Fig. 1

Optimized gene editing workflow enables efficient knock-ins in iPSCs. (a) Overview of optimized gene editing protocol. Critical optimizations are highlighted in blue. (b) Incremental increase of KI efficiency by implementation of each optimization indicated by flow cytometry quantification of GFP + cells 10 days after nucleofection. C = control, T = target. Controls received donor and RNP, but an irrelevant gRNA was used. (c-d) Example results of standard protocol (c) or fully optimized protocol (d) showing flow cytometry and fluorescence microscopy (with 5 × objective) of GFP. Scale bar = 200 µm. (e–g) Flow cytometry quantification of GFP integration comparing Cas12a against Cas9 (e), different target loci (f) or different GMP iPS cell lines (g). Replicate numbers are indicated by dots overlaying bar charts. Error bars denote standard deviation.