Fig. 6. AsCas12a Ultra enables efficient one-step generation of allogenic CAR-NK cells.

A Potential editing strategy for the generation of edited NK cells to overcome tumor microenvironment. Knocking out TGFBR2 (Transforming growth factor-beta receptor type 2) prevents inactivation by tumor-derived TGF-β1. B Editing efficiency of TGFBR2A across a range of RNP concentrations in NK cells derived from three different donors. Data are presented as mean values ± SD. C Tumor spheroid killing mediated by TGFBR2 knock-out NK cells. Engineered NK cells were co-cultured with SK-OV-3 ovarian tumor spheroids at effector-to-target cells ratio of 10:1 (E:T). The spheroid size was monitored over time by Incucyte real-time cell analysis as a proxy for NK cytotoxicity. Two-way ANOVA was performed to evaluate statistical significance (****P-value ≤ 0.0001). Data are representative of five independent experiments using six unique NK cell donors. D Graphical depiction of EGFR CAR NK cell recognition of an EGFR⁺ tumor cell. E Knock-in efficiency of a fluorescent reporter at either TRAC (Target site 1 and Target site 2) or B2M (Target site 3) loci using 4 μM RNP and 2.5 × 10⁴ vg/cell AAV6 titer carrying the transgenes. F Tumor spheroid killing mediated by GFP⁺ or αEGFR CAR⁺ NK cells. Engineered NK cells were co-cultured with EGFR⁺ PC-3 spheroids at effector-to-target cell ratios of 2:1 (E:T). The spheroid size was monitored over time by Incucyte real-time cell analysis as a proxy for NK cytotoxicity. Two-way ANOVA was performed to evaluate statistical significance (****P-value ≤ 0.0001). Raw source data are provided in the Source Data File.