Modified genome detection assay For the HR assay, 2 × 10^6 K562 cells were transfected with 10 µg of each nuclease-encoding plasmid and 50 µg of donor plasmid using the 4D-Nucleofector, SF Cell Line 4D-Nucleofector X Kit, Program FF-120 (Lonza) according to the manufacturer\\\'s protocol. After 96 h, genomic DNA was isolated, and the target locus was amplified with primers that bind outside of the homology arm sequences (Supplemental Table 3) using HiPi DNA polymerase (Elpisbio). PCR amplicons were digested with XbaI; digested fragments were analyzed on a 1% agarose gel. For detection of local indels, genomic DNA of nuclease-treated cells was analyzed using the T7E1 assay as previously described using target-specific primers (Supplemental Table 3). Deep sequencing of on- and off-target sites 2 × 106 K562 cells were nucleofected with 10 µg of each ZFN- or nickase-encoding plasmid and 5 µg of GFP-encoding plasmid using the SF Cell Line 4D-Nucleofector X kit and the FF120/cell line SF program (Lonza) according to the manufacturer\\\'s protocol. One-eighth of the cells were subjected to FACS analysis to confirm transfection, and the rest of the cells were harvested and used for genomic DNA isolation 72 h after transfection.