Figure 5. Highly efficient target gene KO in nonactivated human and mouse primary T cells with an optimized CRISPR/Cas9 RNP transfection approach. (A–D) KO efficiency by flow cytometry of CXCR4, CD127, and CCR7 in nonactivated human CD4 T cells 72 h after transfection (A); PD1, TIGIT, and CTLA4 in nonactivated human CD8 T cells 72 h after transfection followed by 72 h of stimulation with anti-CD3/anti-CD28 (B); CD90 and CTLA4 in IL-7–preconditioned nonactivated mouse CD4 T cells 5 d after transfection and incubation with IL-7 (C); and CD8α and CTLA4 in IL-7–preconditioned nonactivated mouse CD8+ T cells after transfection and 5 d of incubation in the presence of IL-7 followed by 48 h stimulation with anti-CD3/anti-CD28 (D; all compared with nontargeting control Cas9 RNP-transfected T cells). Data are presented as mean ± SD (n = 2) and representative of three (A) or two (B–D) independent experiments. (E) IFN-γ expression by flow cytometry in crIFNγ or NTC-transfected nonactivated human CD8+ T cells cultured for 3 d and restimulated for 4 h with PMA/ionomycin. Data are presented as mean ± SD (n = 2) and representative of two independent experiments. (F) FoxP3 and c-Maf expression by flow cytometry in crFoxp3 or NTC transfected IL-7–preconditioned nonactivated mouse CD4+ T cells 5 d after transfection with IL-7 followed by 3 d of polarization into iTreg with TGF-β and IL-2. Data are presented as mean ± SD (n = 2) and representative of two independent experiments. (G–I) KO efficiency by flow cytometry of double KOs targeting CD127 and CCR7 in nonactivated human CD4+ T cells 72 h after transfection (G), CD90 and CTLA4 in nonactivated mouse CD8 T cells after transfection and 5 d of incubation in the presence of IL-7 followed by 48 h stimulation with anti-CD3/anti-CD28 (H), or CD90 and FoxP3 in nonactivated mouse CD4+ T cells after transfection and 5 d of incubation with IL-7 followed by 3 d of polarization into iTreg with TGF-β and IL-2 with anti-CD3/anti-CD28 (I). Data are presented as mean ± SD (n = 2) and representative of two independent experiments. ***, P < 0.001; **** P < 0.0001 by unpaired Student’s t test.
Figure 5.

Highly efficient target gene KO in nonactivated human and mouse primary T cells with an optimized CRISPR/Cas9 RNP transfection approach. (A–D) KO efficiency by flow cytometry of CXCR4, CD127, and CCR7 in nonactivated human CD4 T cells 72 h after transfection (A); PD1, TIGIT, and CTLA4 in nonactivated human CD8 T cells 72 h after transfection followed by 72 h of stimulation with anti-CD3/anti-CD28 (B); CD90 and CTLA4 in IL-7–preconditioned nonactivated mouse CD4 T cells 5 d after transfection and incubation with IL-7 (C); and CD8α and CTLA4 in IL-7–preconditioned nonactivated mouse CD8+ T cells after transfection and 5 d of incubation in the presence of IL-7 followed by 48 h stimulation with anti-CD3/anti-CD28 (D; all compared with nontargeting control Cas9 RNP-transfected T cells). Data are presented as mean ± SD (n = 2) and representative of three (A) or two (B–D) independent experiments. (E) IFN-γ expression by flow cytometry in crIFNγ or NTC-transfected nonactivated human CD8+ T cells cultured for 3 d and restimulated for 4 h with PMA/ionomycin. Data are presented as mean ± SD (n = 2) and representative of two independent experiments. (F) FoxP3 and c-Maf expression by flow cytometry in crFoxp3 or NTC transfected IL-7–preconditioned nonactivated mouse CD4+ T cells 5 d after transfection with IL-7 followed by 3 d of polarization into iTreg with TGF-β and IL-2. Data are presented as mean ± SD (n = 2) and representative of two independent experiments. (G–I) KO efficiency by flow cytometry of double KOs targeting CD127 and CCR7 in nonactivated human CD4+ T cells 72 h after transfection (G), CD90 and CTLA4 in nonactivated mouse CD8 T cells after transfection and 5 d of incubation in the presence of IL-7 followed by 48 h stimulation with anti-CD3/anti-CD28 (H), or CD90 and FoxP3 in nonactivated mouse CD4+ T cells after transfection and 5 d of incubation with IL-7 followed by 3 d of polarization into iTreg with TGF-β and IL-2 with anti-CD3/anti-CD28 (I). Data are presented as mean ± SD (n = 2) and representative of two independent experiments. ***, P < 0.001; **** P < 0.0001 by unpaired Student’s t test.

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