Journal: Nature Communications

Article Title: Identification of regulators of poly-ADP-ribose polymerase inhibitor response through complementary CRISPR knockout and activation screens

doi: 10.1038/s41467-020-19961-w

Figure Lengend Snippet: a Schematic representation of the CRISPR knockout screen for olaparib sensitivity in wildtype cells. HeLa cells were infected with the Brunello CRISPR knockout library. Infected cells were divided into PARP inhibitor (olaparib)-treated or control (DMSO) arms. Genomic DNA was extracted from cells surviving the drug treatment and single-guide RNAs (sgRNAs) were identified using Illumina sequencing. b Scatterplot showing the results of this screen. Each gene targeted by the library was ranked based on the MAGeCK negative selection score. Several biologically interesting hits are highlighted. c Schematic representation of the CRISPR knockout screen for olaparib resistance in BRCA2 KO cells. HeLa BRCA2 KO cells were infected with the Brunello CRISPR knockout library. Infected cells were divided into PARP inhibitor (olaparib)-treated or control (DMSO) arms. d Scatterplot showing the results of this screen, with several biologically interesting hits highlighted. Each gene targeted by the library was ranked based on the MAGeCK positive selection score. e Schematic representation of the CRISPR activation screen for olaparib resistance in BRCA2 KO cells. HeLa BRCA2 KO cells stably expressing the modified dCas9 enzyme were infected with the Calabrese CRISPR activation library. Infected cells were divided into PARP inhibitor (olaparib)-treated or control (DMSO) arms. f Scatterplot showing the results of this screen, with several biologically interesting hits highlighted. Each gene targeted by the library was ranked based on the MAGeCK positive selection score. Source data are provided as a Source Data file.

Article Snippet: For the CRISPR activation screens, HeLa BRCA2-knockout cells were infected with dCas9 (Addgene, 61425-LV) and selected with blasticidin (3 μg/ml). dCas9-expressing cells were then transduced with the Calabrese Human CRISPR Activation Pooled Library (Set A, Addgene, 92379-LV) using enough cells to obtain a library coverage of 500 cells per sgRNA at an MOI of 0.4 .

Techniques: CRISPR, Knock-Out, Infection, Control, Illumina Sequencing, Selection, Activation Assay, Stable Transfection, Expressing, Modification

Journal: Nature Communications

Article Title: Identification of regulators of poly-ADP-ribose polymerase inhibitor response through complementary CRISPR knockout and activation screens

doi: 10.1038/s41467-020-19961-w

Figure Lengend Snippet: a Western blot showing overexpression of ABCB1 in the cell line containing all three components of the CRISPR lentiviral activation particle (LAP) system (dCas9, activator helper complex, and sgRNA targeting the ABCB1 gene) but not in the control cell line lacking the sgRNA. b Cellular viability assay showing that ABCB1 transcriptional activation rescues PARPi sensitivity in HeLa BRCA2-knockout cells. The averages of four experiments are shown, with standard deviations as error bars. c Olaparib-induced apoptosis in BRCA2-knockout cells is suppressed by ABCB1 overexpression. The averages of four experiments are shown, with standard deviations as error bars. Asterisks indicate statistical significance ( t -test, two-tailed, unequal variance) compared to the No Guide sample. Source data are provided as a Source Data file.

Article Snippet: For the CRISPR activation screens, HeLa BRCA2-knockout cells were infected with dCas9 (Addgene, 61425-LV) and selected with blasticidin (3 μg/ml). dCas9-expressing cells were then transduced with the Calabrese Human CRISPR Activation Pooled Library (Set A, Addgene, 92379-LV) using enough cells to obtain a library coverage of 500 cells per sgRNA at an MOI of 0.4 .

Techniques: Western Blot, Over Expression, CRISPR, Activation Assay, Control, Cell Viability Assay, Knock-Out, Two Tailed Test