CRISPR/Cas9 NHEJ- and HDR-mediated CAR-T modification. CRISPR/Cas9 NHEJ-mediated knock-out and HDR-mediated knock-in can be used for a variety of applications. For example in CAR-T cells, (A) NHEJ has been used to block alloreactivity to allogeneic receptors, to enhance the efficacy through deletion of immune checkpoint and death receptors, to prevent fratricide by deleting pan T-cell markers, and to increase safety by decreasing the potential for cytokine release syndrome. (B) Additionally, donor DNA deliver by AAV or dsDNA can be used for HDR-mediated knock-in of CARs into the TRAC locus, thereby creating CAR-T cells that have endogenous control over their CAR expression profiles. These donor templates contain the gene insert (green) which is flanked by two homologous arms (blue) that are complementary to the target locus.