Enrichment of HLA-A^neg primary T cells after genetic editing with ZFNs. (A) Generation of an HLA-A2^neg T-cell population. HLA-A2^neg T cells were enriched by magnetic bead-based selection. Input dose of mRNA coding for ZFN and 3-day culture conditions (37°C vs 30°C) after electro-transfer of mRNA are indicated. The numbers represent HLA-A2 negative population within the CD4- and CD8-positive population. EL:KK, obligate heterodimer mutant FokI cleavage domain; wt, wild type FokI cleavage domain. (B) Surveyor nuclease assay of the enriched HLA-A2^neg T cells. Analysis of T cells enriched for loss of HLA-A2 expression demonstrates disruption in the HLA-A2 locus by the appearance of a fast-moving band (arrow). (C) Sequencing of the HLA^neg T cells. PCR products using HLA-A2-specific primers from enriched cell (2.5 μg ZFNs, EL:KK FokI domain, 30°C treatment) were cloned into a TOPO vector (Invitrogen), and plasmid products were sequenced. The wild-type sequence is listed at the top, with the expected ZFN binding sites underlined. Shown below are the sequences obtained from the ZFN-treated and enriched cells. Deletions are indicated by hyphens, and sequence changes are highlighted in bold. All 18 sequence changes result in frame shifts predicted to prevent protein translation.