Biological applications of CRISPR perturbations and expanded modalities of bulk CRISPR screens in T cells. (A) Biological applications of CRISPR perturbations include the discovery of master regulator genes, identification of downstream mechanisms through genetic interaction screens, mapping of causal biological circuits via single-cell perturbation screens, and investigation of non–cell-autonomous effects through spatial screening approaches, which collectively help facilitate therapeutic engineering. (B) Bulk CRISPR-based screening approaches, including CRISPR KO, CRISPRi, and CRISPRa screens, have been widely used in T cells to probe key functional readouts, such as activation (e.g., CD69 and CD25 expression), proliferation, mTORC1 signaling, cytokine production, and Foxp3 expression. Emerging screening strategies, including ORF overexpression, targeted knock-in, base editing, prime editing, and RNA editing, are adding new layers of functional interrogation to these platforms. sKO, single knockout; NTC, nontargeting control; dgRNA, catalytically dead guide RNA; pegRNA, prime editing guide RNA; HA-GD2-28ζ CAR, GD2-targeting CAR with HA tag and CD28-CD3ζ signaling domain.