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non targeting sgrna above  (Addgene inc)


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    Addgene inc non targeting sgrna above
    ( A ) Schematic overview of the genome-wide CRISPRi screen. K562 dCas9 cells stably expressing dCas9-KRAB were infected with a pooled genome-scale <t>sgRNA</t> library. After growth in galactose, cells were subjected to four pulses of antimycin A or vehicle treatment followed by a 48 hr recovery period. After the last antimycin A pulse, genomic DNA from each condition was isolated and sgRNA abundance was quantified by deep sequencing. ( B ) Volcano plot showing the statistical significance (y axis) versus phenotype scores (ρ, x axis) of control non-targeting and genome-wide targeting sgRNAs. Knockdown of Complex III structural proteins and assembly factors sensitized cells to antimycin A. Genes were considered a hit if they scored above a threshold of ρ z-score x−log 10 p-value of 7 (dashed line). ( C ) CRISPRi knockdown of ovarian carcinoma immunoreactive antigen domain-containing protein 1 (OCIAD1) expression. Western blot showing the expression level of OCIAD1 in K562 dCas9-KRAB cells stably expressing either a control non-targeting sgRNA or two different sgRNAs against OCIAD1. CRISPRi-based silencing reduced OCIAD1 protein expression by ~90%. ( D ) Validation of the OCIAD1 phenotype. K562 dCas9 cells were mixed with an equal number of K562 dCas9-KRAB BFP + cells stably expressing a non-targeting sgRNA (brown bars) or an sgRNA against OCIAD1 (light blue bars). Cell mixtures were then treated with the drug or a vehicle for 24 hr. The percentage of BFP + cells in the cell mixtures was measured by flow cytometry before and 24 hr after treatment. OCIAD1 silencing selectively sensitized cells to antimycin treatment.
    Non Targeting Sgrna Above, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/non+targeting+sgrna+above/pLKO%2E1-blast-SCRAMBLE+(Plasmid+%2326701)/pmc08154037-274-13-21
    Average 93 stars, based on 28 article reviews
    non targeting sgrna above - by Bioz Stars, 2026-09
    93/100 stars

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    1) Product Images from "Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells"

    Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells

    Journal: eLife

    doi: 10.7554/eLife.67624

    ( A ) Schematic overview of the genome-wide CRISPRi screen. K562 dCas9 cells stably expressing dCas9-KRAB were infected with a pooled genome-scale sgRNA library. After growth in galactose, cells were subjected to four pulses of antimycin A or vehicle treatment followed by a 48 hr recovery period. After the last antimycin A pulse, genomic DNA from each condition was isolated and sgRNA abundance was quantified by deep sequencing. ( B ) Volcano plot showing the statistical significance (y axis) versus phenotype scores (ρ, x axis) of control non-targeting and genome-wide targeting sgRNAs. Knockdown of Complex III structural proteins and assembly factors sensitized cells to antimycin A. Genes were considered a hit if they scored above a threshold of ρ z-score x−log 10 p-value of 7 (dashed line). ( C ) CRISPRi knockdown of ovarian carcinoma immunoreactive antigen domain-containing protein 1 (OCIAD1) expression. Western blot showing the expression level of OCIAD1 in K562 dCas9-KRAB cells stably expressing either a control non-targeting sgRNA or two different sgRNAs against OCIAD1. CRISPRi-based silencing reduced OCIAD1 protein expression by ~90%. ( D ) Validation of the OCIAD1 phenotype. K562 dCas9 cells were mixed with an equal number of K562 dCas9-KRAB BFP + cells stably expressing a non-targeting sgRNA (brown bars) or an sgRNA against OCIAD1 (light blue bars). Cell mixtures were then treated with the drug or a vehicle for 24 hr. The percentage of BFP + cells in the cell mixtures was measured by flow cytometry before and 24 hr after treatment. OCIAD1 silencing selectively sensitized cells to antimycin treatment.
    Figure Legend Snippet: ( A ) Schematic overview of the genome-wide CRISPRi screen. K562 dCas9 cells stably expressing dCas9-KRAB were infected with a pooled genome-scale sgRNA library. After growth in galactose, cells were subjected to four pulses of antimycin A or vehicle treatment followed by a 48 hr recovery period. After the last antimycin A pulse, genomic DNA from each condition was isolated and sgRNA abundance was quantified by deep sequencing. ( B ) Volcano plot showing the statistical significance (y axis) versus phenotype scores (ρ, x axis) of control non-targeting and genome-wide targeting sgRNAs. Knockdown of Complex III structural proteins and assembly factors sensitized cells to antimycin A. Genes were considered a hit if they scored above a threshold of ρ z-score x−log 10 p-value of 7 (dashed line). ( C ) CRISPRi knockdown of ovarian carcinoma immunoreactive antigen domain-containing protein 1 (OCIAD1) expression. Western blot showing the expression level of OCIAD1 in K562 dCas9-KRAB cells stably expressing either a control non-targeting sgRNA or two different sgRNAs against OCIAD1. CRISPRi-based silencing reduced OCIAD1 protein expression by ~90%. ( D ) Validation of the OCIAD1 phenotype. K562 dCas9 cells were mixed with an equal number of K562 dCas9-KRAB BFP + cells stably expressing a non-targeting sgRNA (brown bars) or an sgRNA against OCIAD1 (light blue bars). Cell mixtures were then treated with the drug or a vehicle for 24 hr. The percentage of BFP + cells in the cell mixtures was measured by flow cytometry before and 24 hr after treatment. OCIAD1 silencing selectively sensitized cells to antimycin treatment.

    Techniques Used: Genome Wide, Stable Transfection, Expressing, Infection, Isolation, Sequencing, Control, Knockdown, Western Blot, Biomarker Discovery, Flow Cytometry

    ( A ) Read count distribution of all 10 sgRNAs targeting OCIAD1 (squares) and OCIAD2 (triangles) in untreated and antimycin-treated K562 cells. Gray circles represent non-targeting sgRNAs. Dashed blue lines represent the 95% prediction interval. ( B ) Western blot of U2OS and K562 cell extracts immunoblotted with anti-OCIAD2 and anti-ATP5A1 antibodies. ( C ) Western blot of cell extracts from U2OS cells expressing scramble or OCIAD2 shRNAs. The membrane was immunoblotted with anti-OCIAD2 and anti-β-actin antibodies. ( D ) Western blot of extracts from U2OS cells expressing OCIAD2 shRNA and OCIAD1gRNA. The membrane was immunoblotted with anti-OCIAD2 and anti-SDHA antibodies. SDHA was used as a loading control. ( E ) Blue-native polyacrylamide gel electrophoresis (BN-PAGE) analysis of digitonin-solubilized mitochondrial extracts from U2OS cells expressing OCIAD1 sgRNA#2 and OCIAD2 shRNA#1. ATP5A1 served as a loading control. Values represent normalized intensity ± SEM (n = 3 biological replicates). Asterisks (*p<0.05, **p<0.01, or ***p<0.001) correspond to the adjusted (false discovery rate [FDR]) p-values from the post-ANOVA (analysis of variance) pairwise t-test.
    Figure Legend Snippet: ( A ) Read count distribution of all 10 sgRNAs targeting OCIAD1 (squares) and OCIAD2 (triangles) in untreated and antimycin-treated K562 cells. Gray circles represent non-targeting sgRNAs. Dashed blue lines represent the 95% prediction interval. ( B ) Western blot of U2OS and K562 cell extracts immunoblotted with anti-OCIAD2 and anti-ATP5A1 antibodies. ( C ) Western blot of cell extracts from U2OS cells expressing scramble or OCIAD2 shRNAs. The membrane was immunoblotted with anti-OCIAD2 and anti-β-actin antibodies. ( D ) Western blot of extracts from U2OS cells expressing OCIAD2 shRNA and OCIAD1gRNA. The membrane was immunoblotted with anti-OCIAD2 and anti-SDHA antibodies. SDHA was used as a loading control. ( E ) Blue-native polyacrylamide gel electrophoresis (BN-PAGE) analysis of digitonin-solubilized mitochondrial extracts from U2OS cells expressing OCIAD1 sgRNA#2 and OCIAD2 shRNA#1. ATP5A1 served as a loading control. Values represent normalized intensity ± SEM (n = 3 biological replicates). Asterisks (*p<0.05, **p<0.01, or ***p<0.001) correspond to the adjusted (false discovery rate [FDR]) p-values from the post-ANOVA (analysis of variance) pairwise t-test.

    Techniques Used: Western Blot, Expressing, Membrane, shRNA, Control, Polyacrylamide Gel Electrophoresis

    ( A ) Western blot of wildtype U2OS cells or U2OS cells expressing a non-targeting sgRNA, sgRNA#2 against OCIAD1, or sgRNA#2 and wildtype OCIAD1 rescued by lentivirus expression. The upper band (EGFP-OCIAD1) represents intact fusion gene product. The non-targeting sgRNA used in this study does not affect OCIAD1 expression (compare control and wildtype lanes). ( B ) Blue-native polyacrylamide gel electrophoresis (BN-PAGE) results using two CIII 2 core subunits (UQCRC1, left and UQCRC2, right) showing that OCIAD1 is also required for CIII 2 assembly in U2OS cells grown in glucose-containing media. ( C ) BN-PAGE indicating that silencing OCIAD1, but not OCIAD2, disrupts CIII 2 assembly in U2OS cells grown in glucose-containing media. ATP5A1 served as a loading control. Values represent normalized intensity ± SEM (n = 3 biological replicates). Asterisks (*p<0.05, **p<0.01, or ***p<0.001) correspond to the adjusted (false discovery rate [FDR]) p-values from the post-ANOVA (analysis of variance) pairwise t-test.
    Figure Legend Snippet: ( A ) Western blot of wildtype U2OS cells or U2OS cells expressing a non-targeting sgRNA, sgRNA#2 against OCIAD1, or sgRNA#2 and wildtype OCIAD1 rescued by lentivirus expression. The upper band (EGFP-OCIAD1) represents intact fusion gene product. The non-targeting sgRNA used in this study does not affect OCIAD1 expression (compare control and wildtype lanes). ( B ) Blue-native polyacrylamide gel electrophoresis (BN-PAGE) results using two CIII 2 core subunits (UQCRC1, left and UQCRC2, right) showing that OCIAD1 is also required for CIII 2 assembly in U2OS cells grown in glucose-containing media. ( C ) BN-PAGE indicating that silencing OCIAD1, but not OCIAD2, disrupts CIII 2 assembly in U2OS cells grown in glucose-containing media. ATP5A1 served as a loading control. Values represent normalized intensity ± SEM (n = 3 biological replicates). Asterisks (*p<0.05, **p<0.01, or ***p<0.001) correspond to the adjusted (false discovery rate [FDR]) p-values from the post-ANOVA (analysis of variance) pairwise t-test.

    Techniques Used: Western Blot, Expressing, Control, Polyacrylamide Gel Electrophoresis


    Figure Legend Snippet:

    Techniques Used: Control, Transduction, shRNA, Knockdown, Software

    Related Articles

    Control:

    Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells
    Article Snippet: The U2OS OCIAD1/2 double knockdown cell line was generated by infecting stable U2OS CRISPRi cells stably expressing sgRNA#2 (above) with the lentivirus vector pLKO1-OCIAD2_shRNA1 and selecting infected cells with 15 µg/mL blasticidin for 7 days. .. A control cell line was generated by infecting U2OS cells stably expressing a non-targeting sgRNA (above) with the lentivirus vector pLKO.1-blast-Scramble (Addgene, cat# 26701) expressing a non-targeting shRNA sequence and selected with 15 μg/mL blasticidin for 7 days. ..

    Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells
    Article Snippet: The U2OS OCIAD1/2 double knockdown cell line was generated by infecting stable U2OS CRISPRi cells stably expressing sgRNA#2 (above) with the lentivirus vector pLKO1-OCIAD2_shRNA1 and selecting infected cells with 15µg/ml blasticidin for 7 days. .. A control cell line was generated by infecting U2OS cells stably expressing a non-targeting sgRNA (above) with the lentivirus vector pLKO.1-blast-Scramble (Addgene, cat# 26701) expressing a non-targeting shRNA sequence and selected with 15μg/ml blasticidin for 7 days. ..

    Generated:

    Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells
    Article Snippet: The U2OS OCIAD1/2 double knockdown cell line was generated by infecting stable U2OS CRISPRi cells stably expressing sgRNA#2 (above) with the lentivirus vector pLKO1-OCIAD2_shRNA1 and selecting infected cells with 15 µg/mL blasticidin for 7 days. .. A control cell line was generated by infecting U2OS cells stably expressing a non-targeting sgRNA (above) with the lentivirus vector pLKO.1-blast-Scramble (Addgene, cat# 26701) expressing a non-targeting shRNA sequence and selected with 15 μg/mL blasticidin for 7 days. ..

    Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells
    Article Snippet: The U2OS OCIAD1/2 double knockdown cell line was generated by infecting stable U2OS CRISPRi cells stably expressing sgRNA#2 (above) with the lentivirus vector pLKO1-OCIAD2_shRNA1 and selecting infected cells with 15µg/ml blasticidin for 7 days. .. A control cell line was generated by infecting U2OS cells stably expressing a non-targeting sgRNA (above) with the lentivirus vector pLKO.1-blast-Scramble (Addgene, cat# 26701) expressing a non-targeting shRNA sequence and selected with 15μg/ml blasticidin for 7 days. ..

    Stable Transfection:

    Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells
    Article Snippet: The U2OS OCIAD1/2 double knockdown cell line was generated by infecting stable U2OS CRISPRi cells stably expressing sgRNA#2 (above) with the lentivirus vector pLKO1-OCIAD2_shRNA1 and selecting infected cells with 15 µg/mL blasticidin for 7 days. .. A control cell line was generated by infecting U2OS cells stably expressing a non-targeting sgRNA (above) with the lentivirus vector pLKO.1-blast-Scramble (Addgene, cat# 26701) expressing a non-targeting shRNA sequence and selected with 15 μg/mL blasticidin for 7 days. ..

    Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells
    Article Snippet: The U2OS OCIAD1/2 double knockdown cell line was generated by infecting stable U2OS CRISPRi cells stably expressing sgRNA#2 (above) with the lentivirus vector pLKO1-OCIAD2_shRNA1 and selecting infected cells with 15µg/ml blasticidin for 7 days. .. A control cell line was generated by infecting U2OS cells stably expressing a non-targeting sgRNA (above) with the lentivirus vector pLKO.1-blast-Scramble (Addgene, cat# 26701) expressing a non-targeting shRNA sequence and selected with 15μg/ml blasticidin for 7 days. ..

    Expressing:

    Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells
    Article Snippet: The U2OS OCIAD1/2 double knockdown cell line was generated by infecting stable U2OS CRISPRi cells stably expressing sgRNA#2 (above) with the lentivirus vector pLKO1-OCIAD2_shRNA1 and selecting infected cells with 15 µg/mL blasticidin for 7 days. .. A control cell line was generated by infecting U2OS cells stably expressing a non-targeting sgRNA (above) with the lentivirus vector pLKO.1-blast-Scramble (Addgene, cat# 26701) expressing a non-targeting shRNA sequence and selected with 15 μg/mL blasticidin for 7 days. ..

    Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells
    Article Snippet: The U2OS OCIAD1/2 double knockdown cell line was generated by infecting stable U2OS CRISPRi cells stably expressing sgRNA#2 (above) with the lentivirus vector pLKO1-OCIAD2_shRNA1 and selecting infected cells with 15µg/ml blasticidin for 7 days. .. A control cell line was generated by infecting U2OS cells stably expressing a non-targeting sgRNA (above) with the lentivirus vector pLKO.1-blast-Scramble (Addgene, cat# 26701) expressing a non-targeting shRNA sequence and selected with 15μg/ml blasticidin for 7 days. ..

    shRNA:

    Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells
    Article Snippet: The U2OS OCIAD1/2 double knockdown cell line was generated by infecting stable U2OS CRISPRi cells stably expressing sgRNA#2 (above) with the lentivirus vector pLKO1-OCIAD2_shRNA1 and selecting infected cells with 15 µg/mL blasticidin for 7 days. .. A control cell line was generated by infecting U2OS cells stably expressing a non-targeting sgRNA (above) with the lentivirus vector pLKO.1-blast-Scramble (Addgene, cat# 26701) expressing a non-targeting shRNA sequence and selected with 15 μg/mL blasticidin for 7 days. ..

    Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells
    Article Snippet: The U2OS OCIAD1/2 double knockdown cell line was generated by infecting stable U2OS CRISPRi cells stably expressing sgRNA#2 (above) with the lentivirus vector pLKO1-OCIAD2_shRNA1 and selecting infected cells with 15µg/ml blasticidin for 7 days. .. A control cell line was generated by infecting U2OS cells stably expressing a non-targeting sgRNA (above) with the lentivirus vector pLKO.1-blast-Scramble (Addgene, cat# 26701) expressing a non-targeting shRNA sequence and selected with 15μg/ml blasticidin for 7 days. ..

    Sequencing:

    Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells
    Article Snippet: The U2OS OCIAD1/2 double knockdown cell line was generated by infecting stable U2OS CRISPRi cells stably expressing sgRNA#2 (above) with the lentivirus vector pLKO1-OCIAD2_shRNA1 and selecting infected cells with 15 µg/mL blasticidin for 7 days. .. A control cell line was generated by infecting U2OS cells stably expressing a non-targeting sgRNA (above) with the lentivirus vector pLKO.1-blast-Scramble (Addgene, cat# 26701) expressing a non-targeting shRNA sequence and selected with 15 μg/mL blasticidin for 7 days. ..

    Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells
    Article Snippet: The U2OS OCIAD1/2 double knockdown cell line was generated by infecting stable U2OS CRISPRi cells stably expressing sgRNA#2 (above) with the lentivirus vector pLKO1-OCIAD2_shRNA1 and selecting infected cells with 15µg/ml blasticidin for 7 days. .. A control cell line was generated by infecting U2OS cells stably expressing a non-targeting sgRNA (above) with the lentivirus vector pLKO.1-blast-Scramble (Addgene, cat# 26701) expressing a non-targeting shRNA sequence and selected with 15μg/ml blasticidin for 7 days. ..



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    ( A ) Schematic overview of the genome-wide CRISPRi screen. K562 dCas9 cells stably expressing dCas9-KRAB were infected with a pooled genome-scale <t>sgRNA</t> library. After growth in galactose, cells were subjected to four pulses of antimycin A or vehicle treatment followed by a 48 hr recovery period. After the last antimycin A pulse, genomic DNA from each condition was isolated and sgRNA abundance was quantified by deep sequencing. ( B ) Volcano plot showing the statistical significance (y axis) versus phenotype scores (ρ, x axis) of control non-targeting and genome-wide targeting sgRNAs. Knockdown of Complex III structural proteins and assembly factors sensitized cells to antimycin A. Genes were considered a hit if they scored above a threshold of ρ z-score x−log 10 p-value of 7 (dashed line). ( C ) CRISPRi knockdown of ovarian carcinoma immunoreactive antigen domain-containing protein 1 (OCIAD1) expression. Western blot showing the expression level of OCIAD1 in K562 dCas9-KRAB cells stably expressing either a control non-targeting sgRNA or two different sgRNAs against OCIAD1. CRISPRi-based silencing reduced OCIAD1 protein expression by ~90%. ( D ) Validation of the OCIAD1 phenotype. K562 dCas9 cells were mixed with an equal number of K562 dCas9-KRAB BFP + cells stably expressing a non-targeting sgRNA (brown bars) or an sgRNA against OCIAD1 (light blue bars). Cell mixtures were then treated with the drug or a vehicle for 24 hr. The percentage of BFP + cells in the cell mixtures was measured by flow cytometry before and 24 hr after treatment. OCIAD1 silencing selectively sensitized cells to antimycin treatment.
    Non Targeting Sgrna Above, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/non+targeting+sgrna+above/pLKO%2E1-blast-SCRAMBLE+(Plasmid+%2326701)/pmc08154037-274-13-21
    Average 93 stars, based on 1 article reviews
    non targeting sgrna above - by Bioz Stars, 2026-09
    93/100 stars
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    ( A ) Schematic overview of the genome-wide CRISPRi screen. K562 dCas9 cells stably expressing dCas9-KRAB were infected with a pooled genome-scale sgRNA library. After growth in galactose, cells were subjected to four pulses of antimycin A or vehicle treatment followed by a 48 hr recovery period. After the last antimycin A pulse, genomic DNA from each condition was isolated and sgRNA abundance was quantified by deep sequencing. ( B ) Volcano plot showing the statistical significance (y axis) versus phenotype scores (ρ, x axis) of control non-targeting and genome-wide targeting sgRNAs. Knockdown of Complex III structural proteins and assembly factors sensitized cells to antimycin A. Genes were considered a hit if they scored above a threshold of ρ z-score x−log 10 p-value of 7 (dashed line). ( C ) CRISPRi knockdown of ovarian carcinoma immunoreactive antigen domain-containing protein 1 (OCIAD1) expression. Western blot showing the expression level of OCIAD1 in K562 dCas9-KRAB cells stably expressing either a control non-targeting sgRNA or two different sgRNAs against OCIAD1. CRISPRi-based silencing reduced OCIAD1 protein expression by ~90%. ( D ) Validation of the OCIAD1 phenotype. K562 dCas9 cells were mixed with an equal number of K562 dCas9-KRAB BFP + cells stably expressing a non-targeting sgRNA (brown bars) or an sgRNA against OCIAD1 (light blue bars). Cell mixtures were then treated with the drug or a vehicle for 24 hr. The percentage of BFP + cells in the cell mixtures was measured by flow cytometry before and 24 hr after treatment. OCIAD1 silencing selectively sensitized cells to antimycin treatment.

    Journal: eLife

    Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells

    doi: 10.7554/eLife.67624

    Figure Lengend Snippet: ( A ) Schematic overview of the genome-wide CRISPRi screen. K562 dCas9 cells stably expressing dCas9-KRAB were infected with a pooled genome-scale sgRNA library. After growth in galactose, cells were subjected to four pulses of antimycin A or vehicle treatment followed by a 48 hr recovery period. After the last antimycin A pulse, genomic DNA from each condition was isolated and sgRNA abundance was quantified by deep sequencing. ( B ) Volcano plot showing the statistical significance (y axis) versus phenotype scores (ρ, x axis) of control non-targeting and genome-wide targeting sgRNAs. Knockdown of Complex III structural proteins and assembly factors sensitized cells to antimycin A. Genes were considered a hit if they scored above a threshold of ρ z-score x−log 10 p-value of 7 (dashed line). ( C ) CRISPRi knockdown of ovarian carcinoma immunoreactive antigen domain-containing protein 1 (OCIAD1) expression. Western blot showing the expression level of OCIAD1 in K562 dCas9-KRAB cells stably expressing either a control non-targeting sgRNA or two different sgRNAs against OCIAD1. CRISPRi-based silencing reduced OCIAD1 protein expression by ~90%. ( D ) Validation of the OCIAD1 phenotype. K562 dCas9 cells were mixed with an equal number of K562 dCas9-KRAB BFP + cells stably expressing a non-targeting sgRNA (brown bars) or an sgRNA against OCIAD1 (light blue bars). Cell mixtures were then treated with the drug or a vehicle for 24 hr. The percentage of BFP + cells in the cell mixtures was measured by flow cytometry before and 24 hr after treatment. OCIAD1 silencing selectively sensitized cells to antimycin treatment.

    Article Snippet: A control cell line was generated by infecting U2OS cells stably expressing a non-targeting sgRNA (above) with the lentivirus vector pLKO.1-blast-Scramble (Addgene, cat# 26701) expressing a non-targeting shRNA sequence and selected with 15 μg/mL blasticidin for 7 days.

    Techniques: Genome Wide, Stable Transfection, Expressing, Infection, Isolation, Sequencing, Control, Knockdown, Western Blot, Biomarker Discovery, Flow Cytometry

    ( A ) Read count distribution of all 10 sgRNAs targeting OCIAD1 (squares) and OCIAD2 (triangles) in untreated and antimycin-treated K562 cells. Gray circles represent non-targeting sgRNAs. Dashed blue lines represent the 95% prediction interval. ( B ) Western blot of U2OS and K562 cell extracts immunoblotted with anti-OCIAD2 and anti-ATP5A1 antibodies. ( C ) Western blot of cell extracts from U2OS cells expressing scramble or OCIAD2 shRNAs. The membrane was immunoblotted with anti-OCIAD2 and anti-β-actin antibodies. ( D ) Western blot of extracts from U2OS cells expressing OCIAD2 shRNA and OCIAD1gRNA. The membrane was immunoblotted with anti-OCIAD2 and anti-SDHA antibodies. SDHA was used as a loading control. ( E ) Blue-native polyacrylamide gel electrophoresis (BN-PAGE) analysis of digitonin-solubilized mitochondrial extracts from U2OS cells expressing OCIAD1 sgRNA#2 and OCIAD2 shRNA#1. ATP5A1 served as a loading control. Values represent normalized intensity ± SEM (n = 3 biological replicates). Asterisks (*p<0.05, **p<0.01, or ***p<0.001) correspond to the adjusted (false discovery rate [FDR]) p-values from the post-ANOVA (analysis of variance) pairwise t-test.

    Journal: eLife

    Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells

    doi: 10.7554/eLife.67624

    Figure Lengend Snippet: ( A ) Read count distribution of all 10 sgRNAs targeting OCIAD1 (squares) and OCIAD2 (triangles) in untreated and antimycin-treated K562 cells. Gray circles represent non-targeting sgRNAs. Dashed blue lines represent the 95% prediction interval. ( B ) Western blot of U2OS and K562 cell extracts immunoblotted with anti-OCIAD2 and anti-ATP5A1 antibodies. ( C ) Western blot of cell extracts from U2OS cells expressing scramble or OCIAD2 shRNAs. The membrane was immunoblotted with anti-OCIAD2 and anti-β-actin antibodies. ( D ) Western blot of extracts from U2OS cells expressing OCIAD2 shRNA and OCIAD1gRNA. The membrane was immunoblotted with anti-OCIAD2 and anti-SDHA antibodies. SDHA was used as a loading control. ( E ) Blue-native polyacrylamide gel electrophoresis (BN-PAGE) analysis of digitonin-solubilized mitochondrial extracts from U2OS cells expressing OCIAD1 sgRNA#2 and OCIAD2 shRNA#1. ATP5A1 served as a loading control. Values represent normalized intensity ± SEM (n = 3 biological replicates). Asterisks (*p<0.05, **p<0.01, or ***p<0.001) correspond to the adjusted (false discovery rate [FDR]) p-values from the post-ANOVA (analysis of variance) pairwise t-test.

    Article Snippet: A control cell line was generated by infecting U2OS cells stably expressing a non-targeting sgRNA (above) with the lentivirus vector pLKO.1-blast-Scramble (Addgene, cat# 26701) expressing a non-targeting shRNA sequence and selected with 15 μg/mL blasticidin for 7 days.

    Techniques: Western Blot, Expressing, Membrane, shRNA, Control, Polyacrylamide Gel Electrophoresis

    ( A ) Western blot of wildtype U2OS cells or U2OS cells expressing a non-targeting sgRNA, sgRNA#2 against OCIAD1, or sgRNA#2 and wildtype OCIAD1 rescued by lentivirus expression. The upper band (EGFP-OCIAD1) represents intact fusion gene product. The non-targeting sgRNA used in this study does not affect OCIAD1 expression (compare control and wildtype lanes). ( B ) Blue-native polyacrylamide gel electrophoresis (BN-PAGE) results using two CIII 2 core subunits (UQCRC1, left and UQCRC2, right) showing that OCIAD1 is also required for CIII 2 assembly in U2OS cells grown in glucose-containing media. ( C ) BN-PAGE indicating that silencing OCIAD1, but not OCIAD2, disrupts CIII 2 assembly in U2OS cells grown in glucose-containing media. ATP5A1 served as a loading control. Values represent normalized intensity ± SEM (n = 3 biological replicates). Asterisks (*p<0.05, **p<0.01, or ***p<0.001) correspond to the adjusted (false discovery rate [FDR]) p-values from the post-ANOVA (analysis of variance) pairwise t-test.

    Journal: eLife

    Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells

    doi: 10.7554/eLife.67624

    Figure Lengend Snippet: ( A ) Western blot of wildtype U2OS cells or U2OS cells expressing a non-targeting sgRNA, sgRNA#2 against OCIAD1, or sgRNA#2 and wildtype OCIAD1 rescued by lentivirus expression. The upper band (EGFP-OCIAD1) represents intact fusion gene product. The non-targeting sgRNA used in this study does not affect OCIAD1 expression (compare control and wildtype lanes). ( B ) Blue-native polyacrylamide gel electrophoresis (BN-PAGE) results using two CIII 2 core subunits (UQCRC1, left and UQCRC2, right) showing that OCIAD1 is also required for CIII 2 assembly in U2OS cells grown in glucose-containing media. ( C ) BN-PAGE indicating that silencing OCIAD1, but not OCIAD2, disrupts CIII 2 assembly in U2OS cells grown in glucose-containing media. ATP5A1 served as a loading control. Values represent normalized intensity ± SEM (n = 3 biological replicates). Asterisks (*p<0.05, **p<0.01, or ***p<0.001) correspond to the adjusted (false discovery rate [FDR]) p-values from the post-ANOVA (analysis of variance) pairwise t-test.

    Article Snippet: A control cell line was generated by infecting U2OS cells stably expressing a non-targeting sgRNA (above) with the lentivirus vector pLKO.1-blast-Scramble (Addgene, cat# 26701) expressing a non-targeting shRNA sequence and selected with 15 μg/mL blasticidin for 7 days.

    Techniques: Western Blot, Expressing, Control, Polyacrylamide Gel Electrophoresis

    Journal: eLife

    Article Title: Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells

    doi: 10.7554/eLife.67624

    Figure Lengend Snippet:

    Article Snippet: A control cell line was generated by infecting U2OS cells stably expressing a non-targeting sgRNA (above) with the lentivirus vector pLKO.1-blast-Scramble (Addgene, cat# 26701) expressing a non-targeting shRNA sequence and selected with 15 μg/mL blasticidin for 7 days.

    Techniques: Control, Transduction, shRNA, Knockdown, Software