Figure 4. Combination of multiple gRNAs further increases target gene KO efficiency. (A) Schematic depiction of potential outcome of CRISPR targeting of three regions in the same gene leading either to local InDels or deletion of flanked DNA. (B–D) KO efficiency of targeting CCR7 (B), CD127 (C), or IFN-γ (D) using individual gRNAs or the combination of two or three gRNAs per target in nonactivated human CD4+ T cells 72 h after RNP transfection. Data are presented as mean ± SD (n = 2; one-way ANOVA) and representative of two independent experiments. (E) Comparison of KO efficiency 72 h after RNP transfection targeting CCR7 or CD127 in nonactivated human CD4+ T cells using either recombinant GeneArt Platinum Cas9 or TrueCut Cas9 V2. Data are presented as mean ± SD (n = 2; unpaired Student’s t test) and representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Figure 4.

Combination of multiple gRNAs further increases target gene KO efficiency. (A) Schematic depiction of potential outcome of CRISPR targeting of three regions in the same gene leading either to local InDels or deletion of flanked DNA. (B–D) KO efficiency of targeting CCR7 (B), CD127 (C), or IFN-γ (D) using individual gRNAs or the combination of two or three gRNAs per target in nonactivated human CD4+ T cells 72 h after RNP transfection. Data are presented as mean ± SD (n = 2; one-way ANOVA) and representative of two independent experiments. (E) Comparison of KO efficiency 72 h after RNP transfection targeting CCR7 or CD127 in nonactivated human CD4+ T cells using either recombinant GeneArt Platinum Cas9 or TrueCut Cas9 V2. Data are presented as mean ± SD (n = 2; unpaired Student’s t test) and representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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