Extended Data Fig. 9: Transfection efficiency of HAP1 cells.

Flow cytometry analysis was used for measuring the transfection efficiency of HAP1 cells, since tracrRNA used for generating the RNP complexes for CGE experiment contain ATTO550 fluorochrome. Manual gating was performed using non-transfected control cells (top panel), and similar gates were applied for transfected samples to analyze transfection efficiency (lower panel). Gating strategy from left to right: 1. FSC/SSC: Cells were gated on the main population, excluding clear outliers such as cell debris. 2. FSC/Trigger pulse width: Cells were gated on the main population that represent single cells, excluding the outliers with larger trigger pulse width representing potential duplets. 3. FSC/405 nm (ex. 405 nm, em. 460/50 nm for SYTOX Blue dead cell stain): Cell viability was checked using the SYTOX stain and SYTOX-negative cells were gated to exclude the dead cells with higher fluorescence values. 4. Fluorescence was monitored on two channels: ex. 488 nm, em. 530/40 nm as an extra negative control, and ex. 561 nm, em. 585/29 nm for ATTO550. Gate was set using the non-transfected HAP1 cells so that all cells remained negative for ATTO550. Same gate was maintained to analyze transfected cells to measure the proportion of ATTO550-positive cells. Of note, flow cytometry was used for assessing the transfection efficiency of HAP1 cells, but the cells were not sorted for the CGE experiments.