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.

CRISPR-on system for the activation of the endogenous human INS gene

Gene Therapy volume 23, pages 543–547 (2016)Cite this article

Subjects

Abstract

Advances in the field of epigenetics have allowed the design of new therapeutic strategies to address complex diseases such as type 1 diabetes (T1D). Clustered regularly interspaced short palindromic repeats (CRISPR)-on is a novel and powerful RNA-guided transcriptional activator system that can turn on specific gene expression; however, it remains unclear whether this system can be widely used or whether its use will be restricted depending on cell types, methylation promoter statuses or the capacity to modulate chromatin state. Our results revealed that the CRISPR-on system fused with transcriptional activators (dCas9-VP160) activated endogenous human INS, which is a silenced gene with a fully methylated promoter. Similarly, we observed a synergistic effect on gene activation when multiple single guide RNAs were used, and the transcriptional activation was maintained until day 21. Regarding the epigenetic profile, the targeted promoter gene did not exhibit alteration in its methylation status but rather exhibited altered levels of H3K9ac following treatment. Importantly, we showed that dCas9-VP160 acts on patients’ cells in vitro, particularly the fibroblasts of patients with T1D.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Figure 1
Figure 2
Figure 3
Figure 4

References

  1. Togliatto G, Dentelli P, Brizzi MF . Skewed epigenetics: an alternative therapeutic option for diabetes complications. J Diabetes Res 2015; 2015: 373708.

    Article  Google Scholar 

  2. de Groote ML, Verschure PJ, Rots MG . Epigenetic Editing: targeted rewriting of epigenetic marks to modulate expression of selected target genes. Nucleic Acids Res 2012; 40: 10596–10613.

    Article  CAS  Google Scholar 

  3. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E . A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012; 337: 816–821.

    Article  CAS  Google Scholar 

  4. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE et al. RNA-guided human genome engineering via Cas9. Science 2013; 339: 823–826.

    Article  CAS  Google Scholar 

  5. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013; 152: 1173–1183.

    Article  CAS  Google Scholar 

  6. Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 2014; 159: 647–661.

    Article  CAS  Google Scholar 

  7. Maeder ML, Linder SJ, Cascio VM, Fu Y, Ho QH, Joung JK . CRISPR RNA-guided activation of endogenous human genes. Nat Methods 2013; 10: 977–979.

    Article  CAS  Google Scholar 

  8. Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein LR et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods 2013; 10: 973–976.

    Article  CAS  Google Scholar 

  9. Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res 2013; 10: 1163–1171.

    Article  Google Scholar 

  10. Chakraborty S, Ji H, Kabadi AM, Gersbach CA, Christoforou N, Leong KW . A CRISPR/Cas9-based system for reprogramming cell lineage specification. Stem Cell Rep 2014; 3: 940–947.

    Article  CAS  Google Scholar 

  11. Copeland MF, Politz MC, Pfleger BF . Application of TALEs, CRISPR/Cas and sRNAs as trans-acting regulators in prokaryotes. Curr Opin Biotechnol 2014; 29: 46–54.

    Article  CAS  Google Scholar 

  12. Gersbach CA, Perez-Pinera P . Activating human genes with zinc finger proteins, transcription activator-like effectors and CRISPR/Cas9 for gene therapy and regenerative medicine. Expert Opin Ther Targets 2014; 8: 835–839.

    Article  Google Scholar 

  13. Balboa D, Weltner J, Eurola S, Trokovic R, Wartiovaara K, Otonkoski T . Conditionally stabilized dCas9 activator for controlling gene expression in human cell reprogramming and differentiation. Stem Cell Rep 2015; 5: 448–459.

    Article  CAS  Google Scholar 

  14. Hu J, Lei Y, Wong WK, Liu S, Lee KC, He X et al. Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors. Nucleic Acids Res 2014; 7: 4375–4390.

    Article  Google Scholar 

  15. Liu J, Jia G . Methylation modifications in eukaryotic messenger RNA. J Genet Genomics 2014; 41: 21–33.

    Article  CAS  Google Scholar 

  16. Lee EK, Gorospe M . Minireview: posttranscriptional regulation of the insulin and insulin-like growth factor systems. Endocrinology 2010; 151: 1403–1408.

    Article  CAS  Google Scholar 

  17. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 2013; 31: 827–832.

    Article  CAS  Google Scholar 

  18. Agalioti T, Chen G, Thanos D . Deciphering the transcriptional histone acetylation code for a human gene. Cell 2002; 111: 381–392.

    Article  CAS  Google Scholar 

  19. Ikeda K, Stuehler T, Meisterernst M . The H1 and H2 regions of the activation domain of herpes simplex virion protein 16 stimulate transcription through distinct molecular mechanisms. Genes Cells 2002; 1: 49–58.

    Article  Google Scholar 

  20. Tie F, Banerjee R, Stratton CA, Prasad-Sinha J, Stepanik V, Zlobin A et al. CBP-mediated acetylation of histone H3 lysine 27 antagonizes Drosophila Polycomb silencing. Development 2009; 18: 3131–3141.

    Article  Google Scholar 

  21. Yuan H, Reddy MA, Sun G, Lanting L, Wang M, Kato M et al. Involvement of p300/CBP and epigenetic histone acetylation in TGF-β1-mediated gene transcription in mesangial cells. Am J Physiol Renal Physiol 2013; 304: F601–F613.

    Article  CAS  Google Scholar 

  22. Wang T, Liu H, Ning Y, Xu Q . The histone acetyltransferase p300 regulates the expression of pluripotency factors and odontogenic differentiation of human dental pulp cells. PLoS ONE 2014; 9: e102117.

    Article  Google Scholar 

  23. Puri S, Folias AE, Hebrok M . Plasticity and dedifferentiation within the pancreas: development, homeostasis, and disease. Cell Stem Cell 2015; 16: 18–31.

    Article  CAS  Google Scholar 

  24. Pereyra-Bonnet F, Gimeno ML, Argumedo NR, Ielpi M, Cardozo JA, Giménez CA et al. Skin fibroblasts from patients with type 1 diabetes (T1D) can be chemically transdifferentiated into insulin-expressing clusters: a transgene-free approach. PLoS ONE 2014; 9: e100369.

    Article  Google Scholar 

  25. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F . Genome engineering using the CRISPR-Cas9 system. Nat Protoc 2013; 8: 2281–2308.

    Article  CAS  Google Scholar 

  26. Haring M, Offermann S, Danker T, Horst I, Peterhansel C, Stam M . Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods 2007; 3: 11.

    Article  Google Scholar 

  27. Jiang M, Zhang Y, Fei J, Chang X, Fan W, Qian X et al. Rapid quantification of DNA methylation by measuring relative peak heights in direct bisulfite-PCR sequencing traces. Lab Invest 2010; 90: 282–290.

    Article  CAS  Google Scholar 

  28. Matarazzo MR, Lembo F, Angrisano T, Ballestar E, Ferraro M, Pero R et al. In vivo analysis of DNA methylation patterns recognized by specific proteins: coupling CHIP and bisulfite analysis. Biotechniques 2004; 37: 666–668; 670, 672–673.

    Article  CAS  Google Scholar 

  29. Fradin D, Le Fur S, Mille C, Naoui N, Groves C, Zelenika D et al. Association of the CpG methylation pattern of the proximal insulin gene promoter with type 1 diabetes. PLoS ONE 2012; 7: e36278.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work is dedicated to the memory of Dr Pablo Argibay. CG and FPB were financed by CONICET. We thank Dr Masaru Okabe, Osaka University, for kindly providing the pCX-EGFP plasmid. We thank Nelson Argumedo Rueda, Hospital Italiano, for help in the preparation of this manuscript.

Author contributions

CG, PA and FPB conceived and designed the experiments. CG, MI and FPB performed the experiments. CG and FPB analyzed the data. AM and LG contributed reagents/materials/analysis tools. CG and FPB wrote the paper. FPB is the guarantor of this work and as such had full access to all of the data in the study and assumes responsibility for the integrity of the data and the accuracy of the data analysis.

Author information

Authors and Affiliations

  1. Basic Science and Experimental Medicine Institute, University Institute of the Italian Hospital of Buenos Aires (HIBA), Buenos Aires, Argentina

    C A Giménez, M Ielpi, P Argibay & F Pereyra-Bonnet

  2. Laboratory of Reproductive Biotechnologies and Animal Genetic Improvement, Biotechnology Research Institute, National University of General San Martín, Buenos Aires, Argentina

    A Mutto

  3. Endocrinology and Nuclear Medicine Service, HIBA, Buenos Aires, Argentina

    L Grosembacher

Authors
  1. C A Giménez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M Ielpi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A Mutto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. L Grosembacher
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. P Argibay
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. F Pereyra-Bonnet
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to F Pereyra-Bonnet.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on Gene Therapy website

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Giménez, C., Ielpi, M., Mutto, A. et al. CRISPR-on system for the activation of the endogenous human INS gene. Gene Ther 23, 543–547 (2016). https://doi.org/10.1038/gt.2016.28

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2016.28

This article is cited by

Search

Advanced search

Quick links