RNP transfection enables gene KO before TCR stimulation in nonactivated mouse T cells. (A) Comparison of KO efficiency and cell viability by flow cytometry 48 h after RNP transfection using selected nucleofection pulses and buffers for targeting CD90 in mouse CD8⁺ T cells transfected before anti-CD3/anti-CD28 stimulation. Data are presented as mean ± SD (n = 2) and representative of two independent experiments. (B) Transfection efficiency (ATTO550-labeled tracrRNA) 48 h after transfection of mouse CD4⁺ or CD8⁺ T cells transfected ex vivo or after 24-h preincubation with IL-7 (gated on live cells). (C) Systematic optimization of nucleofection parameters for RNP transfection of IL-7–preincubated nonactivated mouse CD8⁺ T cells. Analysis of cell viability, transfection efficiency (ATTO550 MFI), and CD90 KO frequency 72 h after transfection. Data are from one experiment. (D) Comparison of transfection and KO efficiencies by flow cytometry 72 h after RNP transfection using selected nucleofection pulses and buffers for targeting CD90 in mouse CD4⁺ or CD8⁺ T cells cultured with IL-7. Data are presented as mean ± SD (n = 2) and representative of two independent experiments. (E and F) KO efficiency as measured by flow cytometry using optimized RNP transfection in nonactivated mouse CD8⁺ T cells targeting CD8 or CD90 (E) and nonactivated mouse CD4⁺ T cells targeting CD90 or Foxp3 compared with NTC (F). Data are presented as mean ± SD (n = 2) and representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by one-way ANOVA.