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Nat Commun. 2018 Aug 29;9(1):3509. doi: 10.1038/s41467-018-05766-5.

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

Author information

1
Department of Cardiology and Pneumology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075, Göttingen, Germany.
2
German Center for Cardiovascular Research (DZHK) Partner Site, Göttingen, Germany.
3
Department of Nephrology and Rheumatology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075, Göttingen, Germany.
4
Department of Pathology and Medical Biology, University Medical Center Groningen, Hanzeplein 1, 9713, Groningen, GZ, Netherlands.
5
Institute for Auditory Neuroscience & Inner Ear Lab, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075, Göttingen, Germany.
6
Department of Cancer Biology, Metastasis Research Center, University of Texas, MD Anderson Cancer Center, 1881 East Road, Houston, TX, 77054-1901, USA.
7
Department of Cardiology and Pneumology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075, Göttingen, Germany. elisabeth.zeisberg@med.uni-goettingen.de.
8
German Center for Cardiovascular Research (DZHK) Partner Site, Göttingen, Germany. elisabeth.zeisberg@med.uni-goettingen.de.

Abstract

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.

PMID:
30158531
PMCID:
PMC6115451
DOI:
10.1038/s41467-018-05766-5
[Indexed for MEDLINE]
Free PMC Article

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