CD3ζ-editing strategy. A CAR missing the activation domain (truncCAR) is integrated in the CD3ζ locus through CRISPR/Cas-9 nonviral technology to obtain a fully functional CAR construct. The proposed strategy brings the CAR under the transcriptional regulation of the CD3ζ promoter, reducing the expression of inhibitory receptors, and ablates the expression of the endogenous TCR. This approach can be applied to all T-cell subsets and NK cells. αβT, T-cell receptor-αβ T lymphocytes; CAR, chimeric antigen receptor; H/S, hinge/spacer sequences; scFv, single-chain fragment variable; TCR, T-cell receptor; TM, transmembrane domain; Treg, regulatory T lymphocytes; γδT, TCRγδ T lymphocytes; NK, natural killer.