Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article - Enabling Technologies
  • Published:

Original Article – Enabling Technologies

Enhanced gene disruption by programmable nucleases delivered by a minicircle vector

Gene Therapy volume 21, pages 921–930 (2014)Cite this article

Subjects

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8

Similar content being viewed by others

References

  1. Kim H, Kim JS . A guide to genome engineering with programmable nucleases. Nat Rev Genet 2014; 15: 321–334.

    Article  CAS  Google Scholar 

  2. Kim YG, Cha J, Chandrasegaran S . Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci USA 1996; 93: 1156–1160.

    Article  CAS  Google Scholar 

  3. Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol 2011; 29: 731–734.

    Article  CAS  Google Scholar 

  4. Lieber MR . The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem 2010; 79: 181–211.

    Article  CAS  Google Scholar 

  5. Moynahan ME, Jasin M . Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nat Rev Mol Cell Biol 2010; 11: 196–207.

    Article  CAS  Google Scholar 

  6. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD . Genome editing with engineered zinc finger nucleases. Nat Rev Genet 2010; 11: 636–646.

    Article  CAS  Google Scholar 

  7. Bibikova M, Golic M, Golic KG, Carroll D . Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 2002; 161: 1169–1175.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Lloyd A, Plaisier CL, Carroll D, Drews GN . Targeted mutagenesis using zinc-finger nucleases in Arabidopsis. Proc Natl Acad Sci USA 2005; 102: 2232–2237.

    Article  CAS  Google Scholar 

  9. Bibikova M, Beumer K, Trautman JK, Carroll D . Enhancing gene targeting with designed zinc finger nucleases. Science 2003; 300: 764.

    Article  CAS  Google Scholar 

  10. Doyon Y, McCammon JM, Miller JC, Faraji F, Ngo C, Katibah GE et al. Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat Biotechnol 2008; 26: 702–708.

    Article  CAS  Google Scholar 

  11. Bibikova M, Carroll D, Segal DJ, Trautman JK, Smith J, Kim YG et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol 2001; 21: 289–297.

    Article  CAS  Google Scholar 

  12. Geurts AM, Cost GJ, Freyvert Y, Zeitler B, Miller JC, Choi VM et al. Knockout rats via embryo microinjection of zinc-finger nucleases. Science 2009; 325: 433.

    Article  CAS  Google Scholar 

  13. Wright DA, Townsend JA, Winfrey Jr RJ, Irwin PA, Rajagopal J, Lonosky PM et al. High-frequency homologous recombination in plants mediated by zinc-finger nucleases. Plant J 2005; 44: 693–705.

    Article  CAS  Google Scholar 

  14. Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eichtinger M et al. Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell 2008; 31: 294–301.

    Article  CAS  Google Scholar 

  15. Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Worden SE et al. Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 2009; 459: 437–441.

    Article  CAS  Google Scholar 

  16. Porteus MH, Baltimore D . Chimeric nucleases stimulate gene targeting in human cells. Science 2003; 300: 763.

    Article  Google Scholar 

  17. Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 2005; 435: 646–651.

    Article  CAS  Google Scholar 

  18. Lombardo A, Genovese P, Beausejour CM, Colleoni S, Lee YL, Kim KA et al. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat Biotechnol 2007; 25: 1298–1306.

    Article  CAS  Google Scholar 

  19. Perez EE, Wang J, Miller JC, Jouvenot Y, Kim KA, Liu O et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol 2008; 26: 808–816.

    Article  CAS  Google Scholar 

  20. Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 2011; 39: e82.

    Article  CAS  Google Scholar 

  21. Mahfouz MM, Li L, Shamimuzzaman M, Wibowo A, Fang X, Zhu JK . De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks. Proc Natl Acad Sci USA 2011; 108: 2623–2628.

    Article  CAS  Google Scholar 

  22. Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 2010; 186: 757–761.

    Article  CAS  Google Scholar 

  23. Dong Z, Ge J, Li K, Xu Z, Liang D, Li J et al. Heritable targeted inactivation of myostatin gene in yellow catfish (Pelteobagrus fulvidraco) using engineered zinc-finger nucleases. PLoS One 2011; 6: e28897.

    Article  CAS  Google Scholar 

  24. Wood AJ, Lo TW, Zeitler B, Pickle CS, Ralston EJ, Lee AH et al. Targeted genome editing across species using ZFNs and TALENs. Science 2011; 333: 307.

    Article  CAS  Google Scholar 

  25. Tesson L, Usal C, Menoret S, Leung E, Niles BJ, Remy S et al. Knockout rats generated by embryo microinjection of TALENs. Nat Biotechnol 2011; 29: 695–696.

    Article  CAS  Google Scholar 

  26. Sung YH, Baek IJ, Kim DH, Jeon J, Lee J, Lee K et al. Knockout mice created by TALEN-mediated gene targeting. Nat Biotechnol 2013; 31: 23–24.

    Article  CAS  Google Scholar 

  27. Zou J, Maeder ML, Mali P, Pruett-Miller SM, Thibodeau-Beganny S, Chou BK et al. Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell 2009; 5: 97–110.

    Article  CAS  Google Scholar 

  28. Zou J, Mali P, Huang X, Dowey SN, Cheng L . Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell disease. Blood 2011; 118: 4599–4608.

    Article  CAS  Google Scholar 

  29. Soldner F, Laganiere J, Cheng AW, Hockemeyer D, Gao Q, Alagappan R et al. Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. Cell 2011; 146: 318–331.

    Article  CAS  Google Scholar 

  30. Mussolino C, Morbitzer R, Lutge F, Dannemann N, Lahaye T, Cathomen T . A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res 2011; 39: 9283–9293.

    Article  CAS  Google Scholar 

  31. DeKelver RC, Choi VM, Moehle EA, Paschon DE, Hockemeyer D, Meijsing SH et al. Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome. Genome Res 2010; 20: 1133–1142.

    Article  CAS  Google Scholar 

  32. Yusa K, Rashid ST, Strick-Marchand H, Varela I, Liu PQ, Paschon DE et al. Targeted gene correction of alpha1-antitrypsin deficiency in induced pluripotent stem cells. Nature 2011; 478: 391–394.

    Article  CAS  Google Scholar 

  33. Lombardo A, Cesana D, Genovese P, Di Stefano B, Provasi E, Colombo DF et al. Site-specific integration and tailoring of cassette design for sustainable gene transfer. Nat Methods 2011; 8: 861–869.

    Article  CAS  Google Scholar 

  34. Yuan J, Wang J, Crain K, Fearns C, Kim KA, Hua KL et al. Zinc-finger nuclease editing of human cxcr4 promotes HIV-1 CD4(+) T cell resistance and enrichment. Mol Ther 2012; 20: 849–859.

    Article  CAS  Google Scholar 

  35. Holkers M, Maggio I, Liu J, Janssen JM, Miselli F, Mussolino C et al. Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells. Nucleic Acids Res 2013; 41: e63.

    Article  CAS  Google Scholar 

  36. Handel EM, Gellhaus K, Khan K, Bednarski C, Cornu TI, Muller-Lerch F et al. Versatile and efficient genome editing in human cells by combining zinc-finger nucleases with adeno-associated viral vectors. Hum Gene Ther 2012; 23: 321–329.

    Article  Google Scholar 

  37. Lei Y, Guo X, Liu Y, Cao Y, Deng Y, Chen X et al. Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs). Proc Natl Acad Sci USA 2012; 109: 17484–17489.

    Article  CAS  Google Scholar 

  38. Carbery ID, Ji D, Harrington A, Brown V, Weinstein EJ, Liaw L et al. Targeted genome modification in mice using zinc-finger nucleases. Genetics 2010; 186: 451–459.

    Article  CAS  Google Scholar 

  39. Cui X, Ji D, Fisher DA, Wu Y, Briner DM, Weinstein EJ . Targeted integration in rat and mouse embryos with zinc-finger nucleases. Nat Biotechnol 2011; 29: 64–67.

    Article  CAS  Google Scholar 

  40. Gaj T, Guo J, Kato Y, Sirk SJ, Barbas 3rd CF . Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nat Methods 2012; 9: 805–807.

    Article  CAS  Google Scholar 

  41. Ramakrishna S, Kwaku Dad AB, Beloor J, Gopalappa R, Lee SK, Kim H . Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res 2014; 24: 1020–1027.

    Article  CAS  Google Scholar 

  42. Pan H, Zhang W, Liu GH . Find and replace: editing human genome in pluripotent stem cells. Protein Cell 2011; 2: 950–956.

    Article  CAS  Google Scholar 

  43. Chen ZY, He CY, Ehrhardt A, Kay MA . Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo. Mol Ther 2003; 8: 495–500.

    Article  CAS  Google Scholar 

  44. Darquet AM, Cameron B, Wils P, Scherman D, Crouzet J . A new DNA vehicle for nonviral gene delivery: supercoiled minicircle. Gene Therapy 1997; 4: 1341–1349.

    Article  CAS  Google Scholar 

  45. Vandermeulen G, Marie C, Scherman D, Preat V . New generation of plasmid backbones devoid of antibiotic resistance marker for gene therapy trials. Mol Ther 2011; 19: 1942–1949.

    Article  CAS  Google Scholar 

  46. Darquet AM, Rangara R, Kreiss P, Schwartz B, Naimi S, Delaere P et al. Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer. Gene Therapy 1999; 6: 209–218.

    Article  CAS  Google Scholar 

  47. Chang CW, Christensen LV, Lee M, Kim SW . Efficient expression of vascular endothelial growth factor using minicircle DNA for angiogenic gene therapy. J Control Release 2008; 125: 155–163.

    Article  CAS  Google Scholar 

  48. Osborn MJ, McElmurry RT, Lees CJ, DeFeo AP, Chen ZY, Kay MA et al. Minicircle DNA-based gene therapy coupled with immune modulation permits long-term expression of alpha-L-iduronidase in mice with mucopolysaccharidosis type I. Mol Ther 2011; 19: 450–460.

    Article  CAS  Google Scholar 

  49. Jia F, Wilson KD, Sun N, Gupta DM, Huang M, Li Z et al. A nonviral minicircle vector for deriving human iPS cells. Nat Methods 2010; 7: 197–199.

    Article  CAS  Google Scholar 

  50. Kim HJ, Lee HJ, Kim H, Cho SW, Kim JS . Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. Genome Res 2009; 19: 1279–1288.

    Article  CAS  Google Scholar 

  51. Kim H, Um E, Cho SR, Jung C, Kim JS . Surrogate reporters for enrichment of cells with nuclease-induced mutations. Nat Methods 2011; 8: 941–943.

    Article  CAS  Google Scholar 

  52. Kim H, Kim MS, Wee G, Lee CI, Kim JS . Magnetic separation and antibiotics selection enable enrichment of cells with ZFN/TALEN-induced mutations. PLoS One 2013; 8: e56476.

    Article  CAS  Google Scholar 

  53. Ramakrishna S, Kim YH, Kim H . Stability of zinc-finger nuclease protein is enhanced by the proteasome inhibitor MG132. PLoS One 2013; 8: e54282.

    Article  CAS  Google Scholar 

  54. Cho SW, Kim S, Kim JM, Kim JS . Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 2013; 31: 230–232.

    Article  CAS  Google Scholar 

  55. Kim YH, Ramakrishna S, Kim H, Kim JS . Enrichment of cells with TALEN-induced mutations using surrogate reporters. Methods 2014; doi:10.1016/j.ymeth.2014.04.012

  56. Ramakrishna S, Cho SW, Kim S, Song M, Gopalappa R, Kim JS et al. Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nuclease-induced mutations. Nat Commun 2014; 5: 3378.

    Article  Google Scholar 

  57. Doyon Y, Choi VM, Xia DF, Vo TD, Gregory PD, Holmes MC . Transient cold shock enhances zinc-finger nuclease-mediated gene disruption. Nat Methods 2010; 7: 459–460.

    Article  CAS  Google Scholar 

  58. Ramalingam S, Kandavelou K, Rajenderan R, Chandrasegaran S . Creating designed zinc-finger nucleases with minimal cytotoxicity. J Mol Biol 2011; 405: 630–641.

    Article  CAS  Google Scholar 

  59. Pruett-Miller SM, Reading DW, Porter SN, Porteus MH . Attenuation of zinc-finger nuclease toxicity by small-molecule regulation of protein levels. PLoS Genet 2009; 5: e1000376.

    Article  Google Scholar 

  60. Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, Rupniewski I et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 2007; 25: 778–785.

    Article  CAS  Google Scholar 

  61. Pruett-Miller SM, Connelly JP, Maeder ML, Joung JK, Porteus MH . Comparison of zinc-finger nucleases for use in gene targeting in mammalian cells. Mol Ther 2008; 16: 707–717.

    Article  CAS  Google Scholar 

  62. Szczepek M, Brondani V, Buchel J, Serrano L, Segal DJ, Cathomen T . Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol 2007; 25: 786–793.

    Article  CAS  Google Scholar 

  63. Huang M, Chen Z, Hu S, Jia F, Li Z, Hoyt G et al. Novel minicircle vector for gene therapy in murine myocardial infarction. Circulation 2009; 120: S230–S237.

    Article  CAS  Google Scholar 

  64. Mayrhofer P, Schleef M, Jechlinger W . Use of minicircle plasmids for gene therapy. Methods Mol Biol 2009; 542: 87–104.

    Article  CAS  Google Scholar 

  65. Madeira C, Rodrigues CA, Reis MS, Ferreira FF, Correia RE, Diogo MM et al. Nonviral Gene Delivery to Neural Stem Cells with Minicircles by Microporation. Biomacromolecules 2013; 14: 1379–1387.

    Article  CAS  Google Scholar 

  66. Chabot S, Orio J, Schmeer M, Schleef M, Golzio M, Teissie J . Minicircle DNA electrotransfer for efficient tissue-targeted gene delivery. Gene Therapy 2013; 20: 62–68.

    Article  CAS  Google Scholar 

  67. Riu E, Chen ZY, Xu H, He CY, Kay MA . Histone modifications are associated with the persistence or silencing of vector-mediated transgene expression in vivo. Mol Ther 2007; 15: 1348–1355.

    Article  CAS  Google Scholar 

  68. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000; 408: 740–745.

    Article  CAS  Google Scholar 

  69. Wagner H . Toll meets bacterial CpG-DNA. Immunity 2001; 14: 499–502.

    Article  CAS  Google Scholar 

  70. Hyde SC, Pringle IA, Abdullah S, Lawton AE, Davies LA, Varathalingam A et al. CpG-free plasmids confer reduced inflammation and sustained pulmonary gene expression. Nat Biotechnol 2008; 26: 549–551.

    Article  CAS  Google Scholar 

  71. Thomas CE, Ehrhardt A, Kay MA . Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet 2003; 4: 346–358.

    Article  CAS  Google Scholar 

  72. Mayrhofer P, Blaesen M, Schleef M, Jechlinger W . Minicircle-DNA production by site specific recombination and protein-DNA interaction chromatography. J Gene Med 2008; 10: 1253–1269.

    Article  CAS  Google Scholar 

  73. Ding Q, Lee YK, Schaefer EA, Peters DT, Veres A, Kim K et al. A TALEN genome-editing system for generating human stem cell-based disease models. Cell Stem Cell 2013; 12: 238–251.

    Article  CAS  Google Scholar 

  74. Frank S, Skryabin BV, Greber B . A modified TALEN-based system for robust generation of knock-out human pluripotent stem cell lines and disease models. BMC Genomics 2013; 14: 773.

    Article  CAS  Google Scholar 

  75. Carlson DF, Tan W, Lillico SG, Stverakova D, Proudfoot C, Christian M et al. Efficient TALEN-mediated gene knockout in livestock. Proc Natl Acad Sci USA 2012; 109: 17382–17387.

    Article  CAS  Google Scholar 

  76. Kim Y, Kweon J, Kim A, Chon JK, Yoo JY, Kim HJ et al. A library of TAL effector nucleases spanning the human genome. Nat Biotechnol 2013; 31: 251–258.

    Article  CAS  Google Scholar 

  77. Kay MA, He CY, Chen ZY . A robust system for production of minicircle DNA vectors. Nat Biotechnol 2010; 28: 1287–1289.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF-2014R1A1A1A05006189).

Author information

Author notes

  1. A-BK Dad and S Ramakrishna: These authors contributed equally to this work.

Authors and Affiliations

  1. Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Seoul, South Korea

    A-BK Dad, S Ramakrishna, M Song & H Kim

Authors
  1. A-BK Dad
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S Ramakrishna
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. H Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to H Kim.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dad, AB., Ramakrishna, S., Song, M. et al. Enhanced gene disruption by programmable nucleases delivered by a minicircle vector. Gene Ther 21, 921–930 (2014). https://doi.org/10.1038/gt.2014.76

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2014.76

This article is cited by

Search

Advanced search

Quick links