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

• DOI: 10.1038/s41467-018-05766-5
• Publication Date: 2018-08-23T12:16:37Z
• Authors: Xingbo Xu; Xiaoying Tan; Björn Tampe; Tim Wilhelmi; Melanie S. Hulshoff; Shoji Saito; Tobias Moser; Raghu Kalluri; Gerd Hasenfuss; Elisabeth M. Zeis...; Michael Zeisberg
• Keywords: 0301 basic medicine; Chromatin Immunoprecipitation; Science; KLOTHO EXPRESSION; Article; Cell Line; ACTIVATION; Mice; 03 medical and health sciences; KIDNEY; RASAL1; TARGETED DNA DEMETHYLATION; PROMOTER METHYLATION; BREAST-CANCER; Animals; Humans; TUMOR-SUPPRESSOR; CRISPR-CAS9 SYSTEM; Promoter Regions, Genetic; Klotho Proteins; Glucuronidase; Q; GTPase-Activating Proteins; Lentivirus; High-Throughput Nucleotide Sequencing; DNA Methylation; Fibrosis; Kidney Diseases; CRISPR-Cas Systems; GASTRIC-CANCER

AbstractWhile 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.