Genome-scale CRISPR-Cas9 screen of OVCAR3 and CCL218 cancer cells with or without hydroxychloroquine

A Brunello CRISPR-Cas9 library screen was performed with OVCAR3 and CCL218 cells in the presence or absence of hydroxychloroquine. Drug pulse chases were used, with 48h of drug exposure followed by replacement of fresh media. Cells were allowed to grow to near confluence and then another drug pulse-chase was performed. After each pulse-chase, a pool of cells were collected for sgRNA quantitation. Three pulse chases were performed. Cell pellets were processed for genomic DNA and sgRNA sequences amplified by PCR for NGS-based quantitation. Once quantified by NGS analysis, MAGeCK MLE analysis was performed to determine which sgRNAs were most associated with resistance or sensitization to hydroxychloroquine. Cell pellets were processed for genomic DNA and sgRNA sequences amplified by PCR for NGS-based quantitation.