Correction at the β-globin locus in CD34⁺ cells using an oligonucleotide donor. (A) Schematic of oligonucleotide-directed gene modification. The top sequence is the genomic DNA of the β-globin locus with the site of the sickle mutation in bold and italicized. The sequence of the modified locus is shown on the bottom with the inserted bases shown in italics. (B) Gene modification in mPB CD34⁺ cells with an oligonucleotide donor. PAGE of an AvrII-digested PCR amplicon of the β-globin locus. The fragment contains a native AvrII site, cleavage of which serves as an internal control for AvrII digestion (the lower band on the gel). Arrows indicate AvrII cleavage products. (C) Six possible sites of silent mutation in the SBS 33501 ZFN binding site. Sickle mutation italicized in bold, and all possible silent mutation sites are in bold (including those not discussed). (D) Silent mutations increase gene modification at β-globin. mPB CD34⁺ cells were transfected with ZFNs (30 μg/mL) and the indicated donor oligonucleotide (3 μM). Introduction of the relevant silent mutation was assayed via high-throughput sequencing. White bars indicate gene modification; gray bars indicate indels. (E) Silent mutations block ZFN recleavage. Alleles with indels were examined for evidence of homology-mediated modification. Shown are the percentages of alleles with gene modification that also have evidence of NEHJ-driven indels. (F) Optimization of ZFN concentration and donor type. NHEJ-driven indels (gray bars) and gene modification (white bars) were assayed by high-throughput DNA sequencing. Given the depth of high-throughput DNA sequencing, measurement error is expected to be very low.