CRISPR Screening of CAR T Cells and Cancer Stem Cells Reveals Critical Dependencies for Cell-Based Therapies

Cancer Discov. 2021 May;11(5):1192-1211. doi: 10.1158/2159-8290.CD-20-1243. Epub 2020 Dec 16.


Glioblastoma (GBM) contains self-renewing GBM stem cells (GSC) potentially amenable to immunologic targeting, but chimeric antigen receptor (CAR) T-cell therapy has demonstrated limited clinical responses in GBM. Here, we interrogated molecular determinants of CAR-mediated GBM killing through whole-genome CRISPR screens in both CAR T cells and patient-derived GSCs. Screening of CAR T cells identified dependencies for effector functions, including TLE4 and IKZF2. Targeted knockout of these genes enhanced CAR antitumor efficacy. Bulk and single-cell RNA sequencing of edited CAR T cells revealed transcriptional profiles of superior effector function and inhibited exhaustion responses. Reciprocal screening of GSCs identified genes essential for susceptibility to CAR-mediated killing, including RELA and NPLOC4, the knockout of which altered tumor-immune signaling and increased responsiveness of CAR therapy. Overall, CRISPR screening of CAR T cells and GSCs discovered avenues for enhancing CAR therapeutic efficacy against GBM, with the potential to be extended to other solid tumors. SIGNIFICANCE: Reciprocal CRISPR screening identified genes in both CAR T cells and tumor cells regulating the potency of CAR T-cell cytotoxicity, informing molecular targeting strategies to potentiate CAR T-cell antitumor efficacy and elucidate genetic modifications of tumor cells in combination with CAR T cells to advance immuno-oncotherapy.This article is highlighted in the In This Issue feature, p. 995.

MeSH terms

  • Brain Neoplasms / genetics*
  • Brain Neoplasms / pathology
  • Cell Line, Tumor
  • Cell- and Tissue-Based Therapy
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Glioblastoma / genetics*
  • Glioblastoma / pathology
  • Humans
  • Neoplastic Stem Cells / metabolism*
  • Receptors, Chimeric Antigen / genetics*


  • Receptors, Chimeric Antigen