Aberrant methylation of gene promoters leads to the transcriptional silencing of genes and has been linked to pathologies including renal fibrosis. Such DNA methylation can be inhibited through the use of demethylating drugs, but the nonspecific nature of these agents limits their clinical utility. Michael Zeisberg and colleagues now show that delivery of a gene-specific system can be used to specifically reverse methylation of two genes — Rasal1 and Kl (which encodes Klotho) — and attenuate kidney fibrosis in mice.

Previous work had shown associations between hypermethylation of Rasal1 and Kl with renal fibrosis, indicating that demethylation of these genes might inhibit the fibrotic process. In addition to demethylating drugs, reversal of hypermethylation can be achieved by ten-eleven translocation (TET) family members through a process called hydroxymethylation. To demethylate specific genes, the researchers designed constructs that directed TET3 — the main TET protein in the kidney — to specific promoter sites using CRISPR–Cas9 technology. “We fused the sequence encoding the catalytic domain of TET3 to an endonuclease-deactivated high-fidelity Cas9, and targeted this to Rasal1 and Kl promoters using guide RNAs,” says Zeisberg. “The endonuclease mutation enables Cas9 to maintain its site specificity, but it no longer cleaves its target. It therefore no longer functions as a molecular scissor but rather as a molecular eraser that removes unwanted methylation marks from DNA in a site-specific manner.”

Following confirmation that these constructs could rescue expression of hypermethylated Rasal1 and Kl in vitro, the researchers demonstrated that lentiviral delivery of the Rasal1-targeted fusion protein to interstitial cells and of the Kl-targeted fusion protein to tubular epithelial cells resulted in reduced promoter methylation, reactivation of the specific gene and attenuation of renal fibrosis in mice with unilateral ureteral obstruction. “This study opens the possibility of using a molecularly informed specific epigenetic therapy for fibrosis, in which specific demethylation therapy is based on the detection of aberrant DNA methylation,” says Zeisberg.