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High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis
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High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis
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Journal Article
High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis
2018
Overview
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.
Suppression of gene expression due to aberrant promoter methylation contributes to organ fibrosis. Here, the authors couple a deactivated Cas9 to the TET3 catalytic domain to induce expression of four antifibrotic genes, and show that lentiviral-mediated delivery is effective in reducing kidney fibrosis in mouse models.
Publisher
Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
Subject
/ 42/41
/ Animals
/ Chromatin Immunoprecipitation
/ CRISPR
/ CRISPR-Cas Systems - genetics
/ DNA
/ DNA Methylation - physiology
/ Fibrosis
/ Fidelity
/ Genes
/ GTPase-Activating Proteins - genetics
/ High-Throughput Nucleotide Sequencing
/ Humanities and Social Sciences
/ Humans
/ Kinases
/ Mice
/ Promoter Regions, Genetic - genetics
/ Proteins
/ Science
