Extended Data Fig. 1: Functional CRISPR cRNP screen in primary human NK cells identifies positive regulators of human NK cell function.

(a) Differentially expressed transcription factors between human NK cell developmental subsets. (b) Primary human NK cells are isolated from fresh human PBMCs via negative magnetic bead selection. Following expansion in IL-2/15 for 9 days, cells are electroporated with CRISPR-Cas9 RNP complexes. CRISPR-edited NK cells are further expanded for 6 days before functional and flow cytometric analyses. (c) Left, quantification of percent IFN-γ⁺ in TRAC^cRNP or TCF7^cRNP NK cells after 16 h stimulation with IL-2, IL-15, K562 cells, and IL-12 and/or IL-18. Right, specific lysis of K562 cells after 16 h coculture with IL-2 and IL-15 at indicated effector:target ratios. (d) Left, density of viable TRAC^cRNP or MYC^cRNP NK cells 6 days after cRNP editing expanded with IL-2 and IL-15. Right, quantification of percent IFN-γ⁺ in TRAC^cRNP or MYC^cRNP NK cells after 16 h stimulation with IL-2, IL-15, K562 cells, and IL-12 and/or IL-18. (e) Density of viable TRAC^cRNP or ZEB2^cRNP NK cells 6 days after cRNP editing expanded with IL-2 and IL-15. (f) Quantification of percent IFN-γ⁺ in TRAC^cRNP or RORC^cRNP NK cells after 16 h stimulation with IL-2, IL-15, K562 cells, and IL-12 and/or IL-18. (g) Representative gating strategy for peripheral human NK cells. Data are representative of n = 6-8 independent donors presented as individual paired donors. *p < 0.05, **p < 0.01 by two-sided paired t test. Specific p-values are as follows: c percent IFN-γ⁺ NT = 0.1686, IL-18 = 0.0200, IL-12 = 0.0136, IL-12/18 = 0.8445, % specific lysis 1:8 = 0.0399, 1:4 = 0.0366; d cells/mL = 0.0040, percent IFN-γ⁺ NT = 0.0769, IL-18 = 0.0128, IL-12 = 0.0244, IL-12/18 = 0.4241; e = 0.0216, f = 0.0456.