Designed ZFNs disrupt HLA-A expression on HEK293. (A) Schematic of the HLA-A genetic loci. Arrows indicate the location of the putative ZFN binding sites. (B) ZFN binding sites within the HLA-A alleles expressed by HEK293. A 46-base pair segment of the HLA-A*03:01 and HLA-A*02:01 alleles is shown, with bold and underlined nucleotides highlighting the anticipated binding sites for the ZFN-L and ZFN-R monomers, respectively. Cleavage occurs in the region between these sites. Note that HLA-A*02:01 differs from and HLA-A*03:01 at 3 positions within this region, which are marked with an asterisk. ZFN-L was designed to ignore these polymorphisms and to cleave both alleles. (C) Levels of HLA-A3 and HLA-A2 genetic disruption assessed by the Surveyor nuclease assay. The lower (fast-moving) bands (arrows) are digestion products indicating ZFN-mediated gene modification. The numbers indicate the percentage of modified HLA-A alleles based on densitometry. GFP represents genetically modified HEK293 expressing GFP as a control. Mock represents no DNA used.