Figure 1. Stable knockout of CCR5 in CD4+ T cells mediated by TALEN. A) Schematic of the CCR5 locus with TALEN binding sites in exon 3 (boxes). B) Quantification of TALEN activity. Multiple T7E1 assays were performed in CD4+ T cells of 7 or 3 different donors at day 7/21 post-electroporation. Where indicated, cells were transiently incubation at 32°C for 24 h, or kept at 37°C, respectively. Samples derived from a Δ32 heterozygous donor are highlighted in red (n=3-7; ±SD). C) Expression of endogenous CCR5 in CD4+ T cells. Cell were treated with only left TALEN arm (L+L) or both TALEN arms (L+R). Data is shown relative to untreated (UT) cells. Each dot represents a different donor (n=2-7; ±SD; ns=not significant, nd=not determined, *p ≤0,05; *** p ≤0,001, paired t -test). D) Frequency of various mutations. Targeted amplicon sequencing of the target site was performed after 7 and 21 days of culture in 3 different donors (n=3; ±SD). Displayed are the frequencies of all indels with a frequency >1 in at least one of the three experiments. E) Sequence of induced mutations. Displayed are all sequences quantified in (D).