Fig 2: Targeted hydroxymethylation of four different aberrantly methylated genes by dCas9-TET3CD fusion protein in human kidney cells. a Schematic representing hypermethylated RASAL1 promoter region (upper panel) and reactivated RASAL1 expression through induction of RASAL1 promoter hydroxymethylation by dCas9-TET3CD fusion protein in complex with a sgRNA binding to its target region (lower panel). b Schematic of domain structure of the dCas9-TET3CD (upper panel) and dCas9-TETCDi (lower panel) fusion protein. c–e Locations for RASAL1/EYA1/LRFN2-sgRNAs are indicated by thick lines with corresponding PAM in magenta within the human RASAL1/EYA1/LRFN2 gene locus, respectively. Human fibrotic TK188 fibroblasts were transduced with lentivirus expressing demethylation constructs guided by RASAL1-sgRNAs 1–10, EYA1-sgRNA 1–6, LRFN2-sgRNA 1–8, or by LacZ control sgRNA. Results were normalized to reference gene GAPDH. f, g MeDIP and hMeDIP analysis of TK188 cells were transduced with dCas9-TET3CD-RASAL1-sgRNA3. The results were calculated relative to the input. h Bisulfite sequencing summary of promoter methylation status of the RASAL1 gene in TK188 cells transduced with demethylation constructs guided by RASAL1-sgRNA3, by LacZ control sgRNA or DNA treated with M.SssI serving as positive control. Each data point represents the mean of three independent transduction experiments with error bars indicating the standard error of the mean for six or more bisulfite sequencing results. i Locations for KL-sgRNAs are indicated by thick lines with corresponding PAM in magenta within the human KL gene locus. Three days TGFß1-treated HK2 cells were transduced with lentivirus expressing demethylation constructs guided by KL-sgRNAs 1–8 or by LacZ control sgRNA. j, k MeDIP and hMeDIP analysis of HK2 cells were transduction with dCas9-TET3CD-KL-sgRNA2. The results were calculated relative to the input. All data are presented as mean value; error bars represent S.D.; n = 3 independent biological replicates, n.s. not significant; *p < 0.05, **p < 0.01, ***p < 0.001

Fig 3: Targeted Rasal1 promoter demethylation by dCas9/dHFCas9-TET3CD-Rasal1-sgRNA fusion protein ameliorates kidney fibrosis. a Schematic showing the parenchymal injection of lentiviral particles containing dCas9/dHFCas9-TET3CD fusion protein into UUO-challenged kidneys. b Schedule of UUO mouse surgery, lentivirus injection, and analysis. c qRT-PCR results showing that Rasal1 mRNA expression was significantly induced in UUO-challenged kidneys transduced with dCas9/dHFCas9-TET3CD-Rasal1-sgRNA, but not in UUO-challenged kidneys transduced with control dCas9/dHFCas9-TET3CD-LacZ-sgRNA. There is no significant difference between dCas9-TET3CD and dHFCas9-TET3CD constructs. Results were normalized to reference gene Gapdh (expression is presented as mean value; error bars represent S.D., n ≥ 5 in each group, # not significant, ***p < 0.001). d Western blots showing restored RASAL1 protein expression in UUO-challenged kidneys which were transduced with lentivirus expressing dCas9/dHFCas9-TET3CD-Rasal1-sgRNA. The membranes were restriped and re-probed with α-TUBULIN antibody to serve as equal loading control. e, f UUO-challenged kidneys which were transduced with lentivirus expressing dCas9/dHFCas9-TET3CD-Rasal1-sgRNA show significantly reduced Rasal1 promoter methylation by MeDIP-qPCR assay (e) and increased hydroxymethylation by hMeDIP-qPCR assay (f). There is no significant difference between dCas9-TET3CD and dHFCas9-TET3CD constructs. The results were calculated relative to input. The data are presented as mean value, error bars represent S.D., n ≥ 5; # not significant, ***p < 0.001. g Kidney sections from UUO- and sham-operated mice which were transduced with lentivirus expressing dCas9/dHFCas9-TET3CD-Rasal1-sgRNA or dCas9/dHFCas9-TET3CD-LacZ-sgRNA were stained for Masson’s trichrome (MTS) (representative light microscopy images are shown in the top row), Collagen-1 or α-SMA (representative confocal images are shown in the middle and bottom row, respectively) (Scale bars: 25 μm or 50 μm). h–j Quantification of the percentage of total interstitial fibrosis and immunostained positive cells in each group is depicted (data are presented as mean value, error bars represent S.E.M., n ≥ 5 in each group, # not significant, ***p < 0.001, ****p < 0.0001). Both dCas9-TET3CD and dHFCas9-TET3CD lentivirus transduced UUO-operated kidneys show significantly decreased interstitial fibrosis level and a significantly decreased number of α-SMA- and Collagen-1-positive cells. HFCas9-TET3CD shows significantly better efficacies when compared to dCas9-TET3CD

Fig 4: Rasal1 gene disruption results in aggravated fibrosis level in the UUO model. a Schematic of knockout-first strategy for Rasal1 gene. The gene trapping LacZ cassette is alternatively spliced with exon 2 mediated by a splicing acceptor (SA). A promoter-driven Neomycin cassette is inserted after LacZ. The targeted exon 3 and 4 are flanked by loxP sites. Black arrows indicate the location of genotyping primers. b qRT-PCR analysis shows the Rasal1 mRNA expression in homozygous mice are significantly reduced as compared with wild-type mice, while there is no significant reduction in heterozygous mice. The data are presented as mean value, error bars represent S.D., n.s. not significant, ***p < 0.001. c Western blot analysis shows the RASAL1 protein expression is largely decreased in homozygous mice as compared with wild-type mice. d Kidney sections of wild type and homozygous Rasal1tm1a mutant mouse which were either sham controls (CL) or challenged with UUO were stained for Masson’s Trichrome (MTS) (representative light microscopy images are shown in the top row), Collagen-1 or a-SMA (representative confocal images are shown in the middle and bottom row, respectively.) (Scale bars: 25 µm or 50 µm). Quantification of the percentage of total interstitial fibrosis and immunostained positive cells in each group are depicted (data are presented as mean value, error bars represent S.E.M., # not significant, ****p < 0.0001)