Activities and specificities of HBB-ZFNs that stimulate gene targeting in a GFP reporter assay. (A) The putatitive recognition sequence of HBB-ZFNs in the HBB gene (5′ to 3′, starting from the first codon). The left (12-bp) and left (19-bp) ZFN sites are underlined. The homologous sequences from other β-locus genes (HBE, HBD, and HBG1/2) and their differences from the HBB gene in L-ZFN, R-ZFN, and spacer regions (no. of mismatches) also are shown. Each was inserted into the GFP reporter as ZFN target sequence to test the specificity of HBB-ZFNs. All the inserts start with a STOP codon (red, taa) and end with a HindIII site (blue, aagctt). A short version of the HBB target sequence (called HBB-Short) also was tested. (B) Schematic of the GFP* reporter rescue assay. An EGIP* mutant was created by inserting a ZFN target sequence including a STOP codon and HindIII site into the GFP gene. Only after gene targeting with a tGFP donor (with or without ZFNs), the EGIP* will be corrected to restore wild-type GFP expression. (C) Flow cytometric analysis of GFP correction after HR in 293T cells stably transfected with EGIP*-HBB reporter. Two days after transient transfection of tGFP donor alone or with HBB-ZFNs, numbers of GFP+ cells were measured by dot plot of 1 million collected cell events. (C) Gene correction efficiency of EGIP* mutants with HBB, HBE, HBD, HBG, or HBB-Short ZFN target sequence using the tGFP donor, with or without HBB-ZFNs. Numbers of GFP+ cells per 10⁶ 293T-EGIP* cells are plotted as mean ± SEM, n = 3.