Bt Research 2024, Vol.15, No.1, 53-64 http://microbescipublisher.com/index.php/bt 57 Figure 2 Nucleofection of RNPs leads to highly efficient target gene KO in activated mouse T cells (Adopted from Seki and Rutz, 2018) Image capton: (A) Schematic depiction of RNP components, chemically stabilized crRNA, fluorescently labeled tracrRNA, and recombinant Cas9 protein. (B) KO efficiency as measured by CD90-negative CD8+ T cells 72 h after nucleofection of RNPs (DN100/P3) targeting CD90, and titration of gRNA to Cas9 ratio. Data are presented as mean ± SD (n = 2) and representative of two independent experiments. (C and D) KO efficiency as measured by CD90-negative CD8+ T cells (C) and cell viability 72 h after nucleofection of RNPs (DN100/P3) targeting CD90 (D), and titration Cas9 amount. Data are presented as mean ± SD (n = 2) and representative of two independent experiments. (E) Example of KO efficiency of RNP transfection targeting CD90 with 3:1 RNA/Cas9 ratio and 10 µg Cas9 3 d after transfection. (F) Systematic optimization of nucleofection parameters for RNP transfection of activated mouse CD8+ T cells. Analysis of transfection efficiency (ATTO550 expression and MFI), cell viability, and CD90 KO frequency 48 h after transfection. Data are from one experiment. (G) Comparison of KO efficiency by flow cytometry after RNP transfection using selected nucleofection pulses and buffers for targeting CD90 in CD8+ activated mouse T cells. Data are presented as mean ± SD (n = 2) and representative of two independent experiments. (H) KO efficiency as measured by flow cytometry using optimized RNP transfection in activated mouse CD8 T cells targeting CD90, CTLA4, or PD1 compared with target expression in cells transfected with NTC. Data are presented as mean ± SD (n = 2) and representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by one-way ANOVA (Adopted from Seki and Rutz, 2018)
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