Extended Data Fig. 1: Clonal HAP1-A5 line and experimental protocol optimization improves editing rate.

a. FACS data comparing the activity of Cas9 in a polyclonal Cas9+ LIG4- HAP1 cell line (top row) with a monoclonal line (HAP1-A5) derived from the same polyclonal line (bottom row) at 72hrs post-transduction. BFP+ GFP+ cells (green) gated in top right quadrant are cells in which Cas9 has failed to inactivate GFP by editing. The Cas9-inactive fraction (green) is significantly reduced, and the Cas9-active fraction (blue) is significantly increased in the clonal line. The negative control contains no sgRNA targeting to GFP coding sequence. Non-fluorescent cells in black. 10,000-20,000 cells were assessed per sample. See Supplementary Fig. 1a for representative gating. b. Nocodazole-based metaphase arrest and DAPI staining was performed on cells edited at exon 5 of BAP1 at D3 post-transfection (PT) and D19 (final passage) PT. Unsorted and untransfected wild-type HAP1 cells were included as a control, as were untransfected clonal line cells. A slight increase in ploidy is seen as a result of transfection and culturing. 1n are un-arrested haploid cells, 2n and 4n are metaphase-arrested haploid and diploid cells, respectively. 10,000-20,000 cells were assessed per line. See Supplementary Fig. 1b for representative gating. c. X-fect (Takara) based transfection of 6 million clonal HAP1-A5 cells using the pMax-GFP (Lonza) 3486bp construct, which is of a similar to size to a typical HDR library, and exon 5 sgRNA-A plasmid. Pre-selection sees a transfection rate of 64% and post-selection with puromycin (+PURO) showing 98% GFP+ cells (green) at Day 3 (D3) post-transfection (PT). GFP- cells in black. 50,000 cells assessed per sample. See Supplementary Fig. 1c for representative gating. d. Sanger sequencing of HAP1-A5 clone at LIG4 locus confirms expected frameshifting, 10 base-pair deletion in CDS, creating a null allele (Horizon Bioscience).

Source data