A genome-scale gain-of-function CRISPR screen in CD8 T cells identifies proline metabolism as a means to enhance CAR-T therapy

Cell Metab. 2022 Apr 5;34(4):595-614.e14. doi: 10.1016/j.cmet.2022.02.009. Epub 2022 Mar 10.


Chimeric antigen receptor (CAR)-T cell-based immunotherapy for cancer and immunological diseases has made great strides, but it still faces multiple hurdles. Finding the right molecular targets to engineer T cells toward a desired function has broad implications for the armamentarium of T cell-centered therapies. Here, we developed a dead-guide RNA (dgRNA)-based CRISPR activation screen in primary CD8+ T cells and identified gain-of-function (GOF) targets for CAR-T engineering. Targeted knockin or overexpression of a lead target, PRODH2, enhanced CAR-T-based killing and in vivo efficacy in multiple cancer models. Transcriptomics and metabolomics in CAR-T cells revealed that augmenting PRODH2 expression reshaped broad and distinct gene expression and metabolic programs. Mitochondrial, metabolic, and immunological analyses showed that PRODH2 engineering enhances the metabolic and immune functions of CAR-T cells against cancer. Together, these findings provide a system for identification of GOF immune boosters and demonstrate PRODH2 as a target to enhance CAR-T efficacy.

Keywords: CAR-T; PRODH2; T cell CRISPR activation screen; T cell GOF screen; antitumor efficacy; dead-guide RNA; metabolism; mitochondria; proline metabolism.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CD8-Positive T-Lymphocytes
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • Gain of Function Mutation
  • Humans
  • Neoplasms*
  • Proline
  • Receptors, Chimeric Antigen* / genetics
  • Receptors, Chimeric Antigen* / metabolism


  • Receptors, Chimeric Antigen
  • Proline