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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics*
  • Cell Line
  • Chromatin Immunoprecipitation
  • DNA Methylation / genetics
  • DNA Methylation / physiology*
  • Fibrosis / genetics*
  • Fibrosis / therapy*
  • GTPase-Activating Proteins / genetics
  • Glucuronidase / genetics
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Kidney Diseases / genetics
  • Kidney Diseases / therapy*
  • Lentivirus / genetics
  • Mice
  • Promoter Regions, Genetic / genetics

Substances

  • GTPase-Activating Proteins
  • RASAL1 protein, human
  • Glucuronidase
  • klotho protein