Fig. 1

Constructs and workflow of death receptor-based enrichment of Cas9-expressing cells. a Constructs for different selection strategies. The Cas9 nuclease is linked to the resistance gene by a cleavable 2A peptide allowing stoichiometric expression. As resistance gene, we used death receptors that lack the intracellular death domain, denoted by ∆. CD95 is chosen when using CD95L as selection agent. DR4 and DR5 are death receptors that bind the death ligand TRAIL. To allow simultaneous expression of several death receptors, we linked them to Cas9 via the 2A peptide from Thosea asigna virus (T2A), from foot-and-mouth disease virus (F2A) and porcine teschovirus-1 (P2A). To allow comparison to the puromycin selection strategy, we cloned the puromycin resistance gene puromycin N-acetyl-transferase, denoted here as puro. We used the human cytomegalovirus (CMV) and the 7SK promoters for the protein and gRNA expression, respectively. b Workflow. Cells are transfected with a plasmid encoding the Cas9 nuclease, a selection gene and the gRNA. Two days after transfection, death ligand was added to the cells, before they were washed and expanded