Figure 4.

Double and triple KO in a single nucleofection. (A) A mixture of two Cas9 RNPs, consisting of sgRNAs targeting both CD96 and NKG2A, was nucleofected into NK-92 cells to simultaneously KO CD96 and NKG2A. Representative flow cytometry plots show the expression levels of target proteins in the parental and double KO cells. (B) Double KO of CD96 and NCR1 (encoding NKp46). (C) Triple KO of CD96, NKG2A, and NCR1 by nucleofection of a mixture of three Cas9 RNPs. To increase readability, only the percentages of triple positive and negative cells are shown in the bar graph. In the representative flow cytometry plots, Q2 of the parental plot was gated to determine triple positive cells. Q4 of the triple KO cells was gated to determine triple negative cells. Cell viability was determined by the Precision beads assay and normalized to untreated cells. Data are shown as mean ± SD of three independent experiments.