CRISPR Screens Identify Gene Edits That Supercharge Cancer-Fighting CAR T Cells

CAR T cell therapy has revolutionized blood cancer treatment but faces significant limitations including T cell exhaustion, poor proliferation, and fratricide (CAR T cells killing each other). Researchers developed CELLFIE (Cell Engineering For Immunotherapy Enhancement), a comprehensive CRISPR screening platform to systematically identify genetic modifications that overcome these barriers.

The team performed genome-wide knockout screens in human primary CAR T cells from multiple donors, testing how different gene deletions affected key therapeutic functions. They used both laboratory culture conditions and xenograft mouse models of human leukemia to validate their findings. The platform uniquely combines CAR delivery, CRISPR editing, and multiple functional readouts in a single workflow.

The most significant discovery was that knocking out RHOG dramatically enhances CAR T cell performance. This finding was unexpected because RHOG deficiency causes immunodeficiency in humans, illustrating that engineered therapeutic cells have different biological requirements than natural immune cells. The researchers also identified FAS knockout as beneficial, and showed that combining RHOG and FAS knockouts provides synergistic enhancement.

Validation experiments across multiple CAR designs, patient samples, and animal models confirmed these genetic modifications consistently improve anti-tumor activity. The team further developed base-editing approaches to create safer RHOG variants for clinical translation, avoiding potentially problematic double-strand DNA breaks.

This work establishes a foundational platform for optimizing cell-based immunotherapies through systematic genetic engineering, potentially leading to more effective CAR T cell treatments for cancer patients.