A genome-wide screen identifies E2F6 as critical factor for TMZ chemoresistance in EGFRvIII expressing GBM (DNA-seq). A genome-wide screen identifies E2F6 as critical factor for TMZ chemoresistance in EGFRvIII expressing GBM (DNA-seq)

We conducted a genome-wide screen by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 library to identify genes which are particularly resistant to EGFRvIII GBM cells under the chemo-stress of TMZ. In vitro and in vivo validation of EGFRvIII-specific targets revealed several strong hits, including E2F6. Mechanistic studies demonstrated that E2F6 reveals its resistance to TMZ through NF-κB and TMZ inducement. Overall design: A pooled genome-wide CRISPR-Cas9-based screen using a sgRNA lentiviral library harboring 123,411 sgRNAs which target 20,914 human genes were performed to transduce EGFRwt and EGFRvIII U87 cells ( no replicate), then treated with TMZ for 7 and 14 days. The end time point of each screen was compared to day 0 in order to determine which sgRNAs were overrepresented or underrepresented in the population.

本研究借助成簇规律间隔短回文重复序列(CRISPR)-Cas9文库开展全基因组筛选，旨在鉴定在替莫唑胺(TMZ)化疗应激下，对EGFRvIII突变型胶质母细胞瘤(GBM)细胞具有特异性耐药性的基因。对EGFRvIII特异性靶点的体外与体内验证实验筛选出多个强阳性命中靶点，其中包括E2F6。机制研究表明，E2F6可经TMZ诱导后通过核因子κB(NF-κB)通路介导细胞对TMZ的耐药性。实验设计概况：本研究采用包含123411条单引导RNA(sgRNA)、靶向20914个人类基因的慢病毒文库，开展混合池式全基因组CRISPR-Cas9筛选：将该文库转导至EGFR野生型(EGFRwt)及EGFRvIII突变型U87细胞（无生物学重复），随后用TMZ分别处理7天和14天。将每个筛选实验的终末时间点样本与第0天样本进行比对，以确定细胞群体中sgRNA的富集或缺失情况。