A genome-wide CRISPR screen identifies mediators of ferroptosis sensitivity in clear-cell carcinomas

Clear-cell carcinomas (CCCs) are a histological group of highly aggressive malignancies commonly originating in the kidney and ovary. CCC tumors are distinguished by aberrant lipid and glycogen accumulation and are inherently refractory to a broad range of anti-cancer therapies. In the study associated with this dataset, we identified an intrinsic vulnerability to ferroptosis associated with the unique metabolic state in CCC cells. However, the mediators of this sensitivity to ferroptosis are unknown. Here we perform a genome-wide CRISPR screen to identify genes that mediate the sensitivity to ML210, a selective inhibitor of glutathione peroxidase 4 (GPX4) and a potent inducer of ferroptotic cell death in 786-O, a clear-cell renal cell carcinoma cell line. 786-O cells with Cas9 expression were infected with a genome-wide lentiviral sgRNA library, selected with puromycin, and expanded for treatment with DMSO or ML210 for 4, 6, or 8 days, respectively. sgRNA-barcodes that were enriched in ML210 treated conditions were identified by high-throughput sequencing.

透明细胞癌（Clear-cell carcinomas, CCCs）是一类高度侵袭性的恶性肿瘤组织学类型，通常起源于肾脏与卵巢。该类肿瘤以脂质与糖原异常蓄积为典型特征，且固有地对多种抗癌疗法产生耐药性。
在本数据集关联的研究中，我们发现透明细胞癌细胞独特的代谢状态与铁死亡（ferroptosis）的内在易感性相关，但该铁死亡敏感性的介导因子仍不明确。
本研究通过全基因组CRISPR筛选，在786-O细胞——一种透明细胞肾癌细胞系——中鉴定介导对ML210敏感性的基因：ML210是谷胱甘肽过氧化物酶4（glutathione peroxidase 4, GPX4）的选择性抑制剂，同时也是铁死亡性细胞死亡的强效诱导剂。具体实验流程如下：将表达Cas9的786-O细胞感染全基因组慢病毒sgRNA（single guide RNA）文库，经嘌呤霉素筛选后扩增，再分别用二甲基亚砜（dimethyl sulfoxide, DMSO）或ML210处理4、6或8天。最终通过高通量测序，鉴定在ML210处理组中富集的sgRNA条形码序列。