ORIGINAL ARTICLE - ENABLING TECHNOLOGIES Enhanced gene disruption by programmable nucleases delivered by a minicircle vector A-BK Dad 1 , S Ramakrishna 1 , M Song and H Kim Targeted genetic modification using programmable nucleases such as zinc finger nucleases (ZFNs) and transcription activator- like effector nucleases (TALENs) is of great value in biomedical research, medicine and biotechnology. Minicircle vectors, which lack extraneous bacterial sequences, have several advantages over conventional plasmids for transgene delivery. 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. Surrogate reporter assays and T7 endonuclease analyses revealed that cells in the minicircle vector group displayed significantly higher mutation frequencies at the target sites than those in the conventional plasmid group. Quantitative PCR and reverse transcription-PCR showed higher vector copy number and programmable nuclease transcript levels, respectively, in 293T cells after minicircle versus conventional plasmid vector transfection. In addition, 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. Taken together, our results show that gene disruption 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. Gene Therapy (2014) 21, 921–930; doi:10.1038/gt.2014.76; published online 21 August 2014 INTRODUCTION Targeted genetic modifications are of great value in biomedical research, medicine and biotechnology. 1 Currently, engineered nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are widely used as a promising tool for targeted genome engineering. ZFNs and TALENs have a modular structure and are composed of DNA- binding domain linked to a nuclease domain derived from FokI restriction enzyme 2,3 The DNA-binding domains are zinc finger proteins and transcription activator-like effector in ZFNs and TALENs, respectively, and can be custom-designed to bind to specific genomic sequences, enabling targeted genetic modifica- tion at those sites. When introduced into cells, ZFNs and TALENs make double-strand breaks at their target sites, which can be repaired either through error-prone non-homologous-end- joining 4,5 or homologous recombination in the presence of donor DNA. 6–8 Non-homologous-end-joining can result in gene knockout and homologous recombination can lead to insertion of transgenes or correction of abnormal sequences at the target site. The efficiency of homologous recombination is usually more than 10 000 times higher in the presence of engineered nuclease activity. ZFN has been successfully used for targeted genome editing in many systems, such as Drosophila, 7,9 nema- tode, fish, 10 frog oocytes, 11 rodents, 12 plants 8,13–15 and human cells. 16–19 TALEN-mediated gene targeting has also been success- fully demonstrated in several systems including plants, 20,21 zebrafish, 22 yellow catfish, 23 Caenorhabditis elegans, 24 rats, 25 mice 26 and human cells. 3 To date, conventional plasmids, 3,20,27–32 integrase-defective lentiviral vectors, 18,33 adenoviral vectors, 34,35 adeno-associated viral vectors, 36 direct microinjection into embryos, 12,37–39 and recombinant proteins 40,41 have been used to deliver engineered nucleases. Among these approaches, plasmid-mediated delivery has been predominant because it is easy to generate the necessary components and vector integration into the host genome is relatively rare. However, the overall efficiency of obtaining gene-modified cells with this plasmid-based approach is often low. 29,42 A minicircle vector is a new generation of DNA vectors that lack extraneous bacterial sequences encoding antibiotic resistance genes and a bacterial origin of replication. 43,44 Several lines of evidence suggest that the minicircle possesses merits such as robust transgene expression, 43,45 high efficiency in gene transfer 46–48 and biosafety, 49 all of which indicate that the minicircle could be an efficient and safe delivery system for programmable nucleases. Because of these advantages, we postulated that ZFN and TALEN delivery via the minicircle vector might facilitate targeted genetic modification. In this study, for the first time, we cloned ZFN and TALEN sequences into a minicircle vector and a conventional plasmid vector and compared the resulting nuclease activities and toxicities in cells. Our studies revealed that minicircle delivery of programmable nucleases resulted in significantly higher levels of target gene modification and reduced cytotoxicity compared with delivery by conventional plasmid vectors, support- ing the use of the minicircle vector as an advanced nonviral delivery method for programmable nucleases. Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Seoul, South Korea. Correspondence: Dr H Kim, Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, FTC 12th floor Rm1209-12, Haengdangdong 17, Seoul 133-791, South Korea. E-mail: hkim1@hanyang.ac.kr 1 These authors contributed equally to this work. Received 30 December 2013; revised 18 June 2014; accepted 9 July 2014; published online 21 August 2014 Gene Therapy (2014) 21, 921 – 930 © 2014 Macmillan Publishers Limited All rights reserved 0969-7128/14 www.nature.com/gt