Generation of knockout cell lines using CRISPR/Cas9

Wild-type HL-60s were transduced with vectors containing puromycin-selectable gRNAs targeting HEM1/NCKAPL1 or ARP2/ACTR2. Following selection, cells were then transduced with an S. pyrogenes Cas9 sequence fused to tagBFP. Cells expressing high levels of Cas9-tagBFP were collected using FACS, after which a heterogeneous population was obtained, as assessed by immunoblot and by sequencing of the genomic DNA flanking the Cas9 cut site. These cells were then diluted into 96-well plates at a density of approximately 1 cell per well to generate clonal lines, which were again verified by genomic DNA sequencing and immunoblot. We verified that candidate clonal lines arose from single cells as previously described [42].