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

Nature Communications
Xingbo XuMichael Zeisberg

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.

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Citations

Sep 14, 2018·Nature Reviews. Nephrology·Susan J Allison
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Methods Mentioned

BETA
transgenic
PCR
ChIP-seq
immunoprecipitation
gene-trap
gene trapping
genotyping
light microscopy
transfection
gene knockout

Software Mentioned

IGV browser
CCTop
MACSII
ImageLab
Blueheronbio
CellSens

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