TY - JOUR
T1 - High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis
AU - Xu, Xingbo
AU - Tan, Xiaoying
AU - Tampe, Björn
AU - Wilhelmi, Tim
AU - Hulshoff, Melanie S.
AU - Saito, Shoji
AU - Moser, Tobias
AU - Kalluri, Raghu
AU - Hasenfuss, Gerd
AU - Zeisberg, Elisabeth M.
AU - Zeisberg, Michael
N1 - Publisher Copyright:
© 2018, The Author(s).
PY - 2018/12/1
Y1 - 2018/12/1
N2 - 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.
AB - 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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U2 - 10.1038/s41467-018-05766-5
DO - 10.1038/s41467-018-05766-5
M3 - Article
C2 - 30158531
AN - SCOPUS:85052662332
SN - 2041-1723
VL - 9
JO - Nature communications
JF - Nature communications
IS - 1
M1 - 3509
ER -