Plasmid-based CRISPR gene editing was completely ineffective in primary NK cells. (A) Effect of pmaxGFP dosage on cell viability and GFP expression. 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. *, P ≤ 0.05. ns, not significant; NT, untreated cells; std, standard for comparison. (B) Plasmid-based gene editing was performed to target CD96, TIGIT, and KLRC1 genes in NK cells and the HEK293T cell line as a positive control. No indel was detected by ICE analysis in NK cells from three donors (n = 3). HEK293T results were from three independent experiments (n = 3). (C) Western blotting and RT-PCR were performed to detect the expression of Cas9-FLAG and sgRNA, respectively. Cell lysates were sampled immediately (time = 0) and 24 h after nucleofection. GAPDH protein and β-tubulin cDNA served as internal controls. The results were from one donor (n = 1).