Fig. 5

Death receptor-based and puromycin-based enrichment of Cas9-expressing cells. In two independent experiments, HT-1080, LN-18 and HeLa cells were transfected with plasmid Cas9-2A-CD95∆DD or Cas9-2A-puro encoding gRNA-3 targeting the IRF3 gene or gRNA targeting gfp. Cell lines were tested against wild type cells for presence of indels in the gene IRF3 using primer IRF3 p1 (Table 1). Indels were detected by Sanger sequencing and TIDE analysis. a Selection using IZsCD95L or 5 μg/ml puromycin treatment. b No selection. c gfp gRNA cells treated with IZsCD95L or 5 μg/ml puromycin. Mutation = 0 represents wild type sequence. The non-interpretable fraction (n.i.) relates to the correlation coefficient with R² = 100 - n.i. d The relative frequency of indels in enriched cells was calculated by dividing their amplitude from panel 5a by the sum of all indels