Encapsulating the hCas9 transgene on a piggyBac transposon enabled its efficient excision. To illustrate this, we transiently transfected PGP1-hCas9-PB-TAZc.517delG with an excision competent, integration defective piggyBac expression plasmid15 and assessed hCas9 transgene excision by loss of puromycin resistance, encoded on the piggyBac transposon. PiggyBac transposase reduced the frequency of puromycin resistant clones, as assessed by crystal violet visualization of puromycin-resistant clones, demonstrating efficient transposon excision (Fig. 3a). Most individual clones recovered after transient piggyBac transposase expression were negative for the hCas9 transgene, as determined by PCR genotyping. For establishment of the PGP1-TAZc.517delG line lacking the hCas9 transgene, we genotyped 34 clones and 22 (64%) had undergone successful transgene removal (Fig. 3b). We have further streamlined the protocol by introducing piggyBac transposase into Dox-induced cells in the same transfection as gRNA and donor DNA. We found that co-transfection of the excision-only piggyBac mutant did not substantially reduce the yield of genome-edited clones, yet most of the recovered clones had still successfully undergone piggyBac transgene excision (Suppl. Fig. 2). Thus, including the excision-only piggyBac mutant into the transfection mix with gRNA and donor DNA permits efficient, single step genome editing and transgene excision.
