Figure 2.

Episomal-Based MMEJ Assay

(A) The plasmids used in this assay. pSQT1313 harbors gRNAs from therapeutic relevant target sequences. The px459 plasmid encodes only the Cas9 protein in the episomal-based assays. For the genomic editing experiments it harbors gRNA3 resp. gRNA4. (B) Schematic overview of plasmid combinations to induce MMEJ recombination events. The donor plasmid (DB) harbors a complete BFP plasmid without promotor. BFP is flanked by 18-bp microhomology regions (orange and purple), and the plasmid can be linearized (DB1) or the insert can be cleaved out (DB2). The AP was generated following the same principle. The Cas9 cleavage occurs in a PAMout manner (cf. Figure 3). (C) Episomal gene-editing efficiencies measure by quadruple transfections of the reporter plasmids. Depicted is the number of BFP-positive cells after DNA repair. Error bars indicate the SD of the data set.