Fig. 1

Establishment of the knockout-rescue system with double episomal vectors. a Schematic of the krCRISPR system design. This system consists of two plasmids, KO and Rescue. KO plasmid is for gene knockout, and Rescue plasmid is for gene rescue. b Schematic of the plasmid design. KO plasmid contains a hU6 promoter-driven gRNA and an EF1α promoter-driven Cas9 nuclease for gene knockout, a tTA gene for inducible gene expression, and OriP/EBNA1 elements for the episomal maintenance of the plasmid in the cells. Cas9 and the tTA gene used the same promoter but were separated by a P2A peptide. Rescue plasmid contains a TRE promoter-driven rescue gene, puromycin resistance gene and copGFP separated with P2A peptides, and an OriP element for the plasmid replication. c An MfeI restriction site was removed from the Rescue plasmid by digestion and religation. d Representative gel pictures of RFLP analysis of double plasmids at day 1 and day 10 after transfection in A375 and H9 cells. KO and Rescue plasmids contain a common region that can be amplified by a pair of primers. An MfeI restriction site is only present on the KO plasmid and digestion of the PCR products resulted in two bands (481 + 148 bp). A 633 bp fragment amplified from Rescue plasmid could not be digested by MfeI. Two lanes on the left are the PCR products amplified from plasmid DNA and used as a control. Each lane on the middle and right gels presented an independent transfection. e The ratio of the two plasmids was quantified based on RFLP analysis (n = 3, error bars showed mean ± SEM). f Representative images of the cells transfected with single or double plasmids. Cells transfected with single plasmid could not survive with puromycin selection, while cells transfected with double plasmids could survive and express GFP. GFP expression was inhibited by addition of Dox for 3 days