ETS1 deletion in human embryonic stem cells severely impairs NK cell development. (A) Three homozygous ETS1 knockout (ETS1^−/−) hESC clones were generated by using CRISPR/Cas9 technology to introduce mutations in exon A/1 by non-homologous end joining. The wild-type (WT) clone underwent the same selection processes. The sequence of the targeted region of exon A/1 is indicated for both alleles, showing biallelic frameshifts in the knockout clones. (B) WT and mutated hESCs were differentiated into hematopoietic CD34⁺CD43⁺ progenitor cells. Cell numbers are indicated (mean ± SEM; n = 4-6). (C-D) CD34⁺CD43⁺ cells were subsequently cultured in NK cell–differentiating conditions. (C) The ratio of the cell numbers of generated NK cells (gated as CD45⁺CD56⁺CD94⁺) over the input precursor stem cell number was determined (mean ± SEM; n = 4-6) on d14 and d21. (D) Representative dot plots of d21 cultures are shown, with the indicated NK cell percentage in the CD45⁺ gated population. Significant difference compared with the WT clone, *P < .05 (Student t test).