All experiments were performed using HAP1 myeloid leukemia cells (29 (link)) grown in IMDM (GE Healthcare Bio-Sciences, Pittsburgh, PA) supplemented with 10% FBS, 4 mM L-glutamine, and 1% penicillin/streptomycin. PCR primers used to generate the linear HDR donors are listed in the Supplementary Material, and the genomic regions used for designing the homology arms are shown in the Supplementary Material. Two phosphorothioate linkages were included at the 5′ end of each HDR PCR primer. HAP1 cells were seeded into 10 cm dishes at 70% confluency. Prior to transfection, 10 μM trimethoprim was added from a 10 mM stock in DMSO. HAP1 cells were co-transfected with 5 μg each of pX330 gRNA plasmid and linear HDR donor using FuGENE HD (Promega, Madison, WI) or Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA). Puromycin (750 ng/mL) was added 72 h after transfection, and the medium was changed every 2–3 days. After 1–2 weeks, colonies were transferred to 24-well plates, and genomic DNA was isolated for PCR genotyping.