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High-fidelity CRISPR/Cas9-based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis

Xu, X., Tan, X., Tampe, B., Wilhelmi, T., Hulshoff, M. S., Saito, S., Moser, T., Kalluri, R., Hasenfuss, G., Zeisberg, E. M. & Zeisberg, M., 29-Aug-2018, In : Nature Communications. 9, 15 p., 3509.

Research output: Contribution to journalArticleAcademicpeer-review

  • Xingbo Xu
  • Xiaoying Tan
  • Bjoern Tampe
  • Tim Wilhelmi
  • Melanie S. Hulshoff
  • Shoji Saito
  • Tobias Moser
  • Raghu Kalluri
  • Gerd Hasenfuss
  • Elisabeth M. Zeisberg
  • Michael Zeisberg

While suppression of specific genes through aberrant promoter methylation contributes to different diseases including organ fibrosis, gene-specific reactivation technology is not yet available for therapy. TET enzymes catalyze hydroxymethylation of methylated DNA, reactivating gene expression. We here report generation of a high-fidelity CRISPR/Cas9-based gene-specific dioxygenase by fusing an endonuclease deactivated high-fidelity Cas9 (dHFCas9) to TET3 catalytic domain (TET3CD), targeted to specific genes by guiding RNAs (sgRNA). We demonstrate use of this technology in four different anti-fibrotic genes in different cell types in vitro, among them RASAL1 and Klotho, both hypermethylated in kidney fibrosis. Furthermore, in vivo lentiviral delivery of the Rasal1-targeted fusion protein to interstitial cells and of the Klotho-targeted fusion protein to tubular epithelial cells each results in specific gene reactivation and attenuation of fibrosis, providing gene-specific demethylating technology in a disease model.

Original languageEnglish
Article number3509
Number of pages15
JournalNature Communications
Volume9
Publication statusPublished - 29-Aug-2018

    Keywords

  • TARGETED DNA DEMETHYLATION, TUMOR-SUPPRESSOR, PROMOTER METHYLATION, CRISPR-CAS9 SYSTEM, KLOTHO EXPRESSION, GASTRIC-CANCER, BREAST-CANCER, KIDNEY, RASAL1, ACTIVATION

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