K562 cells

K562 cells (ATCC, Manassas, VA; catalog number CCL-243) were engineered to have an NC CCR5 target site with either a 6 or 7 bp spacing or a canonical CCR5 site inserted into the gene bearing the AAVS1 safe harbor^{43 (link)}. Analogous cells lines were also generated that inserted an NC AAVS1 target site with either a 6 or 7 bp spacing or a canonical AAVS1 site into the CCR5 target site. Transfections were performed by combining 200 ng of DNA for each expression plasmid with 2E5 K562 cells and 2 µM of a single-stranded oligo donor using the Amaxa 96-well Shuttle System (Lonza, Allendale, NJ) as per the manufacturer’s protocol (oligo sequences in Supplementary Table 1). DNA-PK inhibitor NU7441 (Cayman Chemical, Ann Arbor, MI) was added at a concentration of 2 µM at 4 and 20 h post transfection. Three days post transfection, single-cell clones were generated by pelleting and resuspending cells at a concentration of 0.3 cells/200 µl media and 200 µl was put into each well of two 96-well plates. After 2 weeks of incubation, a sample was taken from each single-cell clone for sequencing the modified loci via PCR followed by deep sequencing using an Illumina MiSeq (Illumina, San Diego, CA). The cell lines with the correct DNA sequence inserted were consolidated, frozen with 5% dimethyl sulfoxide, and stored in liquid nitrogen.

ZFNs designed to target the human CCR5 locus have been described previously [24 (link)]. The barcode targeting vector was created by cloning barcode oligos and the P5 Illumina adapter, similar to the lentiviral barcode library, downstream of the UBC-GFP within the homology arms of a CCR5 targeting vector described previously [24 (link)]. Included outside the homology arms was an HSV-TK domain for negative selection.
K562 cells were nucleofected (Lonza, Basel, Switzerland) with 10 μg targeting vector and 1 μg of each ZFN plasmid using program T-016 and nucleofection buffer containing 100 mM KH₂PO₄, 15 mM NaHCO₃, 12 mM MgCl₂•6 H₂0, 8 mM adenosine 5′-triphosphate, 2 mM glucose, pH 7.4. Negative selection was performed with two pulses of 5 μM ganciclovir at days 5 and 11 post-transfection.