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

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

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