue and dark red. Top genes and some genes in TCR signaling pathway are annotated. The p-value was calculated using the Wald test, and p.adjust was calculated using Benjamini–Hochberg with the R package DESeq2 (version 1.22.2). ( j ) Bar graph showing KEGG pathways significantly changed in B4galt1 knockout mouse OT-I CD8 + T-cells after co-culture. The p-value was calculated using the clusterProfiler (version 3.12.0) R package. All of these functional effects were biologically replicated at least twice. Data are shown as the mean ± SEM. *p<0.05; **p<0.01; ***p<0.001.

Figure Legend Snippet: ( a ) CRISPR/Cas9 knockout of B4galt1 (sgB4galt1) (sg2) in CD8 + T-cells increases expression of PD-1 before and after co-culture with B16F10-OVA cells. The MFIs of PD-1 were measured by FACS (n=6). The relative mRNA levels of PD-1 were measured by quantitative RT-qPCR (n=6). The p-values were calculated using a two-tailed Student’s t -test. ( b ) The effect of B4galt1 knockout on PD-1 surface expression could be rescued by overexpression of either long- or short-isoform B4galt1 (n=3). The p-values were calculated using a two-tailed Student’s t -test. ( c ) CRISPR/Cas9 knockout of B4galt1 in CD8 + T-cells increases expression of TNFα and IFNγ after co-culture with B16F10-OVA cells. The relative mRNA levels were measured by quantitative RT-qPCR (n=3). The secreted TNFα and IFNγ in medium were measured by ELISA (n=6). The p-values were calculated using a two-tailed Student’s t -test. ( d ) CRISPR/Cas9 knockout of B4galt1 in OT-I CD8 + T-cells increases in vitro specific killing activities on B16F10-OVA cells (n=3). The p-values were calculated using a two-tailed Student’s t -test. ( e ) Schematic view of B4GALT1 knockdown in human NY-ESO-1 TCR-T-cells. ( f ) Knockdown of B4GALT1 in human NY-ESO-1 TCR-T-cells by shRNA increases in vitro killing activities on A375 cells (n=5). The p-values were calculated using a two-tailed Student’s t -test. ( g ) Knockdown of B4GALT1 in human NY-ESO-1 TCR-T-cells increases expression of TNFα and IFNγ after co-culture with A375 cells. The secreted TNFα and IFNγ in medium were measured by ELISA (n=3). The p-values were calculated using a two-tailed Student’s t -test. ( h ) Heatmap demonstrating differentially expressed genes (DEGs) between B4galt1 knockout and control mouse OT-I CD8 + T-cells after co-culture. The genes in TCR signaling pathway are labeled on the left side. ( i ) Volcano plot showing upregulated and downregulated genes (p-value <0.01) in B4galt1 knockout mouse OT-I CD8 + T-cells after co-culture. The genes in TCR signaling pathway are labeled with dark blue and dark red. Top genes and some genes in TCR signaling pathway are annotated. The p-value was calculated using the Wald test, and p.adjust was calculated using Benjamini–Hochberg with the R package DESeq2 (version 1.22.2). ( j ) Bar graph showing KEGG pathways significantly changed in B4galt1 knockout mouse OT-I CD8 + T-cells after co-culture. The p-value was calculated using the clusterProfiler (version 3.12.0) R package. All of these functional effects were biologically replicated at least twice. Data are shown as the mean ± SEM. *p<0.05; **p<0.01; ***p<0.001.

Techniques Used: CRISPR, Knock-Out, Expressing, Co-Culture Assay, Quantitative RT-PCR, Two Tailed Test, Over Expression, Enzyme-linked Immunosorbent Assay, In Vitro, Knockdown, shRNA, Control, Labeling, Functional Assay

CRISPR/Cas9 knockout of B4GALT1 in hCD19-CAR-T-cells does not affect in vitro killing of Nalm6 target cells (n=3). The killing assays were biologically replicated three times. Data are shown as the mean ± SEM. NS, not significant.

Figure Legend Snippet: CRISPR/Cas9 knockout of B4GALT1 in hCD19-CAR-T-cells does not affect in vitro killing of Nalm6 target cells (n=3). The killing assays were biologically replicated three times. Data are shown as the mean ± SEM. NS, not significant.

Techniques Used: CRISPR, Knock-Out, In Vitro

( a ) Schematic view of B4galt1 functional test in tumor microenvironment. ( b ) CRISPR/Cas9 knockout of B4galt1 in OT-I T-cells enhances growth control of B16F10-OVA tumors in vivo. The p-value was calculated using two-way ANOVA. ( c ) Compared with control OT-I T-cells, the tumors were significantly smaller when B4galt1 knockout OT-I T-cells were transplanted (n=5 for control, n=7 for sgB4galt1). The p-value was calculated using a two-tailed Student’s t -test. ( d ) CRISPR/Cas9 knockout of B4galt1 increases numbers of OT-I T-cells in B16F10-OVA tumors (n=5 for control, n=7 for sgB4galt1). The p-value was calculated using a two-tailed Student’s t -test. The in vivo functional effects were biologically replicated at least twice. Data are shown as the mean ± SEM. *p<0.05; **p<0.01.

Figure Legend Snippet: ( a ) Schematic view of B4galt1 functional test in tumor microenvironment. ( b ) CRISPR/Cas9 knockout of B4galt1 in OT-I T-cells enhances growth control of B16F10-OVA tumors in vivo. The p-value was calculated using two-way ANOVA. ( c ) Compared with control OT-I T-cells, the tumors were significantly smaller when B4galt1 knockout OT-I T-cells were transplanted (n=5 for control, n=7 for sgB4galt1). The p-value was calculated using a two-tailed Student’s t -test. ( d ) CRISPR/Cas9 knockout of B4galt1 increases numbers of OT-I T-cells in B16F10-OVA tumors (n=5 for control, n=7 for sgB4galt1). The p-value was calculated using a two-tailed Student’s t -test. The in vivo functional effects were biologically replicated at least twice. Data are shown as the mean ± SEM. *p<0.05; **p<0.01.

Techniques Used: Functional Assay, CRISPR, Knock-Out, Control, In Vivo, Two Tailed Test

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Journal: eLife

Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

doi: 10.7554/eLife.108724

Figure Lengend Snippet: ( a ) Schematic view of ex vivo CRISPR/Cas9 screening in mouse primary CD8 + T-cells. ( b ) Volcano plot showing results of ex vivo CRISPR/Cas9 genome-wide screenings. The screenings were repeated independently once. The p-values were calculated using the α-robust rank aggregation (α-RRA) algorithm in MAGeCK. ( c ) Verification of candidate genes by individual single gRNAs. The relative expression levels of surface PD-1 protein and PD-1 mRNA were measured by FACS as mean fluorescent intensity (MFI) and RT-qPCR, respectively. The verification assays were biologically replicated twice. ( d ) GSEA of significantly enriched KEGG pathways in genome-wide screening. The enrichment score (ES) and statistical significance were calculated using the clusterProfiler (version 3.12.0) R package.

Article Snippet: A whole-genome CRISPR knockout gRNA library (1000000096) was purchased from Addgene.

Techniques: Ex Vivo, CRISPR, Genome Wide, Expressing, Quantitative RT-PCR

Journal: eLife

Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

doi: 10.7554/eLife.108724

Figure Lengend Snippet: ( a ) Schematic view of in vivo CRISPR/Cas9 screening in mouse primary CD8 + T-cells. ( b ) Volcano plot showing results of ex vivo CRISPR/Cas9 screening. The screenings were repeated independently once. The p-values were calculated using the α-RRA algorithm in MAGeCK. ( c ) Volcano plot showing results of in vivo CRISPR/Cas9 screenings. The screenings were repeated independently once. The p-values were calculated using the α-RRA algorithm in MAGeCK.

Article Snippet: A whole-genome CRISPR knockout gRNA library (1000000096) was purchased from Addgene.

Techniques: In Vivo, CRISPR, Ex Vivo

Journal: eLife

Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

doi: 10.7554/eLife.108724

Figure Lengend Snippet: ( a ) CRISPR/Cas9 knockout of B4galt1 (sgB4galt1) (sg2) in CD8 + T-cells increases expression of PD-1 before and after co-culture with B16F10-OVA cells. The MFIs of PD-1 were measured by FACS (n=6). The relative mRNA levels of PD-1 were measured by quantitative RT-qPCR (n=6). The p-values were calculated using a two-tailed Student’s t -test. ( b ) The effect of B4galt1 knockout on PD-1 surface expression could be rescued by overexpression of either long- or short-isoform B4galt1 (n=3). The p-values were calculated using a two-tailed Student’s t -test. ( c ) CRISPR/Cas9 knockout of B4galt1 in CD8 + T-cells increases expression of TNFα and IFNγ after co-culture with B16F10-OVA cells. The relative mRNA levels were measured by quantitative RT-qPCR (n=3). The secreted TNFα and IFNγ in medium were measured by ELISA (n=6). The p-values were calculated using a two-tailed Student’s t -test. ( d ) CRISPR/Cas9 knockout of B4galt1 in OT-I CD8 + T-cells increases in vitro specific killing activities on B16F10-OVA cells (n=3). The p-values were calculated using a two-tailed Student’s t -test. ( e ) Schematic view of B4GALT1 knockdown in human NY-ESO-1 TCR-T-cells. ( f ) Knockdown of B4GALT1 in human NY-ESO-1 TCR-T-cells by shRNA increases in vitro killing activities on A375 cells (n=5). The p-values were calculated using a two-tailed Student’s t -test. ( g ) Knockdown of B4GALT1 in human NY-ESO-1 TCR-T-cells increases expression of TNFα and IFNγ after co-culture with A375 cells. The secreted TNFα and IFNγ in medium were measured by ELISA (n=3). The p-values were calculated using a two-tailed Student’s t -test. ( h ) Heatmap demonstrating differentially expressed genes (DEGs) between B4galt1 knockout and control mouse OT-I CD8 + T-cells after co-culture. The genes in TCR signaling pathway are labeled on the left side. ( i ) Volcano plot showing upregulated and downregulated genes (p-value <0.01) in B4galt1 knockout mouse OT-I CD8 + T-cells after co-culture. The genes in TCR signaling pathway are labeled with dark blue and dark red. Top genes and some genes in TCR signaling pathway are annotated. The p-value was calculated using the Wald test, and p.adjust was calculated using Benjamini–Hochberg with the R package DESeq2 (version 1.22.2). ( j ) Bar graph showing KEGG pathways significantly changed in B4galt1 knockout mouse OT-I CD8 + T-cells after co-culture. The p-value was calculated using the clusterProfiler (version 3.12.0) R package. All of these functional effects were biologically replicated at least twice. Data are shown as the mean ± SEM. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: A whole-genome CRISPR knockout gRNA library (1000000096) was purchased from Addgene.

Techniques: CRISPR, Knock-Out, Expressing, Co-Culture Assay, Quantitative RT-PCR, Two Tailed Test, Over Expression, Enzyme-linked Immunosorbent Assay, In Vitro, Knockdown, shRNA, Control, Labeling, Functional Assay

Journal: eLife

Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

doi: 10.7554/eLife.108724

Figure Lengend Snippet: CRISPR/Cas9 knockout of B4GALT1 in hCD19-CAR-T-cells does not affect in vitro killing of Nalm6 target cells (n=3). The killing assays were biologically replicated three times. Data are shown as the mean ± SEM. NS, not significant.

Article Snippet: A whole-genome CRISPR knockout gRNA library (1000000096) was purchased from Addgene.

Techniques: CRISPR, Knock-Out, In Vitro

Journal: eLife

Article Title: Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

doi: 10.7554/eLife.108724

Figure Lengend Snippet: ( a ) Schematic view of B4galt1 functional test in tumor microenvironment. ( b ) CRISPR/Cas9 knockout of B4galt1 in OT-I T-cells enhances growth control of B16F10-OVA tumors in vivo. The p-value was calculated using two-way ANOVA. ( c ) Compared with control OT-I T-cells, the tumors were significantly smaller when B4galt1 knockout OT-I T-cells were transplanted (n=5 for control, n=7 for sgB4galt1). The p-value was calculated using a two-tailed Student’s t -test. ( d ) CRISPR/Cas9 knockout of B4galt1 increases numbers of OT-I T-cells in B16F10-OVA tumors (n=5 for control, n=7 for sgB4galt1). The p-value was calculated using a two-tailed Student’s t -test. The in vivo functional effects were biologically replicated at least twice. Data are shown as the mean ± SEM. *p<0.05; **p<0.01.

Article Snippet: A whole-genome CRISPR knockout gRNA library (1000000096) was purchased from Addgene.

Techniques: Functional Assay, CRISPR, Knock-Out, Control, In Vivo, Two Tailed Test

( A ) Schematic overview of the CRISPR screens performed to identify genes and pathways involved in resistance to SAC inhibition. The illustration was created using www.biorender.com . ( B ) Cellular pathways enriched in the top ranking 10% of genes in both CRISPR screens. ( C ) Correlation between the top-ranked 25% of genes in both CRISPR screens, based on their statistical significance. Cdc20 (red) is the top hit in both screens. P values were calculated using the RRA method (see “Methods”). ( D , E ) Quantification ( D ) and representative images ( E ) of an EdU incorporation assay in 3T3 cells after Cdc20 depletion by shRNA and treatment with 250 nM Reversine. Two-way ANOVA ( N , number of biological replicates. N = 3; ns, P value = 0.9998; ****, P value < 0.0001). Error bars represent the standard deviation (SD) of the mean. .

Journal: EMBO Reports

Article Title: High CDC20 levels increase sensitivity of cancer cells to MPS1 inhibitors

doi: 10.1038/s44319-024-00363-8

Figure Lengend Snippet: ( A ) Schematic overview of the CRISPR screens performed to identify genes and pathways involved in resistance to SAC inhibition. The illustration was created using www.biorender.com . ( B ) Cellular pathways enriched in the top ranking 10% of genes in both CRISPR screens. ( C ) Correlation between the top-ranked 25% of genes in both CRISPR screens, based on their statistical significance. Cdc20 (red) is the top hit in both screens. P values were calculated using the RRA method (see “Methods”). ( D , E ) Quantification ( D ) and representative images ( E ) of an EdU incorporation assay in 3T3 cells after Cdc20 depletion by shRNA and treatment with 250 nM Reversine. Two-way ANOVA ( N , number of biological replicates. N = 3; ns, P value = 0.9998; ****, P value < 0.0001). Error bars represent the standard deviation (SD) of the mean. .

Article Snippet: To introduce the sgRNA library, 377 million iCas9-expressing 3T3 cells were transduced with the Mouse Two Plasmid Activity-Optimized CRISPR Knockout Library (Wang et al, ) (Addgene; 1000000096) using spinfection at an MOI of 0.3 to ensure a final library coverage of 500×.

Techniques: CRISPR, Inhibition, shRNA, Standard Deviation

( A ) Western blot validation for Cas9 expression in 3T3 cells used for the CRISPR screen. ( B ) Cleaved and uncleaved PCR product in a T7 assay as a readout of Cas9 activity in 3T3 cells used for the CRISPR screen. ( C ) Correlation between the top-ranked 25% of genes in both CRISPR screens based on their statistical significance with all APC/C-related genes highlighted, showing that Cdc20 is by far the most significant outlier of all APC/C extended complex members. P values were calculated using the RRA method (see “Methods”). ( D , E ) qPCR ( D ) or western blot ( E ) validation of Cdc20 knockdown by shRNA. Paired t test ( N , number of biological replicates; N = 3; ****, P value < 0.0001). Error bars represent the standard deviation (SD) of the mean. .

Journal: EMBO Reports

Article Title: High CDC20 levels increase sensitivity of cancer cells to MPS1 inhibitors

doi: 10.1038/s44319-024-00363-8

Figure Lengend Snippet: ( A ) Western blot validation for Cas9 expression in 3T3 cells used for the CRISPR screen. ( B ) Cleaved and uncleaved PCR product in a T7 assay as a readout of Cas9 activity in 3T3 cells used for the CRISPR screen. ( C ) Correlation between the top-ranked 25% of genes in both CRISPR screens based on their statistical significance with all APC/C-related genes highlighted, showing that Cdc20 is by far the most significant outlier of all APC/C extended complex members. P values were calculated using the RRA method (see “Methods”). ( D , E ) qPCR ( D ) or western blot ( E ) validation of Cdc20 knockdown by shRNA. Paired t test ( N , number of biological replicates; N = 3; ****, P value < 0.0001). Error bars represent the standard deviation (SD) of the mean. .

Techniques: Western Blot, Biomarker Discovery, Expressing, CRISPR, Activity Assay, Knockdown, shRNA, Standard Deviation

$( A , B ) Correlation between mRNA expression of an extended APC/C signature (left), an APC/C signature without CDC20 (middle), or CDC20 alone (right) and sensitivity to genetic disruption of the core SAC components BUB1B ( A ) and MAD2L1 ( B ) in human cancer cell lines from the DepMap. The “APC/C subunit-only” signature contains the 14 core APC/C subunits (Yamano, ), while the “extended APC/C signature” described by Thu et al (Thu et al, ) contains three additional APC/C co-factors, including CDC20. The genes included in each signature are listed in Table . Shown are Spearman’s correlation rho and P values. Spearman correlation ( N , number of cell lines; N = 661 for MAD2L1 or BUB1B vs extended APC/C or subunit-only APC/C, N = 662 for MAD2L1 or BUB1B vs extended APC/C or subunit-only APC/C). RNAi dependency scores were obtained from the Achilles genome-wide RNAi screen, DepMap 22Q2 (Tsherniak et al, ). ( C , D ) Comparison of the sensitivity to two chemical MPS1 inhibitors—MPI-0479605 ( C ) and AZ3146 ( D ) between cell lines in the top vs. bottom mRNA expression quartiles of CDC20. Drug sensitivity data were obtained from PRISM repurposing primary CRISPR screen, DepMap 23Q2 (Corsello et al, ). Two-sided t test ( N = 260 for CDC20 vs MPI-0479605, N = 198 for APC/C vs MPI-0479605, N = 274 for CDC20 vs AZ3146, N = 276 for APC/C vs AZ3146; *, P value = 0.0435; **, P value = 0.006). ( E ) Distribution of the correlation between CDC20 expression and sensitivity to ~6500 different drugs taken from the PRISM primary repurposing screen 2023, ordered by gene ranking percentile (see methods). Comparison between drug classes was performed by Student’s t test. The ranking of CDC20 compared to all protein-coding genes in the response to MPS1 inhibitors is much higher than its average ranking in response to all other drugs. ( F , G ) Percent of EdU-incorporating HCT116 cells following SAC inhibition (125 nM Reversine), with and without CDC20 depletion by shRNA ( F ) or siRNA ( G ). CDC20 depletion increased the fraction of proliferating cells following drug treatment. Two-way ANOVA ( N , number of biological replicates; N = 3; ****, P value < 0.0001). Error bars represent the standard deviation (SD) of the mean. .$

Journal: EMBO Reports

Article Title: High CDC20 levels increase sensitivity of cancer cells to MPS1 inhibitors

doi: 10.1038/s44319-024-00363-8

Figure Lengend Snippet: ( A , B ) Correlation between mRNA expression of an extended APC/C signature (left), an APC/C signature without CDC20 (middle), or CDC20 alone (right) and sensitivity to genetic disruption of the core SAC components BUB1B ( A ) and MAD2L1 ( B ) in human cancer cell lines from the DepMap. The “APC/C subunit-only” signature contains the 14 core APC/C subunits (Yamano, ), while the “extended APC/C signature” described by Thu et al (Thu et al, ) contains three additional APC/C co-factors, including CDC20. The genes included in each signature are listed in Table . Shown are Spearman’s correlation rho and P values. Spearman correlation ( N , number of cell lines; N = 661 for MAD2L1 or BUB1B vs extended APC/C or subunit-only APC/C, N = 662 for MAD2L1 or BUB1B vs extended APC/C or subunit-only APC/C). RNAi dependency scores were obtained from the Achilles genome-wide RNAi screen, DepMap 22Q2 (Tsherniak et al, ). ( C , D ) Comparison of the sensitivity to two chemical MPS1 inhibitors—MPI-0479605 ( C ) and AZ3146 ( D ) between cell lines in the top vs. bottom mRNA expression quartiles of CDC20. Drug sensitivity data were obtained from PRISM repurposing primary CRISPR screen, DepMap 23Q2 (Corsello et al, ). Two-sided t test ( N = 260 for CDC20 vs MPI-0479605, N = 198 for APC/C vs MPI-0479605, N = 274 for CDC20 vs AZ3146, N = 276 for APC/C vs AZ3146; *, P value = 0.0435; **, P value = 0.006). ( E ) Distribution of the correlation between CDC20 expression and sensitivity to ~6500 different drugs taken from the PRISM primary repurposing screen 2023, ordered by gene ranking percentile (see methods). Comparison between drug classes was performed by Student’s t test. The ranking of CDC20 compared to all protein-coding genes in the response to MPS1 inhibitors is much higher than its average ranking in response to all other drugs. ( F , G ) Percent of EdU-incorporating HCT116 cells following SAC inhibition (125 nM Reversine), with and without CDC20 depletion by shRNA ( F ) or siRNA ( G ). CDC20 depletion increased the fraction of proliferating cells following drug treatment. Two-way ANOVA ( N , number of biological replicates; N = 3; ****, P value < 0.0001). Error bars represent the standard deviation (SD) of the mean. .

Techniques: Expressing, Disruption, Genome Wide, Comparison, CRISPR, Inhibition, shRNA, Standard Deviation

Journal: EMBO Reports

Article Title: High CDC20 levels increase sensitivity of cancer cells to MPS1 inhibitors

doi: 10.1038/s44319-024-00363-8

Figure Lengend Snippet: Reagents and tools table

Techniques: Recombinant, Plasmid Preparation, Sequencing, Control, Software, Activity Assay, CRISPR, Knock-Out, Isolation, SYBR Green Assay, Imaging

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