Genome-wide CRISPR screening identifies the E3 ubiquitin ligase HERC1 as a modulator of the response to nucleoside analogs in acute myeloid leukemia

We identified the Ubiquitin Ligase HERC1 in the context of cytarabine response in a genome-wide CRISPR screen in murine AML. We hypothesized to detect early transciptomic changes in cytarabine treated murine AML cells genetically targeted by using single guide RNAs targeting HERC1 and Non-targeting Control. Early Detection of transcriptomic changes in cytarabine treated (200nm) murine MLL-AF9 transformed progenitor cells which were genetically modified with single guide RNA targeting HERC1 or Non-Targeting Ctrl RNA. AML clones targeted for HERC1 were sorted by using BD FACSAria II, and clone E5 was chosen for RNA sequencing. sgHERC1 - and sgCtrl MLL/AF9 cells were treated for six hours with 200nM cytarabine or H20 and RNA was subsequently isolated by using TRIzol (Invitrogen) + RNeasy mini kit (Qiagen, #74106).

我们在小鼠急性髓系白血病（murine AML）的全基因组CRISPR筛选实验中，针对阿糖胞苷（cytarabine）应答情境鉴定出泛素连接酶（Ubiquitin Ligase）HERC1。我们提出假说：通过使用靶向HERC1的单引导RNA（single guide RNA, sgRNA）与非靶向对照单引导RNA，可检测阿糖胞苷处理后的小鼠AML细胞中的早期转录组变化。本研究旨在检测经200nM阿糖胞苷处理、且通过靶向HERC1或非靶向对照RNA的单引导RNA完成遗传修饰的小鼠MLL-AF9转化祖细胞的转录组早期变化。我们采用BD FACSAria II流式细胞分选仪对靶向HERC1的AML克隆进行分选，并选取克隆E5进行RNA测序。将sgHERC1组与sgCtrl组的MLL-AF9细胞用200nM阿糖胞苷或双蒸水（H₂O）处理6小时后，通过TRIzol（Invitrogen）与RNeasy 小型试剂盒（Qiagen，货号#74106）提取总RNA。