CRISPR screen identifies loss of IFNγR signaling and downstream adhesion as a resistance mechanism to CAR T-cell cytotoxicity in solid tumors. CRISPR screen identifies loss of IFNγR signaling and downstream adhesion as a resistance mechanism to CAR T-cell cytotoxicity in solid tumors

U87-CBG-GFP cells selected with blasticidin to stably express Cas9 were transduced with an MOI (< 1) of Brunello guide library (with puromycin resistance in the guide backbone). Cells were rested for 2 days then selected with puromycin antibiotic until the control, untransduced arm was killed (6-7 days). The guide-transduced cells were then pooled and replated in preparation for the screen. There were 12 arms to the screen, two control arms performed in technical duplicate and 8 sample arms from 6 normal donor CAR T-cells (2 were run in technical duplicate). At the time of CAR T-cell addition 2 arms were taken down as technical duplicates to measure guide frequency at the beginning of the screen. 2 additional arms received no CAR T-cells to serve as proliferation controls and were passaged the same as the sample CAR T conditions about to be described. CAR T-cells were added at a 1:10 effector:target cell ratio. After 18 hours of co-culture the CAR T-cells were removed by aspiration and the tumor cells were gently rinsed 4 times with PBS to remove any remaining CAR T-cells. The tumor cells were then passaged so that any remaining dying cells would not survive. After a 2-day recovery period, the tumor cells were rinsed once with PBS and harvested. Their DNA was isolated (NucleoSpin Blood XL, Macherey-Nagel) and prepared for PCR (Onestep PCR Inhibitor, Zymo Research) prior to processing for guide PCR amplification and sequencing by the Genetic Perturbation Platform (GPP) facility at the Broad Institute based on Broad recommended preparation protocols. Overall design: Genome wide CRISPR knockout screen in glioblastoma was utilized to identify mechanisms of resistance to CAR T cell cytotoxicity via loss of a single gene.

将经杀稻瘟菌素筛选以稳定表达Cas9的U87-CBG-GFP细胞，以感染复数（Multiplicity of Infection, MOI）<1的比例转导Brunello向导文库（该向导骨架携带嘌呤霉素抗性基因）。细胞静置恢复2天后，采用嘌呤霉素抗生素进行筛选，直至未转导的对照组细胞完全死亡（耗时6-7天）。随后将转导了向导序列的细胞混合并重铺，为后续筛选实验做好准备。本次筛选共设置12个实验组：其中2个为对照组，均进行技术重复；8个为样本实验组，来源于6名健康供者的嵌合抗原受体T细胞（Chimeric Antigen Receptor T-Cell, CAR-T），其中2组开展了技术重复。在加入CAR-T细胞时，取出2个实验组作为技术重复样本，用于检测筛选起始阶段的向导RNA频率。另有2个实验组未添加CAR-T细胞，作为增殖对照组，其传代操作与后续所述的CAR-T共培养样本组保持一致。将CAR-T细胞以效应细胞与靶细胞比例1:10的比例加入共培养体系。共培养18小时后，通过吸弃法移除CAR-T细胞，再用PBS轻柔冲洗肿瘤细胞4次，以彻底去除残留的CAR-T细胞。随后对肿瘤细胞进行传代培养，确保残留的凋亡细胞无法存活。经过2天的恢复期后，用PBS冲洗肿瘤细胞1次并收集样本。提取收集到的肿瘤细胞的基因组DNA（使用NucleoSpin Blood XL试剂盒，Macherey-Nagel品牌），并通过Onestep PCR Inhibitor试剂盒（Zymo Research品牌）去除PCR反应抑制物，完成PCR前样本制备。随后按照博德研究所（Broad Institute）遗传扰动平台（Genetic Perturbation Platform, GPP）推荐的实验方案，开展向导RNA的PCR扩增与高通量测序。整体实验设计：本研究采用全基因组CRISPR敲除筛选策略，在胶质母细胞瘤细胞中筛选可通过单基因缺失介导CAR-T细胞毒性耐药的分子机制。