Double KO revealed that CD96 was not crucial for NK cell activation. (A) Cytotoxicity assay was performed using MDA-MB-231 and U-251 MG cancer cell lines. Both cells expressed high levels of CD155, which is a ligand for CD96 and DNAM-1 (encoded by the CD226 gene). (B) Representative flow cytometry plots show the distributions of single- and double-KO cells. (C) Workflow of the double-KO nucleofection, FACS, and functional assays. (D) Cytotoxicity of the KO cells against the cancer cells at the indicated ratios. Data are shown as mean ± SD of three donors and each in triplicate (n = 9). (E) Degranulation markers of the KO cells after incubating with the cancer cells. Data are shown as mean ± SD of three donors (n = 3). Two-tailed Welch’s unequal variances t test was used to test for statistical significance. ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.