Enhanced gene editing by programmable nucleases delivered by a minicircle vector

Abstract

Targeted genetic modification using programmable nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are of great value in biomedical research, medicine, and biotechnology. Transient transfection using conventional plasmids has been the predominant way to deliver programmable nucleases into cells. Minicircle vectors, which lack extraneous bacterial sequences such as an antibiotic resistance gene and a bacterial origin of replication, have been reported to have advantages such as more robust transgene expression, higher efficiency of gene transfer, and greater biosafety as compared to conventional plasmids. Here, for the first time, we delivered programmable nucleases into human cells using transient transfection of a minicircle vector and compared the results with those obtained using a conventional plasmid. A surrogate reporter assay showed that nuclease activity was significantly higher in the minicircle vector group of cells than in the conventional plasmid group (43 ± 1% for ZFN group and 30 ± 1% for TALEN group). A T7 endonuclease assay revealed that cells in the minicircle vector group displayed significantly higher mutation frequencies at the endogenous target sites of the programmable nucleases than those in the conventional plasmid group (P < 0.001). Tryphan blue staining and flow cytometry after annexin V and propidium iodide staining showed that cell viability was also significantly higher in the minicircle group than in the conventional plasmid group (P < 0.001). Taken together, our results show that gene editing using minicircle vector-mediated delivery of ZFNs and TALENs is a more efficient, safer, and less toxic method than using a conventional plasmid, and indicate that the minicircle vector could serve as an advanced delivery method for programmable nucleases.