Fig. 5

Phenotypic analysis and specificity assessment of ZFNs targeting TRAC. Across the top are shown names of the distinct sites targeted by each pair of ZFNs (Supplementary Fig. 21). Underneath each label are two panels containing the data for the indicated pair (e.g., a and f for TRAC 1, b and g for TRAC 2, etc.). Architecture information is indicated at the bottom. a–e Phenotypic characterization of ZFN-treated T-cells. Histograms for each pair were generated via analysis of 10,000 cells and plots are shown for each of the indicated pairs with the percentage of cells that are negative for CD3 annotated on each graph. CD3 is a component of the T-cell receptor complex queried as a proxy for TRAC surface disruption. Also shown on each plot are the corresponding % indels induced by each ZFN pair in this transfection. Plots for mock transfected T-cells are shown in Supplementary Fig. 22. f–j Specificity assessment of TRAC ZFNs. Candidate off-target loci were first identified for each pair using an unbiased oligonucleotide duplex-capture assay in K562 cells. In a follow-up study, ZFN-encoding mRNA for each pair was transfected into activated T-cells via BTX transfection. Modification was monitored at each on- and off-target locus by PCR followed by deep sequencing (MiSeq). The off-target characterization for each pair is shown under the corresponding flow cytometry data. On the left of each bottom panel is a table with the locus being monitored (on-target locus at top) and the number of sequences recovered in the capture assay. To the right, log-scale bar graphs are shown that summarize off-target modification in the follow-up indel study. The red bars indicate the % indels for the ZFN-treated T-cells and the gray bars indicate the % indels observed in T-cells treated with mRNA expressing GFP. Candidate loci that were significantly modified in ZFN-treated cells as compared with GFP-treated controls are marked with an asterisk. Statistical significance was determined as previously described⁷⁷ combined with a Bonferroni correction. Each bar represents a single measurement. An expanded dataset for this study is provided in Supplementary Fig. 23. The source data for this figure is available in the Source Data file