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

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.

qRT-PCR results showing that only overexpression of TET3 catalytic domain does not induce the expression of 3 different aberrantly methylated genes RASAL1, EYA1, LRFN2 in TK188 fibrotic human kidney fibroblasts and does not induce the expression of KL in TGFβ1-treated HK2 cells. Results were normalized to reference gene GAPDH (expression is presented as mean value; error bars represent S.E.M.; n=3 independent biological replicates).

Supplementary Figure 4. Sanger sequencing of bisulfite PCR products of RASAL1 promoter region in TK188 cells.
Bisulfite sequencing analysis showing the methylation status of CpGs in two different regions (region 1: from -350 bp to +1 bp; region 2: from + 755 bp to +1106 bp) within the RASAL1 promoter in TK188 cells transduced with lentivirus expressing dCas9-RASAL1-sgRNA and control dCas9-LacZ-sgRNA constructs. M.SssI treated cell genomic DNA served as bisulfite conversion control. Each panel shows at least six different sequencing results derived from three independent biological replicates. Closed circles indicate methylated, open circles indicate un-methylated and grey circles indicate undetermined CpGs.

Supplementary Figure 5. Detection of hydroxymethylation in RASAL1 promoter induced by dCas9-TET3CD fusion protein by gRES-PCR assay.
(a) DNA sequence of the RASAL1 promoter region with exons highlighted in yellow, MspI/ HapII restriction sites in blue, CG dinucleotide highlighted in bold and the start codon in red. The primers used for the glucosylation-mediated restriction enzyme sensitive PCR (gRES-PCR) are framed. (b-e) Hydroxymethylation is detected by glucosylation-mediated restriction enzyme sensitive PCR (gRES-PCR) assay in TK188 cells transduced by lentivirus expressing dCas9-TET3CD-LacZ/RASAL1-sgRNA or dCas9-TET3CDi-RASAL1-sgRNA demethylation constructs. DNA virtual gel images present the RASAL1 PCR products from different combinations of enzymatic treatment. T4-BGT transfers glucose specifically to hydroxymethylated DNA, MspI cleaves hydroxymethylated DNA without but not with glucose and HpaII cleaves only native DNA. Only cells transduced with the dCas9-TET3CD-RASAL1-sgRNA3 construct show a RASAL1 PCR band after treatment with T4-BGT/MspI, but not in dCas9-TET3CD-LacZ-sgRNA nor in dCas9-TET3CDi-RASAL1-sgRNA3 transduced cells, confirming specific hydroxymethylation of RASAL1 is dependent on RASAL1-sgRNA3 and TET3CD activity. Figure 6. Assessment of the off-target effects of the dCas9-TET3CD-RASAL1-sgRNA3 demethylation in TK188 cells.

Supplementary
Human fibrotic TK188 cells were transduced with lentivirus expressing demethylation constructs guided by RASAL1-sgRNA3 or by LacZ-sgRNA. The mRNA expression of predicted off-target genes was assessed by qRT-PCR analysis, but there is a not significant difference between RASAL1-sgRNA3 and LacZ-sgRNA transduced cells for all tested genes. Results were normalized to reference gene GAPDH (expression is presented as mean value; error bars represent S.D.; n = 3 independent biological replicates; n.s., not significant). Figure 7. Assessment of the off-target effects of the dCas9-TET3CD-KL-sgRNA2 demethylation in HK2 cells.

Supplementary
TGFβ1-treated HK2 cells were transduced with lentivirus expressing demethylation constructs guided by KL-sgRNA2 or by LacZ-sgRNA. The mRNA expression of predicted off-target genes was assessed by qRT-PCR analysis, but there is no significant difference in gene expression between KL-sgRNA2 and LacZ-sgRNA transduced cells for all tested genes. Results were normalized to reference gene GAPDH (expression is presented as mean value; error bars represent S.D.; n = 3 independent biological replicates; n.s., not significant).

Supplementary Figure 8. TGFβ1 induces decreased Rasal1 expression through promoter hypermethylation in mKF.
(a) Mouse kidney fibroblasts (mKF) were treated with TGFβ1 for 6 and 10 days. The Rasal1 mRNA expression was analysed by qRT-PCR assay. Results were normalized to reference gene Gapdh (expression is presented as mean value; error bars represent S.D.; n = 3 independent biological replicates; ***, p < 0.001). (b) MeDIP-qPCR analysis showing a significant increase of Rasal1 promoter methylation in TGFβ1-treated mKF compared to non-treated control mKF. The results were calculated relative to input. The data is presented as mean value; error bars represent S.D.; n=3 independent biological replicates; ***, p < 0.001. (c) hMeDIP-qPCR analysis shows no significant change in Rasal1 promoter hydroxymethylation levels in TGFβ1-treated mKF in comparison to nontreated control mKF. (d) Western blot analysis shows the restored RASAL1 protein expression in TGFβ1-treated mKF which are transduced with lentivirus expressing Rasal1-sgRNA but not with control LacZ-sgRNA.