C6 sampler

Cas12a protein expression on an individual cell basis was determined by detecting a C’terminal HA tag. To prepare samples, cells were fixed (ab185917) for 15 minutes at room temperature, washed with chilled PBS, permeabilized (ab185917) and stained with PE anti-HA (Biolegend #901518) for 30 minutes at room temperature, washed with chilled PBS twice to remove residual antibody, and resuspended in flow buffer (PBS, 2% FBS, 5μM EDTA). Cells were visualized by flow cytometry on the BDAccuri C6 Sampler. Gates were set off of the A375 stained parental population.

A375 cells stably expressing enCas12a were transduced with 6 triple knockout arrays, 3 single knockout constructs, and one empty control vector. Two days after transduction, cells were selected with puromycin (1μg/mL), and selected on puromycin for 7 days. Cells were visualized by flow cytometry on the BDAccuri C6 Sampler. To prepare samples for visualization, cells were stained with FITC anti-human CD47 (Biolegend # 323106), PE anti-human CD63 (Biolegend #353004) and APC anti-human β2-microglobulin (Biolegend #316312) antibodies, diluted 1:100 in flow buffer (PBS, 2% FBS, 5μM EDTA), incubated for 30 min on ice, washed with flow buffer twice to remove residual antibody, and resuspended in flow buffer. Flow cytometry data were analyzed using FlowJo (v10). Gates were set such that ~1% of cells score as knockout in the control condition. Compensation was applied using single stained empty vector control and triple stained empty vector control cell populations. Zero, single, double, and triple knockout populations were quantified using boolean gating in FlowJo.