Objective To establish a single-plasmid genome editing method composed of piggyBac transposase (PB) with excision ability but deficient integration ability and Cas9, that is, to simultaneously express the Cas9-PB fusion protein and carry the inserted gene.

Methods Restriction enzyme digestion was performed on the plasmid pU6-(BbsI)_CBh-Cas9-T2A-mCherry. The sequence encoding piggyBac transposase with excision ability but integration ability defect, 3'PB terminal repeat sequence (3'ITR) and 5'PB terminal repeat sequence (5'ITR) were inserted, and the sgRNAs were inserted into between the U6 promoter and sgRNA scaffold to obtain the final plasmid. Human embryonic kidney 293 cells were transfected to screen monoclonal cells, the PCR was used to identify and the Sanger sequencing was used to analyzed.

Results A single plasmid system was constructed, including a fusion protein of piggyBac transposase with excision ability but deficient integration ability, Cas9 and 3'PB (3'ITR) -CMV promoter -5'PB (5'ITR) piggyBac transposon; Targeted integration was achieved in the AAVS1 gene, and no off-target insertion regions was detected.

Conclusions This study establishes a single plasmid genome editing method based on CRISPR-Cas9 and piggyBac transposase, which can perform effective and accurate gene integration with almost no off-target or random insertion. It is expected to become a potential candidate tool for future gene therapy applications.