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

Abstract

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.

Fig. 1

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 TGFβ1-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. 2

dCas9-TET3CD and dHFCas9-TET3CD fusion proteins induce targeted Rasal1/ Kl promoter demethylation in mouse kidney cells and dHFCas9-TET3CD largely reduced off-target effects. a Locations for Rasal1-sgRNAs are indicated by thick lines with corresponding PAM in magenta within the mouse Rasal1 gene locus. 10 days TGFβ1-treated mKF were transduced with dCas9-TET3CD-Rasal1-sgRNAs1-8 or by LacZ control sgRNA. b Locations for Klotho-sgRNAs are indicated by thick lines with corresponding PAM in magenta within the mouse Klotho gene locus. Three days TGFβ1-treated MCT were transduced with dCas9-TET3CD-Klotho-sgRNAs1-6 or by LacZ control sgRNA. c MeDIP and hMeDIP analysis of TGFβ1-treated mKF were transduced with dCas9-TET3CD-Rasal1-sgRNA4. d Bisulfite sequencing summary of promoter methylation status of the Rasal1 gene in TGFβ1-treated cells transduced with dCas9-TET3CD-Rasal1-sgRNA4 or by LacZ control sgRNA. e MeDIP and hMeDIP analysis of TGFβ1-treated MCT cells transduced with dCas9-TET3CD-Kl1-sgRNA2 or with LacZ control sgRNA. f Bisulfite sequencing summary of promoter methylation status of the Klotho gene in TGFβ1-treated MCT cells transduced with dCas9-TET3CD-sgRNA2 or by LacZ control sgRNA. g Schematic of domain structure of the dHFCas9-TET3CD fusion protein. h TGFβ1-treated mKFs were transduced with dCas9/dHFCas9-TET3CD-Rasal1-sgRNA4 or LacZ control sgRNA (left panel). TGFβ1-treated MCT cells were transduced with dCas9/dHFCas9-TET3CD-Klotho-sgRNA2 or LacZ control sgRNA (right panel). i MeDIP-qPCR analysis of TGFβ1-treated mKFs transduced with dCas9/dHFCas9-TET3CD-Rasal1-sgRNA4 or LacZ control sgRNA (left panel) and TGFβ1-treated MCT cells transduced with dCas9/dHFCas9-TET3CD-Klotho-sgRNA2 or LacZ control sgRNA (right panel). j Venn diagram summarizes the common off-targets identified by ChIP-seq analysis between dCas9-TET3CD and dHFCas9-TET3CD transduced mKF (left panel). Tracks indicate the binding regions and the enrichment of dCas9/dHFCas9-TET3CD-Rasal1/Klotho/LacZ sgRNA protein-RNA complexes in mKF cells as visualized in the IGV browser (right panel). Genomic coordinates are shown below the tracks (build mm9). 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. qRT-PCR results were normalized to reference gene Gapdh. MeDIP and hMeDIP results were calculated relative to input. For bisulfate sequencing each data point represents the mean of three independent biological replicates with error bars indicating the standard error of the mean for six or more bisulfite sequencing results

Fig. 3

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 Rasal1^tm1a 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 α-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)

Fig. 4

In vivo gene delivery to mouse kidney by Lentivirus transduction. a, b Immunofluorescence pictures show that mouse kidney fibroblasts and kidney epithelial cells were efficiently transduced with lentivirus containing a RFP (a) or EGFP (b) gene, respectively. c–f Immunohistochemistry pictures show RFP-/EGFP-positive cells in kidney sections which were transduced with lentivirus containing RFP (c, e) or EGFP (d, f) through different delivery routes: renal artery (c), parenchyma (d), renal vein (e), and infusion into retrograde ureter (f) with 80 μl (10⁸TU) virus particles for each method. n = 6 in each injection group. g Representative confocal photomicrographs of UUO kidneys transduced with GFP-labeled lentivirus via parenchymal injection. The sections were double stained for GFP (in green) and myofibroblast marker α-SMA (in red). Nuclei were counterstained with DAPI (in blue). The box-whisker plot (right panel) shows the percentage of GFP and α-SMA double positive cells out of all α-SMA-positive cells. n = 3 mice

Fig. 5

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. 6

Induction of Klotho promoter hydroxymethylation by dHFCas9-TET3CD-Klotho-sgRNA in tubular epithelial cells ameliorates kidney fibrosis. a Schematic shows retrograde ureter injection of lentiviral particles containing dHFCas9-TET3CD-Klotho-sgRNA into UUO-operated kidneys. b Schedule of UUO mouse surgery, lentivirus injection, and analysis. c qRT-PCR results showing that Kl mRNA expression was significantly induced in UUO-operated kidneys transduced with dHFCas9-TET3CD-Kl-sgRNA but not in UUO-challenged kidneys transduced with LacZ-sgRNA control. Results were normalized to reference gene Gapdh (expression is presented as mean value, error bars represent S.E.M., n = 6 in each group, ***p < 0.001). d UUO-operated kidneys which were transduced with lentivirus expressing dHFCas9-TET3CD-Kl-sgRNA show significantly reduced Kl promoter methylation level by MeDIP-qPCR assay. The results were calculated relative to input. The data are presented as mean value, error bars represent S.D., n ≥ 5, ***p < 0.001. e Kidney sections from UUO- and sham-operated mice which were transduced with lentivirus expressing dHFCas9-TET3CD-Kl-sgRNA or 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). f–h 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., n ≥ 5 in each group, # not significant, *p < 0.05, **p < 0.01, ****p < 0.0001). UUO-challenged kidneys transduced with lentivirus expressing dHFCas9-TET3CD-Kl-sgRNA show significantly decreased interstitial fibrosis level and a significantly decreased number of α-SMA- and Collagen-1-positive cells