Skip to main content

Advertisement

SpringerLink
Log in

Applications of TALENs and CRISPR/Cas9 in Human Cells and Their Potentials for Gene Therapy

  • Reviews
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

The newly developed TALENs and emerging CRISPR/Cas9 have spurred interests in the field of genome engineering because of their ease of customization and high-efficient site-specific cleavages. Although these novel technologies have been successfully used in many types of cells, it is of great importance to apply them in human-derived cells to further observe and evaluate their clinical potentials in gene therapy. Here, we review the working mechanism of TALEN and CRISPR/Cas9, their effectiveness and specificity in human cells, and current methods to enhance efficiency and reduce off-target effects. Besides, CCR5 gene was chosen as a target example to illustrate their clinical potentials. Finally, some questions are raised for future research and for researchers to consider when making a proper choice bases on different purposes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Verma, I. M., & Weitzman, M. D. (2005). Gene therapy: Twenty-first century medicine. Annual Review of Biochemistry, 74, 711–738.

    Article  CAS  Google Scholar 

  2. Perez, E. E., Wang, J., Miller, J. C., Jouvenot, Y., Kim, K. A., Liu, O., et al. (2008). Establishment of HIV-1 resistance in CD4+T cells by genome editing using zinc-finger nucleases. Nature Biotechnology, 26, 808–816.

    Article  CAS  Google Scholar 

  3. Li, L., Krymskaya, L., Wang, J., Henley, J., Rao, A., Cao, L.-F., et al. (2013). Genomic editing of the HIV-1 coreceptor CCR5 in adult hematopoietic stem and progenitor cells using zinc finger nucleases. Molecular Therapy, 21, 1259–1269.

  4. Mussolino, C., Morbitzer, R., Lütge, F., Dannemann, N., Lahaye, T., & Cathomen, T. (2011). A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Research, 39, 9283–9293.

    Article  CAS  Google Scholar 

  5. Ding, Q., Lee, Y.-K., Schaefer, E. A., Peters, D. T., Veres, A., Kim, K., et al. (2012). A TALEN genome-editing system for generating human stem cell-based disease models. Cell Stem Cell, 12, 238–251.

  6. Takata, M., Sasaki, M. S., Sonoda, E., Morrison, C., Hashimoto, M., Utsumi, H., et al. (1998). Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. The EMBO Journal, 17, 5497–5508.

    Article  CAS  Google Scholar 

  7. Christian, M., Cermak, T., Doyle, E. L., Schmidt, C., Zhang, F., Hummel, A., et al. (2010). Targeting DNA double-strand breaks with TAL effector nucleases. Genetics, 186, 757–761.

    Article  CAS  Google Scholar 

  8. Miller, J. C., Tan, S., Qiao, G., Barlow, K. A., Wang, J., Xia, D. F., et al. (2010). A TALE nuclease architecture for efficient genome editing. Nature Biotechnology, 29, 143–148.

    Article  Google Scholar 

  9. Hockemeyer, D., Wang, H., Kiani, S., Lai, C. S., Gao, Q., Cassady, J. P., et al. (2011). Genetic engineering of human pluripotent cells using TALE nucleases. Nature Biotechnology, 29, 731–734.

    Article  CAS  Google Scholar 

  10. Li, T., Huang, S., Jiang, W. Z., Wright, D., Spalding, M. H., Weeks, D. P., et al. (2011). TAL nucleases (TALNs): Hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Research, 39, 359–372.

    Article  Google Scholar 

  11. Santiago, Y., Chan, E., Liu, P.-Q., Orlando, S., Zhang, L., Urnov, F. D., et al. (2008). Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proceedings of the National Academy of Sciences, 105, 5809–5814.

    Article  CAS  Google Scholar 

  12. Bogdanove, A. J., & Voytas, D. F. (2011). TAL effectors: Customizable proteins for DNA targeting. Science, 333, 1843–1846.

    Article  CAS  Google Scholar 

  13. Moehle, E. A., Rock, J. M., Lee, Y.-L., Jouvenot, Y., DeKelver, R. C., Gregory, P. D., et al. (2007). Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proceedings of the National Academy of Sciences, 104, 3055–3060.

    Article  CAS  Google Scholar 

  14. Gaj, T., Gersbach, C. A., & Barbas III, C. F. (2013). ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology, 31, 397–405.

  15. Boch, J., Scholze, H., Schornack, S., Landgraf, A., Hahn, S., Kay, S., et al. (2009). Breaking the code of DNA binding specificity of TAL-type III effectors. Science, 326, 1509–1512.

    Article  CAS  Google Scholar 

  16. Bogdanove, A. J., Schornack, S., & Lahaye, T. (2010). TAL effectors: Finding plant genes for disease and defense. Current Opinion in Plant Biology, 13, 394–401.

    Article  CAS  Google Scholar 

  17. Moscou, M. J., & Bogdanove, A. J. (2009). A simple cipher governs DNA recognition by TAL effectors. Science, 326, 1501.

    Article  CAS  Google Scholar 

  18. Ishino, Y., Shinagawa, H., Makino, K., Amemura, M., & Nakata, A. (1987). Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. Journal of Bacteriology, 169, 5429–5433.

    CAS  Google Scholar 

  19. Horvath, P., & Barrangou, R. (2010). CRISPR/Cas, the immune system of bacteria and archaea. Science, 327, 167–170.

    Article  CAS  Google Scholar 

  20. Bhaya, D., Davison, M., & Barrangou, R. (2011). CRISPR-Cas systems in bacteria and archaea: Versatile small RNAs for adaptive defense and regulation. Annual Review of Genetics, 45, 273–297.

    Article  CAS  Google Scholar 

  21. Hwang, W. Y., Fu, Y., Reyon, D., Maeder, M. L., Tsai, S. Q., Sander, J. D., et al. (2013). Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature Biotechnology, 31, 227–229.

    Article  CAS  Google Scholar 

  22. Jiang, W., Bikard, D., Cox, D., Zhang, F., & Marraffini, L. A. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology, 31, 233–239.

    Article  CAS  Google Scholar 

  23. Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., et al. (2013). RNA-guided human genome engineering via Cas9. Science, 339, 823–826.

    Article  CAS  Google Scholar 

  24. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science, 337, 816–821.

    Article  CAS  Google Scholar 

  25. Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., et al. (2013). Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152, 1173–1183.

    Article  CAS  Google Scholar 

  26. Gilbert, L. A., Larson, M. H., Morsut, L., Liu, Z., Brar, G. A., Torres, S. E., et al. (2013). CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell, 154, 442–451.

    Article  CAS  Google Scholar 

  27. Stroud, D. A., Formosa, L. E., Wijeyeratne, X. W., Nguyen, T. N., & Ryan, M. T. (2013). Gene knockout using transcription activator-like effector nucleases (TALENs) reveals that human NDUFA9 protein is essential for stabilizing the junction between membrane and matrix arms of complex I. Journal of Biological Chemistry, 288, 1685–1690.

    Article  CAS  Google Scholar 

  28. Hu, R., Wallace, J., Dahlem, T. J., Grunwald, D. J., & O’Connell, R. M. (2013). Targeting human MicroRNA genes using engineered Tal-effector nucleases (TALENs). PLoS ONE, 8, e63074.

    Article  CAS  Google Scholar 

  29. Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., & Doudna, J. (2013). RNA-programmed genome editing in human cells. Elife, 2, e00471.

  30. Cho, S. W., Kim, S., Kim, J. M., & Kim, J.-S. (2013). Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nature Biotechnology, 31, 230–232.

  31. Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science, 339, 819–823.

    Article  CAS  Google Scholar 

  32. Fu, Y., Foden, J. A., Khayter, C., Maeder, M. L., Reyon, D., Joung, J. K., et al. (2013). High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology, 31, 822–826.

    Article  CAS  Google Scholar 

  33. Kim, Y., Kweon, J., Kim, A., Chon, J. K., Yoo, J. Y., Kim, H. J., et al. (2013). A library of TAL effector nucleases spanning the human genome. Nature Biotechnology, 31, 251–258.

  34. Ding, Q., Regan, S. N., Xia, Y., Oostrom, L. A., Cowan, C. A., & Musunuru, K. (2013). Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell, 12, 393–394.

    Article  CAS  Google Scholar 

  35. Wei, C., Liu, J., Yu, Z., Zhang, B., Gao, G., & Jiao, R. (2013). TALEN or Cas9–rapid, efficient and specific choices for genome modifications. Journal of Genetics and Genomics, 40, 281–289.

    Article  CAS  Google Scholar 

  36. Yang, L., Guell, M., Byrne, S., Yang, J. L., De Los Angeles, A., Mali, P., et al. (2013). Optimization of scarless human stem cell genome editing. Nucleic Acids Research, 41, 9049–9061.

    Article  CAS  Google Scholar 

  37. Streubel, J., Blücher, C., Landgraf, A., & Boch, J. (2012). TAL effector RVD specificities and efficiencies. Nature Biotechnology, 30, 593–595.

    Article  CAS  Google Scholar 

  38. Sakuma, T., Ochiai, H., Kaneko, T., Mashimo, T., Tokumasu, D., Sakane, Y., et al. (2013). Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity. Scientific Reports, 3, 3379.

  39. Wang, T., Wei, J. J., Sabatini, D. M., & Lander, E. S. (2014). Genetic screens in human cells using the CRISPR-Cas9 system. Science, 343, 80–84.

    Article  CAS  Google Scholar 

  40. Kim, E., Kim, S., Kim, D. H., Choi, B.-S., Choi, I.-Y., & Kim, J.-S. (2012). Precision genome engineering with programmable DNA-nicking enzymes. Genome Research, 22, 1327–1333.

    Article  CAS  Google Scholar 

  41. Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., et al. (2013). DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotechnology, 31, 827–832.

    Article  CAS  Google Scholar 

  42. Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nature Protocols, 8, 2281–2308.

    Article  CAS  Google Scholar 

  43. Cho, S. W., Kim, S., Kim, Y., Kweon, J., Kim, H. S., Bae, S., et al. (2013). Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Research, 24, 132–141.

  44. Ran, F., Hsu, P. D., Lin, C.-Y., Gootenberg, J. S., Konermann, S., Trevino, A. E., et al. (2013). Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell, 154, 1380–1389.

    Article  CAS  Google Scholar 

  45. Pattanayak, V., Lin, S., Guilinger, J. P., Ma, E., Doudna, J. A., & Liu, D. R. (2013). High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nature Biotechnology, 31, 839–843.

    Article  CAS  Google Scholar 

  46. Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M., & Joung, J. K. (2014). Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nature Biotechnology, 32, 279–284.

  47. Smith, A. M., Takeuchi, R., Pellenz, S., Davis, L., Maizels, N., Monnat, R. J., et al. (2009). Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease. Proceedings of the National Academy of Sciences, 106, 5099–5104.

    Article  Google Scholar 

  48. Cade, L., Reyon, D., Hwang, W. Y., Tsai, S. Q., Patel, S., Khayter, C., et al. (2012). Highly efficient generation of heritable zebrafish gene mutations using homo-and heterodimeric TALENs. Nucleic Acids Research, 40, 8001–8010.

    Article  CAS  Google Scholar 

  49. Mali, P., Aach, J., Stranges, P. B., Esvelt, K. M., Moosburner, M., Kosuri, S., et al. (2013). CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology, 31, 833–838.

    Article  CAS  Google Scholar 

  50. Pan, Y., Xiao, L., Li, A. S., Zhang, X., Sirois, P., Zhang, J., et al. (2013). Biological and biomedical applications of engineered nucleases. Molecular Biotechnology, 55, 54–62.

    Article  CAS  Google Scholar 

  51. Bloom, K., Ely, A., Mussolino, C., Cathomen, T., & Arbuthnot, P. (2013). Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases. Molecular Therapy, 21, 1889–1897.

    Article  CAS  Google Scholar 

  52. Ousterout, D. G., Perez-Pinera, P., Thakore, P. I., Kabadi, A. M., Brown, M. T., Qin, X., et al. (2013). Reading frame correction by targeted genome editing restores dystrophin expression in cells from duchenne muscular dystrophy patients. Molecular Therapy, 21, 1718–1726.

    Article  CAS  Google Scholar 

  53. Osborn, M. J., Starker, C. G., McElroy, A. N., Webber, B. R., Riddle, M. J., Xia, L., et al. (2013). TALEN-based gene correction for epidermolysis bullosa. Molecular Therapy, 21, 1151–1159.

    Article  CAS  Google Scholar 

  54. Xu, L., Zhao, P., Mariano, A., Han, R. (2013). Targeted myostatin gene editing in multiple mammalian species directed by a single pair of TALE nucleases. Molecular Therapy—Nucleic Acids, 2, 112.

  55. Bacman, S. R., Williams, S. L., Pinto, M., Peralta, S., & Moraes, C. T. (2013). Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs. Nature Medicine, 19, 1111–1113.

    Article  CAS  Google Scholar 

  56. Schwank, G., Koo, B.-K., Sasselli, V., Dekkers, J. F., Heo, I., Demircan, T., et al. (2013). Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell, 13, 653–658.

    Article  CAS  Google Scholar 

  57. Choe, H., Farzan, M., Sun, Y., Sullivan, N., Rollins, B., Ponath, P. D., et al. (1996). The β-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell, 85, 1135–1148.

    Article  CAS  Google Scholar 

  58. Liu, R., Paxton, W. A., Choe, S., Ceradini, D., Martin, S. R., Horuk, R., et al. (1996). Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell, 86, 367–377.

    Article  CAS  Google Scholar 

  59. Kim, H. J., Lee, H. J., Kim, H., Cho, S. W., & Kim, J.-S. (2009). Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. Genome Research, 19, 1279–1288.

    Article  CAS  Google Scholar 

  60. Sakuma, T., Hosoi, S., Woltjen, K., Suzuki, K. I., Kashiwagi, K., Wada, H., et al. (2013). Efficient TALEN construction and evaluation methods for human cell and animal applications. Genes to Cells, 18, 315–326.

  61. Doench, G., & Zhang, F. (2014). Genome-scale CRISPR-Cas9 knockout screening in human cells. Science, 343, 84–87.

Download references

Acknowledgments

This study is supported by The “863 Projects” of Ministry of Science and Technology of PR China (No. 2013AA020103); National Science and Technology Major Project (No. 2013ZX1001003).

Author information

Authors and Affiliations

  1. Department of Hematopoietic Stem Cell Transplantation, Affiliated Hospital of Academy of Military Medical Sciences, No. 8 Dongda Street, Fengtai District, Beijing, 100071, People’s Republic of China

    Jingwen Niu, Bin Zhang & Hu Chen

  2. Cell and Gene Therapy Center, Academy of Military Medical Sciences, Beijing, 100071, People’s Republic of China

    Jingwen Niu, Bin Zhang & Hu Chen

Authors
  1. Jingwen Niu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bin Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hu Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Bin Zhang or Hu Chen.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Niu, J., Zhang, B. & Chen, H. Applications of TALENs and CRISPR/Cas9 in Human Cells and Their Potentials for Gene Therapy. Mol Biotechnol 56, 681–688 (2014). https://doi.org/10.1007/s12033-014-9771-z

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-014-9771-z

Keywords

Search

Navigation