GSCs were acquired from patient specimens at City of Hope under protocols approved by the IRB, and maintained as tumorspheres in GSC media as previously described (4 (link),91 (link)). GSC lines used in this study to test CAR T cell function are IDH1/2-wildtype. The sgRNA library and single-targeted sgRNA lentiviral plasmids (containing a puromycin-resistance gene) for GSC transduction were purchased from Addgene (#73179 and #52961, respectively). Lentiviral particles were generated as previously described (92 (link)). For lentiviral transduction, GSC tumorspheres were dissociated into single cells using Accutase (Innovative Cell Technologies), resuspended in GSC media and lentivirus was added at a 1:50 v/v ratio. GSCs were then washed once after 12 hours, resuspended in fresh GSC media and cultured for 3 days. To ensure that only transduced cells were expanded for further assays, GSCs were selected by puromycin (Thermo Fisher Scientific) for 7 continuous days, with a 1:10000 v/v ratio into GSC media.