Disruption of CD7 expression with CRISPR/Cas9 restores expansion of CD7 CAR T cells. (A) Representative histogram showing ablation of CD7 expression in T cells after electroporation with CRISPR/cas9 and CD7-specific gRNAs 3 days after electroporation. Numbers denote frequency of CD7-negative cells. T cells electroporated with Cas9 only were used as a negative control. (B) Downregulation of surface CD7 expression in T cells after electroporation with CRISPR/cas9 and gRNA-85. A CD7-negative cell line Raji was used as a negative control. (C) Schematic outline of the optimized protocol for generating CD7-knockout (CD7^KO) CD7 CAR T cells. (D) Representative dot plots showing expression of CD7 and CD7 CAR in T cells generated with the optimized protocol. Numbers indicate percentage of cells in each quadrant. (E) Total expansion of CD7 CAR T cells with and without CD7 knockout after 14 days of in vitro culture. (F) Viability of CD7 CAR T cells with and without CD7 gene disruption measured at day 6 after transduction by flow cytometry. Lines denote individual donors. Data represent 3 independent experiments with 3 donors in each. h, hour. *P < .05.