Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation.

In this study, we demonstrate that fusion of dCas9 with the Tet1 enzymatic domain or Dnmt3a allows for targeted erasure or establishment of DNA methylation, respectively. As a proof of principle, we first induced alterations to DNA methylation in two synthetic methylation reporters integrated in mouse embryonic stem cells (mESCs). We further show that targeted demethylation of BDNF promoter IV is sufficient to activate its expression in mouse cortical neurons, and that targeted demethylation of a MyoD distal enhancer promotes reprogramming of fibroblasts into myoblasts and facilitates myotube formation. With dCas9-Dnmt3a, we demonstrate that targeted methylation at CTCF binding sites is able to block CTCF recruitment and to alter the expression of genes in the neighborhood loop by increasing their interaction frequencies with the super-enhancers insulated in the targeted loops. Furthermore, lentiviral delivery of dCas9-Tet1 with target gRNAs into mice enabled in vivo activation of a methylation reporter by demethylation of its promoter. Thus, dCas9-Tet1 and dCas9-Dnmt3a provide powerful tools to investigate the functional significance of DNA methylation in a locus-specific manner.

Clustered regularly interspaced palindromic repeats (CRISPR), a type II bacterial adaptive immune system, has been modified to target the Cas9 nuclease to the desired genomic loci with sequence-specific guide RNAs for genome editing (). Importantly, a catalytically inactive Cas9 (dCas9) was generated and engineered in several systems as a DNA targeting module to bring effector proteins such as transcriptional activator/suppressor, chromatin modifier, and green fluorescence protein to regulate gene expression, to modify chromatin, and to image genomic loci respectively ().

We set out to establish such a toolbox by hybridization of the key enzymes in the DNA methylation pathway with reprogrammable sequence-specific DNA-targeting molecular machinery. DNA methylation is established by two de novo DNA methyltransferases (Dnmt3a/b) and is maintained by Dnmt1 (). Gene activation during development is associated with demethylation of promoter and enhancer sequences with the best-understood mechanism being passive demethylation by inhibition of Dnmt1. In addition, demethylation can be achieved through oxidation of the methyl group by TET (ten-eleven translocation) dioxygenases to form 5-hydroxymethylcytosine (5-hmC), and then restoration into unmodified cytosines by either DNA replication-dependent dilution or DNA glycosylase-initiated base excision repair (BER), a process termed as active demethylation and proposed to operate during specific developmental stages such as preimplantation embryos or in post-mitotic neurons ().

Mammalian DNA methylation at 5-cytosine plays critical roles in many biological processes, including genomic imprinting, cell-fate determination, chromatin architecture organization, and regulation of gene expression (). Genetic studies have revealed that DNA methylation is essential for mammalian development and adaptation to environmental signals (). Abnormal DNA methylation has been observed in cancer and neurological disorders (). Owing to the advancement in sequencing technologies, single-nucleotide resolution methylation maps for many types of human and mouse cells and tissues have been depicted (). Importantly, these maps have allowed for the identification of differentially methylated regions (DMRs) at base pair resolution during different stages of normal development as well as disease (). However, investigation of the functional significance of these DMRs remains a challenge due to lack of appropriate molecular tools that enable efficient editing of DNA methylation in a targeted manner.

To investigate whether the DNA methylation status can be modified in vivo, we infected three epidermal sites on the ventral side of an IG-DMRtransgenic mouse with the dCas9-Tet1 and Snrpn gRNAs ( Figure S7 D). Cells were sparsely infected with cherry expression seen only in some of the hair follicles. dCas9-Tet1 with Snrpn gRNAs, but not dCas9-Tet1 with the scrambled gRNA or dC-dT with Snrpn gRNAs, was able to activate GFP reporter expression in about 85% infected skin dermal cells in vivo ( Figures 7 H, S7 E, and S7F). In addition, we infected the brain of an IG-DMRtransgenic mouse with lentiviral vectors using a stereotaxic setup and analyzed the effect on targeted DNA methylation in brain slices by confocal microscopy. To eliminate possible inter-individual variability, we injected lentiviral vectors expressing dCas9-Tet1 and Snrpn gRNAs, as well as the two negative control vector combinations into different regions of the same brain ( Figure 7 D). As shown in Figures 7 E and 7F, after infection with all three lentiviral combinations, only lentiviral vectors expressing dCas9-Tet1 with Snrpn gRNAs, but not vectors expressing dCas9-Tet1 with sc gRNA or dC-dT with Snrpn gRNAs, activated the GFP reporter with an activation efficiency of about 70% ( Figure 7 G).

To test whether the dCas9-mediated DNA methylation-editing tools could be used to alter methylation in vivo, we utilized a methylation sensitive reporter mouse previously generated ( Figure 7 A;). In these transgenic mice, a methylation sensitive Snrpn-GFP cassette was inserted into the Dlk1-Dio3 locus to report the methylation status of its intergenic-differentially methylated region (IG-DMR). As the IG-DMR of this locus acquires paternal methylation during spermatogenesis, the GFP reporter (IG-DMR) is constitutively repressed in heterozygous mice carrying the paternal Snrpn-GFP allele (). As shown above, the GFP reporter in the Dazl locus was activated by targeted promoter demethylation in mES cells ( Figure 1 ). To assess whether the Dlk1-Dio3 locus GFP reporter could be activated by dCas9-Tet1 in differentiated cells, we derived adult mouse skin fibroblast cells from the tails of IG-DMRtransgenic mice, which were then transduced by lentiviruses expressing dCas9-Tet1 with Snrpn target gRNAs or a scrambled gRNA or a catalytically dead form of Tet1 (dC-dT) with Snrpn target gRNAs ( Figure 7 A). The results in Figures 7 B and 7C reveal GFP reporter activation in about 80% of Cherry (gRNA) positive fibroblasts but only when transduced by both dCas9-Tet1 and Snrpn gRNAs lentiviruses. FACS analysis of these cells further confirmed this notion ( Figures S7 A–S7C).

(E) Representative confocal micrographs for the IG-DMR GFP/Pat mouse skin infected with dCas9-Tet1 and sc gRNA, an inactive form of Tet1 (dC-dT) and the Snrpn gRNAs, and dCas9-Tet1 with Snrpn gRNAs. Arrowheads indicate that only dC-T with Snrpn gRNAs activated the GFP expression. Note red auto-fluorescence on the left edges of the epidermis.

(A) Schematic diagram illustrating the experimental procedure for ex vivo activation of the silenced GFP reporter in IG-DMR GFP/Pat mouse fibroblasts. The cultured fibroblasts were infected with lentiviral vectors expressing dCas9-Tet1 and gRNAs to demethylate the Snrpn promoter and activate the GFP reporter.

(G and H) Quantification of the percentage of IG-DMR GFP/Pat cells with GFP activation in Cherry (gRNAs) positive cells in the in vivo lentiviral delivery experiment in the brain (G) and in the skin epidemis (H). Bars represent mean ± SD of more than four representative images from two animals.

(D) Schematic diagram illustrating the experimental procedure for in vivo activation of GFP reporter in the IG-DMR GFP/Pat mouse brain. Lentiviral vectors expressing dC-T and sc gRNA, dC-dT and Snrpn target gRNAs, and dC-T and Snrpn target gRNAs were delivered with a stereotaxic microinjection approach. Brains were sliced and analyzed by immunohistochemical approaches.

(B) Representative immunohistochemical images of IG-DMR GFP/Pat fibroblasts infected with lentiviruses expressing dCas9-Tet1 (dC-T) with a sc gRNA, an inactive form of dCas9-Tet1 (dC-dT) with Snrpn target gRNA, or dC-T with Snrpn target gRNA. Stained in red for Cherry, green for GFP and DAPI for nuclei. Scale bar, 100 μm.

(A) Schematic diagram illustrating the experimental procedure for the ex vivo activation of a silenced GFP reporter in mouse fibroblast cells. Mouse tail fibroblast cells were derived from a genetically modified mouse line carrying a paternal IG-DMR-Snrpn-GFP allele (IG-DMR GFP/Pat ) in the Dlk1-Dio3 locus. The IG-DMR-Snrpn promoter on the paternal allele is hypermethylated so that the GFP reporter is constitutively silenced. The cultured fibroblast cells were infected with lentiviral vectors expressing dCas9-Tet1 and gRNAs to demethylate the Snrpn promoter and activate the GFP reporter.

In summary, our results demonstrate that the dCas9-Dnmt3a system can be used to change the methylation state of specific CTCF anchor sites and thus to interfere with the CTCF looping function.

To test whether targeted methylations of CTCF binding sites would result in increased interaction frequencies between insulated super-enhancers and activated genes, Chromosome Conformation Capture (3C) assay was performed at these loci. As shown in Figure 6 A, the interaction frequency between super-enhancers in the miR290 loop and the newly activated gene (Nlrp12) in the neighboring loop was significantly increased but the interaction between Nlrp12 and Myadm genes remained the same, indicating an open conformation for this targeted CTCF loop. To confirm that the increased interaction frequency was due to blocking CTCF anchoring, we performed a CTCF ChIP assay. Binding of CTCF to the targeted genomic site was significantly reduced in the sample with miR290 target gRNAs as compared to the sample with a scrambled gRNA, gRNAs targeting other CTCF binding sites, or a catalytically inactive dC-dD with miR290 target gRNAs ( Figure 6 B), supporting the notion that DNA methylation blocks CTCF anchoring and thus alters the CTCF loop conformation. A similar set of experiments was performed for the second CTCF loop (Pou5f1 loop) demonstrating increased interaction frequency between the insulated super-enhancers and the newly activated gene (H2Q10) and decreased binding of CTCF after targeted methylation of its binding site ( Figures 6 C and 6D).

(A) Quantitative Chromosome Conformation Capture (3C) analysis of cells described in Figure 5 C at the miR290 locus. The super-enhancer domain is indicated as a red bar. The targeted CTCF site is highlighted with a box. Arrows indicate the chromosomal positions between which the interaction frequency was assayed. Asterisk indicates the 3C anchor site. ChIP-seq binding profiles (reads per million base pair) for CTCF in black and H3K27Ac (super-enhancer) in red, and methylation track in yellow with DMR in blue are also shown. The interaction frequencies between the indicated chromosomal positions and the 3C anchor sites are displayed as a bar chart (mean ± SD) on the bottom panel. qPCRs were run in duplicates, and values are normalized against the mean interaction frequency in cells with a scrambled gRNA. (p < 0.05 for all three regions; Student’s t test; ns, non-significant; NC, negative control.)

Targeting of dCas9-Dnmt3a to the CTCF binding site bordering the miR290 loop that harbors a super-enhancer ( Figure 5 B) induced de novo methylation of CpGs at this site ( Figures 5 D and 5E). Gene expression analysis of transduced cells showed a significant elevation of Nlrp12 gene, which is outside of this super-enhancer-containing insulated neighborhood and next to the targeted CTCF site, but did not affect the expression of genes that are located inside the miR290 loop or of genes in other neighboring loops including AU018091 and Myadm ( Figure 5 C). Similarly, targeting of dCas9-Dnmt3a to the CTCF binding site bordering the Pou5f1 gene loop that harbors another super-enhancer ( Figure 5 F) induced methylation of CpGs in the CTCF binding sequence ( Figures 5 H and 5I) and increased the expression of H2Q10, which is located in a neighboring loop and next to the targeted CTCF site but did not affect the expression of the Pou5f1 gene itself or the Tcf19 gene in the other neighboring loop ( Figure 5 G). For either targeted CTCF sites, a catalytically inactive Dnmt3a form (dC-dD) did not induce changes in methylation level or gene expression as did by dC-D ( Figures 5 C–5E and 5G–5I). These observations are consistent with the results obtained when these CTCF sites were deleted () and support the notion that methylation of the CTCF binding site interferes with its insulator function.

CTCF is a highly conserved zinc finger protein that plays a primary role in the global organization of chromatin architecture (). Transcriptional enhancers normally interact with their target genes through the formation of DNA loops (), which typically are constrained within larger CTCF-mediated loops called insulated neighborhoods (), which, in turn, can form clusters of loops that contribute to topologically associating domains (TADs) (). Deletion of the CTCF loop anchor sites of insulated neighborhoods can cause enhancers to interact inappropriately with genes located outside the loop and thus increase their expression (). Interestingly, methylation of the DNA recognition site of CTCF has been reported to block CTCF binding (). To study whether methylation of specific CTCF sites could alter CTCF-mediated gene loops, we applied the dCas9-Dnmt3a system to target CTCF anchor sites ( Figure 5 A). We designed specific gRNAs ( Figure S6 ) targeting dCas9-Dnmt3a to two CTCF sites to investigate whether de novo methylation would interfere with the looping function of CTCF ( Figures 5 B and 5F). Doxycycline-inducible dCas9-Dnmt3a mES cells ( Figure S2 H) were infected with lentiviruses expressing the gRNAs and transduced cells were FACS sorted for subsequent analysis.

(G–I) The same set of experiments was performed as described in C–E for CTCF target-2, and cells were harvested for RT-qPCR analysis in (G) and for bisulfite sequencing in (H) and (I). Bars represent mean ± SD of three experimental replicates.

(C–E) Doxycycline-inducible dCas9-Dnmt3a mESCs were infected with lentiviruses expressing a scrambled gRNA or CTCF target-1 gRNAs. Cherry-positive cells were FACS sorted, cultured in the presence of Doxycycline, and then harvested for RT-qPCR analysis in (C), for bisulfite-sequencing analysis in (D) and (E). Bars represent mean ± SD of three experimental replicates.

(B) Schematic representation of CTCF target-1 (miR290 locus) with super-enhancer and miR290 in the loop, AU018091 gene in the left neighboring loop, and Nlrp12 gene in the right neighboring loop (close to the targeted CTCF binding site). The Myadm gene is in the adjacent loop right to the loop containing Nlrp12. The super-enhancer domain is indicated as a red bar. The targeted CTCF site is highlighted with a box. ChIP-seq binding profiles (reads per million per base pair) for CTCF in black and H3K27Ac (super-enhancer) in red, and methylation track in yellow with DMR in blue are also shown.

We then investigated whether targeted demethylation of DMR-5 would synergize with 5-Aza treatment to induce myotube formation ( Figure S5 B). To follow the process of myotube formation after 5-Aza treatment, a time-course experiment was performed. Multi-nucleated myotubes (MHC-positive) with heterogeneous sizes began to form 14 days post-treatment, and both MyoD-positive cell ratio and myotube density and size then increased up to day 25 ( Figures S5 C–S5E). Co-expression of dCas9-Tet1 with gRNAs targeting MyoD DMR-5 facilitated the myotube formation 14 days post-treatment as evidenced by significantly more mature, multi-nucleated MHCclusters (>2 nuclei per MHCcluster) compared to cells expressing only dCas9-Tet1 or dC-dT with MyoD DMR-5 gRNAs ( Figures 4 E, 4G, and 4H). A similar observation was made when the cells were analyzed at a later time point (16 days) post-treatment ( Figures S5 G–S5J). Our results suggest that demethylation of the MyoD distal enhancer by dCas9-Tet1/gRNA synergizes with 5-Aza in C3H10T1/2 cells to substantially facilitate myoblast conversion and myotube formation.

The role of MyoD as a master regulator for muscle development was initially defined by the observations that demethylation of DNA in fibroblasts by 5-Aza (5-Aza-2′-deoxycytidine) treatment resulted in activation of MyoD and subsequent myoblast conversion and myotube formation (). Six muscle-specific DMRs have been described within the 50-kb upstream region of MyoD gene (), and DMR-5 overlaps with a known distal enhancer of MyoD () as shown in Figure 4 A. To test whether demethylation of DMR-5 would activate MyoD in fibroblasts, we designed four gRNAs targeting this DMR ( Figure S5 A). Co-expression of dCas9-Tet1 with these gRNAs in C3H10T1/2 cells, a sub-clone from mouse embryonic fibroblasts previously used for 5-Aza mediated MyoD activation (), resulted in a moderate induction of MyoD expression (3-fold) as shown in Figure 4 B. Combination of dCas9-Tet1/MyoD DMR-5 gRNAs with 5-Aza treatment resulted in a higher induction of MyoD as shown in Figure S5 F. Bisulfite sequencing showed a substantial reduction of methylation in the DMR-5 region of sorted infection-positive cells transduced with dCas9-Tet1 and target gRNAs lentiviruses, but not with a catalytically dead Tet1 (dC-dT) or a scrambled gRNA ( Figures 4 C and 4D). To investigate whether demethylation of the MyoD distal enhancer region could reprogram fibroblasts into muscle cells, we infected C3H10T1/2 cells with lentiviruses expressing dCas9-Tet1 and gRNAs. The cells were cultured for 14 days and analyzed for MyoD and MHC (Myosin Heavy chain, a myotube specific marker) expression. As shown in Figures 4 E and 4F, co-expression of dCas9-Tet1 with gRNAs targeting DMR-5 induced a moderate expression level of MyoD but was not sufficient to induce myotube formation in the absence of 5-Aza treatment.

(J) Myotube density of MHC positive clusters with more than 2 or 5 nuclei 16 days after infection. Addition of 5-Aza induces MHC+ myotube formation. Co-expression of dC-T and gRNAs significantly induced more and larger myotubes (> 5 nuclei MHC+ clusters). Data are quantified from 3-5 representative images for H-J. Bars represent mean ± SD.

(I) Number of nuclei in MHC+ cell clusters (grouped as 2-5, 6-10, 11-20 and > 20 nuclei per MHC+ cluster) 16 days after infection. When treated with 5-Aza, co-expression of gRNAs and dC-T significantly facilitated formation of larger and more maturated MHC+ clusters compared to mock control or dC-T alone.

(G) Representative images for C3H10T1/2 cells 16 days after infection with lentiviruses expressing dC-T and gRNAs targeting DMR-5 (MyoD distal enhancer) in a fibroblast-to-myoblast conversion assay as described in B. Note that a modest level of MyoD activation (compared to the cells treated by 5-Aza) was observed in cells with dC-T and target gRNA, but not myosin heavy chain (MHC) expression or myotube formation. Stained in magenta for MHC, green for MyoD and blue for DAPI. Scale bar: 200 um.

(F) C3H10T1/2 cells were infected with lentiviruses expressing dC-T with MyoD gRNAs for 24 hr, and treated with or without 5-Aza for 48 hr before harvested for qPCR analysis. Bars represent mean ± SD of three experimental replicates.

(E) Number of nuclei in MHC+ cell clusters (grouped as 1-2 and > 2 nuclei per MHC+ cluster). Formation of larger myotubes was observed at later time points after 5-Aza treatment. Bars represent mean ± SD. Data was quantified from 3-5 representative images for each group in D and E.

(D) Fraction of mock C3H10T1/2 cells expressing MyoD at different times after 5-Aza treatment. The fraction of cells expressing MyoD increases from around 6% at day 14 to around 13% at day 16 and reached around 20% at day 25. Bars represent mean ± SD.

(C) Representative confocal micrographs of myotube formation for C3H10T1/2 fibroblast cells after 5-Aza treatment. Upper panel: a clonal field contains sparsely distributed small and mid size myotubes. Middle panel: a clonal field contains sparsely distributed large size myotubes. Bottom panel: a clonal field contains high density of myotubes with heterogeneous size. Stained in green for MHC, red for MyoD and blue for DAPI. Scale bar: 200 um.

(B) Experimental scheme of the fibroblast-to-myoblast conversion assay. Briefly, C3H10T1/2 mouse embryonic fibroblast cells were plated as 1 × 10 4 cells per well in 6-well plate, and then infected with lentiviruses expressing dCas9-Tet1 and target gRNAs. 24 hr post infection, cells were optionally treated with 5-Aza (1 uM) for 24 hr (labeled in red), and harvested for immunofluorescence staining at different time points (day-14, −16 and −25, labeled in dark blue) with medium change every other day. Scale bar: 100 um.

(H) Quantification of myotube density in MHC positive clusters with more than two or five nuclei at 14 days after infection. Data are quantified from three to five representative images for (F)–(H). Bars represent mean ± SD.

To test whether endogenous Tet activity was required to regulate BDNF expression upon neuronal activity stimulation, we treated DIV3 neurons with 2-hydroxygluterate, a competitive inhibitor for α-ketoglutarate-dependent dioxygenases including Tet enzymes (). As shown in Figure S4 K, pharmacological inhibition of Tet enzymatic activity completely abolished the induction of BDNF expression by KCl treatment. Furthermore, mouse primary cortical neurons carrying a Tet1-null mutant showed significantly attenuated activation kinetics of BDNF ( Figure S4 L), supporting a role of endogenous Tet for induction of neuronal activity.

Our results demonstrate that demethylation of the BDNF promoter IV can be induced by dCas9-Tet1/gRNAs and is sufficient to activate BDNF expression. Because post-mitotic neurons were used for these experiments, loss of methylation was likely due to active demethylation. To further support this conclusion, we examined 5-hmC levels in the BDNF promoter IV during the time course of dCas9-Tet1 induced demethylation by Tet-assisted Bisulfite sequencing (TAB-seq) analysis. As shown in Figure S4 H, 5-hmC was detected 40 hr post-infection with dCas9-Tet1 and gRNA lentiviruses and diminished after 60 hr. Similarly, 5-hmC was also detected after KCl treatment ( Figure S4 I). As the bisulfite sequencing method does not distinguish unmethylated 5-cytosine (5-C) and 5-formlycytosine/5-carboxylcytosin (5-fC/5-caC) generated from 5-hmC, it is possible that some CpGs were 5-fC/5-caC modified after targeting with dCas9-Tet1/gRNA. Nevertheless, inhibition of the base excision repair pathway by treatment with ABT-888 (an inhibitor of PARP) reduced the activation of BDNF by KCl treatment ( Figure S4 J), suggesting that demethylation of BDNF promoter IV contributes to BDNF activation.

DNA replication-independent active demethylation has been proposed to operate in post-mitotic neurons (). To test whether active demethylation can be induced in post-mitotic neurons, we applied the dCas9-Tet1 system to study the regulation of the BDNF gene. BDNF expression can be induced by neuronal activity accompanied by demethylation of its promoter IV (). We designed four gRNAs targeting 11 CpGs in the BDNF promoter IV ( Figure S4 A) to determine whether dCas9-Tet1 can activate BDNF by inducing demethylation of this promoter ( Figure 3 A). Mouse cortical neurons were isolated from E17.5 embryos and cultured for 2 days in vitro (DIV2) following a well-established experimental procedure for producing primary neuronal culture (). As shown in Figures S4 B–S4D, KCl treatment induced BDNF expression in these neurons with no detectable cell proliferation. Neurons at day 3 in culture (DIV3) were infected with lentiviral vectors expressing dCas9-Tet1 with or without the four gRNAs at almost 100% transduction efficiency ( Figure S4 E). At 48 hr post-infection some of the cultures were treated with KCl to induce neuronal activity. As shown in Figures 3 B and 3C, dCas9-Tet1/gRNAs induced BDNF expression by about 6-fold, whereas dCas9-Tet1 in the absence of gRNAs showed only a slight induction (less than 2-fold) and a catalytically dead form of Tet1 (dC-dT) showed no induction. Importantly, the same group of dCas9-Tet1/gRNAs did not induce Npas4 expression ( Figure S4 F), another neuronal activity-inducible gene (). Co-transduction of dCas9-Tet1 with each individual gRNA targeting the BDNF promoter IV showed a 2- to 3-fold induction of BDNF ( Figure S4 G). We performed bisulfite sequencing to examine the methylation state of BDNF promoter IV. As shown in Figures 3 D and 3E, dCas9-Tet1/gRNAs significantly reduced methylation in this region in contrast to gRNA negative controls while KCl treatment also induced demethylation of CpGs at positions of −148, −66 and, −19 (relative to transcription start site).

(B) Mouse cortical neurons cultured in vitro for 3 days (DIV3) were infected with lentiviruses expressing dC-T with or without gRNAs targeting the BDNF promoter IV or a catalytically dead form of Tet1 (dC-dT) with BDNF gRNAs for 2 days, and then treated with or without KCl (50 mM) for 6 hr before harvesting for RT-qPCR analysis. Bars are mean ±SD of three biological replicates.

(E) Confocal micrographs of BDNF induction by ectopic expression of dCas9-Tet1 and a set of 4 gRNAs targeting BDNF promoter IV. Stained in green for BDNF, magenta for MAP2, red for Cherry and blue for DAPI. Note that the lentiviral infection efficiency is close to 100% in these neurons. Scale bar: 50 um.

(D) Upper panel, quantification of neuronal density over the course of KCl treatment. The post-mitotic neuron density remains steadily around 4.5 × 10 4 /cm 2 over time. Bars represent mean ± SD of three experimental replicates. Lower panel, quantification of the EDU positive cells over the course of KCl treatment. Less than 2% of the cells are EDU-pistive over 24 hr. Bars represent mean ± SD of three experimental replicates.

(C) EDU labeling analysis for the mouse primary neurons over the course of KCl treatment for 24 hr. Note that extremely few EDU positive cells were observed. Stained in red for EDU, green for MAP2 and DAPI for nuclei. Scale bar: 500 um.

(B) Left panel, schematic diagram depicting the KCl treatment and lentiviral delivery experiment on E17.5 mouse primary cortical neurons to investigate BDNF expression. Note that cultured neurons were treated with AraC on DIV2 to halt cell division in glial cells and neural progenitors. Right panel: DIV3 mouse cortical neurons were treated with 50 mM of KCl, and harvested at different time points for BDNF expression analysis by RT-qPCR.

To evaluate the specificity of dCas9-Tet1/Dnmt3a-mediated methylation editing, we performed a dCas9 chromatin immunoprecipitation sequencing (ChIP-seq) assay and identified nine binding sites in the presence of gRNAs targeting the Dazl-Snrpn region described in Figure S2 A and 18 binding sites in the presence of gRNAs targeting CTCF binding sites adjacent to the miR290 locus (see below Figure S6 A). Figure S3 D shows that among the identified binding sites for each group of gRNAs, the targeted locus (Dazl-Snrpn or miR290) showed the highest level of binding for dCas9-Tet1/Dnmt3a ( Table S1 ). The second and third strongest binding sites for each targeted locus were illustrated in Figure S3 E, and bisulfite sequencing analysis of these loci showed only marginal change in methylation level ( Figures S3 F and S3G), likely due to the significantly lower binding affinity of dCas9-Dnmt3a/Tet1 at these off-target loci compared to the targeted loci. These results indicate that dCas9-based epigenetic editing can be highly specific.

To compare the methylation editing efficacy and effective range by dCas9-Tet1/Dnmt3a with TALE-based methods, we chose two previously reported loci edited by TALE-based method () and designed a single gRNA targeting dCas9-Tet1/Dnmt3a to the same site bound by the TALE-Tet1/Dnmt3a. As shown in Figures S3 A and S3C, dCas9-Dnmt3a with one single gRNA targeting the p16 locus induced an average of a 25% increase of methylation within a 320-bp region of the p16 promoter, whereas TALE-Dnmt3a only induced a 13% increase within a 650-bp region. Similarly, dCas9-Tet1 with one single gRNA targeting the RHOXF2 locus induced an average of a 28% decrease of methylation within a 150-bp region of the RHOXF2 promoter, whereas TALE-Tet1 only induced a 14% decrease within a 200-bp region ( Figures S3 B and S3C). These results suggest that the dCas9-Tet1/Dnmt3a system has higher efficacy and resolution for methylation editing than the TALE-based method.

(G) Genomic DNA from cells used in Figure 1 D and Figure 2 C was subject to bisulfite sequencing of the off-target binding sites at Vrk1 and Gm42619 loci. Shown is the mean percentage ± SD of two biological replicates.

(F) Genomic DNA from cells used in Figure 5 C was subject to bisulfite sequencing of the off-target binding sites at Vac14 and Tenm4 loci. Shown is the mean percentage ± SD of two biological replicates.

(E) ChIP-seq peaks at the targeted loci (miR290 or Dazl-Snrpn) with the highest level of signal and at two off-target loci with the second and third highest signals (Vac14 and Tenm4 loci for miR290 gRNAs; Vrk1 and Gm42619 loci for Dazl-Snrpn gRNAs) are illustrated with the nearby genes listed below. Note that the 4 Dazl-Snrpn gRNAs recognize the promoter sequences of Dazl and Snrpn as described in Figure S2 A, so the peaks for this group of gRNAs were mapped to both loci.

(D) Dox-inducible dCas9-Dnmt3a expression mouse ES cells described in Figure S2 H were infected with a scrambled gRNA or gRNAs targeting the miR290 locus or Dazl-Snrpn locus. FACS sorted Cherry-positive cells were cultured with Dox (2 ug/ml) for 3 days. Then these cells were harvested for anti-dCas9 ChIP-seq analysis. Peaks were called with the pairwise peak calling procedure described previously (), and presented in a Manhattan plot depicting genome-wide ChIP-seq peaks. All peaks with p < 0.001 are shown. Each dot represents a peak, with the x axis showing genomic location and y axis showing the peak summit height output by Model-based Analysis of ChIP-Seq (MACS) (). The size of each dot is proportional to its y axis value, and individual chromosome is colored differently for visualization.

(C) Summary of methylation level analysis in A and B. The effective range was determined by the distance of CpGs that were significantly edited by dCas9-Dnmt3a/Tet1 (change of methylation greater than 10%) from the site of gRNA targeting. The resolution is defined as the effective range of dCas9-Dnmt3a/Tet1 with one single gRNA, and better resolution is referred to the shorter effective range of dCas9-Dnmt3a/Tet1 which will allow for more precise editing of DNA methylation.

(B) HEK293T cells were co-transfected with dCas9-Tet1 and one RHOXF2 target gRNA (with puro cassette) or TALE-Tet1 with a puro cassette expressing plasmid. Puromycin (2 ug/ml) was added to the culture medium to select for transfection positive cells. Cells were harvested after 2-day selection for analysis of methylation levels for individual CpGs in the RHOXF2 promoter region by bisulfite sequencing. Shown is the mean percentage ± SD of two biological replicates with a total of 34 single colonies sequenced for dCas9-Tet1 and 38 single colonies sequenced for TALE-Tet1. Red arrow indicates the position of RHOXF2 target gRNA, and purple arrow indicates the binding site for TALE-Dnmt3a.

(A) HeLa cells were transfected with dCas9-Dnmt3a and one p16 target gRNA (cherry) or TALE-Dnmt3a-GFP. Transfection positive cell populations (cherry+) or (GFP+) were FACS sorted 48 hr post-transfection. Methylation levels of each individual CpG in the p16 promoter region were analyzed by bisulfite sequencing. Shown is the mean percentage ± SD of two biological replicates with a total of 34 single colonies sequenced for dCas9-Dnmt3a and 31 single colonies sequenced for TALE-Dnmt3a. Red arrow indicates the position of p16 target gRNA, and purple arrow indicates the binding site for TALE-Dnmt3a.

(H) Methylation level of each individual CpGs in the Snrpn promoter region and the adjacent Gapdh locus from cells in (G). Shown is the mean percentage ±SD of two biological replicates.

(G) Left, representative fluorescence images of the sorted Cherry-positive population in (F) after culturing for 1 week with or without Doxycycline. Scale bar, 250 μm. Right, percentages of GFP negative colonies were quantified and are shown as the mean percentages of GFP negative colonies ±SD of two biological replicates.

(F) Gapdh-Snrpn-GFP mESCs with Doxycycline-inducible dCas9-Dnmt3a were infected with lentiviruses expressing gRNAs targeting the Snrpn promoter region in the presence of Doxycycline (2 μg/ml). Percentages of GFP negative cells were calculated by flow cytometric analysis 3 days after infection and are shown as the mean percentages of GFP negative cells ±SD of two biological replicates. Note that the percentages of GFP-negative cells are expressed as the fraction of infected Cherry-positive cells.

(C) Left, representative fluorescence images of the sorted Cherry-positive cells in (B) after culturing for 1 week. Scale bar, 250 μm. Right, percentages of GFP negative colonies were quantified and are shown as the mean percentages of GFP negative colonies ±SD of two biological replicates.

(B) Gapdh-Snrpn-GFP mESCs were infected with lentiviruses expressing dCas9-Dnmt3a (dC-D) with a scrambled gRNA (sc gRNA) or gRNAs targeting the Snrpn promoter region (target gRNA). Percentage of GFP negative cells was calculated by flow cytometric analysis 3 days after infection and is shown as the mean percentages of GFP negative cells ±SD of two biological replicates. Note that the percentages of GFP-positive cells are expressed as the fraction of infected Cherry-positive cells.

To assess whether a dCas9-Dnmt3a fusion protein could de novo methylate promoter sequences and silence gene expression, we used cells carrying the Snrpn-GFP reporter in the Gapdh promoter. These cells are GFP positive because Gapdh is unmethylated and expressed in ES cells (). We infected the Gapdh-Snrpn-GFP ESCs with lentiviruses expressing dCas9-Dnmt3a and gRNAs targeting the Snrpn promoter or a scrambled gRNA ( Figures 2 A and S2 E), followed by FACS analysis. Among infection-positive (Cherry-positive) population, about 12% of cells with target gRNAs inactivated GFP, whereas only 2% of cells with the scrambled gRNA were GFP negative ( Figures 2 B and S2 F). When the Cherry positive cells were grown in culture, GFP expression of cells with target gRNAs remained off whereas cells with the scrambled gRNA and mock controls remained GFP positive ( Figure 2 C). Furthermore, bisulfite sequencing showed that transduction of dCas9-Dnmt3a/gRNAs resulted in a significant increase of DNA methylation in the Snrpn promoter region but not in the adjacent Gapdh region ( Figures 2 D and 2E). Further analysis of the GFP-positive and -negative populations within infected Cherry-positive cells showed a more robust methylation of the Snrpn promoter region in Cherry;GFPcells ( Figure S2 G). To overcome the possible limitation caused by low co-transduction efficiency of both dCas9-Dnmt3a and gRNA lentiviruses, a Doxycycline-inducible dCas9-Dnmt3a expression cassette was integrated into the Gapdh-Snrpn-GFP mES cell line by using a PiggyBac transposon system ( Figure S2 H). After delivery of the same group of target gRNAs, FACS analysis showed that GFP inactivation efficiency was increased to 25% ( Figures 2 F and S2 I). Sorted Cherry-positive cells showed loss of GFP expression upon Doxycycline treatment ( Figure 2 G) and were robustly methylated in the Snrpn promoter region ( Figure 2 H). We also generated a new construct of dCas9-Dnmt3a-P2A-BFP, which enables isolation of dCas9-Dnmt3a-expressing cells by FACS. ∼70% of GFP inactivation efficiency was achieved in FACS sorted double-positive cells (BFP;Cherry) after lentiviral delivery of this construct together with gRNAs ( Figure S2 J).

(J) Left panel: Schematic diagram of dCas9-Dnmt3a-P2A-BFP construct and gRNA-Cherry constructs. Middle panel: Percentages of BFP-positive only, Cherry-positive only, and double positive cell populations by FACS analysis of Gapdh-Snrpn-GFP mESCs after infection with lentiviruses expressing dCas9-Dnmt3a-P2A-BFP and Snrpn gRNAs. Right panel: FACS analysis of the percentages of GFP- or GFP+ cells within BFP+;Cherry+ cell population.

(I) Flow cytometric analysis of Gapdh-Snrpn-GFP mESCs with Doxycycline-inducible dCas9-Dnmt3a after 3-day infection with lentiviruses expressing the same 3 gRNAs as in F in the presence of Doxycycline (2 ug/ml). Inactivation efficiency was calculated as shown at the bottom and is expressed as the mean percentage of Cherry positive and GFP negative cells ± SD of two biological replicates.

(F) Flow cytometric analysis of Gapdh-Snrpn-GFP mESCs at 3-days after infection with lentiviruses to express dCas9-Dnmt3a (dC-D) and 3 gRNAs targeting the Snrpn promoter region. Inactivation efficiency was calculated by the listed equation and shown as the mean percentage of Cherry positive and GFP negative cells ± SD of two biological replicates.

(E) Genomic sequence of the Gapdh-Snrpn locus with gRNA sequences labeled in yellow and CpGs in green. PAM for each gRNA is highlighted by a red box. The Gapdh sequence is in lower case, and the Snrpn sequence is in upper case.

(C) Flow cytometric analysis of Dazl-Snrpn-GFP mESCs 3-day after infection with lentiviruses to express dCas9-Tet1 (dC-T) with a scrambled gRNA or 4 gRNAs targeting the Snrpn promoter region. Activation efficiency was calculated by the listed equation and shown as the mean percentages of Cherry and GFP double positive cells ± SD of two biological replicates.

(A) Genomic sequence of the Dazl-Snrpn locus with gRNA sequences labeled in yellow and CpGs in green. PAM for each gRNA is highlighted by a red box. The Dazl sequence is in lower case, and the Snrpn sequence is in upper case.

To test whether defined sequences could be demethylated, we introduced the dCas9-Tet1 construct in combination with gRNAs to target the Snrpn-GFP reporter inserted into the Dazl promoter ( Figure 1 B). Dazl is a germ cell specific gene, which is hypermethylated and not active in ES cells, and thus the GFP reporter is not expressed. To activate GFP expression by dCas9-Tet1, we designed four gRNAs targeting all 14 CpGs in the Snrpn promoter region ( Figure S2 A). After infection with lentiviral vectors co-expressing dCas9-Tet1 and the four gRNAs for 3 days, some infection-positive cells as labeled by Cherry positive signal expressed from gRNA construct began to turn on GFP ( Figure S2 B). To assess the activation efficiency by dCas9-Tet1 with target gRNAs, we analyzed the cells infected by both viruses using FACS. Among the Cherry positive population, about 26% of cells with target gRNAs activated GFP, whereas only 1% of cells with a scrambled gRNA were GFP positive ( Figures 1 C and S2 C). These Cherry positive single cells were further cultured to allow for formation of ES cell colonies. Cells with target gRNAs, but not the scrambled gRNA, expressed GFP ( Figure 1 D). To confirm that the activation of GFP in these cells is caused by demethylation of the Snrpn promoter, we performed bisulfite sequencing of genomic DNA from these samples. As illustrated in Figures 1 E and 1F, samples from cells with target gRNAs showed robust demethylation only in the Snrpn promoter region but not the adjacent Dazl locus, and samples from the cells with the scrambled gRNA showed a similar methylation status to the uninfected (mock) control. We further analyzed the GFP-positive and -negative populations within infected Cherry-positive cells. As shown in Figure S2 D, a more robust demethylation of the Snrpn promoter region was observed in double-positive cells (Cherry;GFP). These results confirm the targeted erasure of DNA methylation by dCas9-Tet1 with gRNAs in proliferative cells.

To assess whether the dCas9-Tet1 and -Dnmt3a fusion constructs would induce demethylation or de novo methylation, respectively, of specific sequences, we utilized a methylation reporter system previously developed in our laboratory (). This reporter system consists of a synthetic methylation-sensing promoter (conserved sequence elements from the promoter of an imprinted gene, Snrpn) that controls the expression of a GFP. Insertion of this reporter construct into a genomic locus was shown to faithfully report on the methylation state of the adjacent sequences ().

To achieve targeted editing of DNA methylation, we fused dCas9 with enzymes in the methylation/demethylation pathway ( Figure 1 A). Based on previous studies using the TALE system to target specific CpGs (), Tet1 and Dnmt3a were chosen as the effectors in our system. Co-expression of sequence-specific guide RNA (gRNA) would be expected to target dCas9-Tet1 or dCas9-Dnmt3a to the specific locus and mediate modification of DNA methylation status without altering the DNA sequence. To optimize this chimeric CRISPR/dCas9-effector system, we tested two types of dCas9-Tet1 lentiviral constructs with nuclear localization signal (NLS) at different positions: dCas9-NLS-Tet1 and NLS-dCas9-NLS-Tet1 ( Figures S1 A and S1B). We also tested two types of gRNA lentiviral constructs, a widely used chimeric single-guide RNA referred to as gRNA () and a modified guide RNA with enhanced capacity to guide Cas9 to the designed genomic locus referred to as E-gRNA (). Both gRNA constructs contain a puro selection cassette and a Cherry fluorescence protein cassette driven by an independent CMV promoter that allows for fluorescence activated cell sorting (FACS) of gRNA-expressing cells after lentiviral transduction ( Figure S1 A). Characterization of these constructs showed a robust gRNA-induced nuclear translocation for the dCas9-NLS-Tet1 construct ( Figures S1 C–S1E), and thus this construct was chosen for all experiments in order to minimize non-specific modifications of DNA. Two types of gRNA behaved similarly ( Figures S1 C–S1E) and thus were used interchangeably.

(E) Quantification of induction index (defined as the nuclear-cytoplasmic ratio with sgRNA normalized to that without sgRNA) for dCas9-NLS-Tet1 and NLS-dCas9-NLS-Tet1. gRNA and E-gRNA induced 3.17 and 3.22 folds of nuclear localization for dCas9-NLS-Tet1, and 1.73 and 1.77 folds for NLS-dCas9-NLS-Tet1, respectively. We reasoned that the combination with the highest induction index would result in the best signal-to-noise ratio for targeted DNA methylation editing.

(D) Quantification of the nuclear-cytoplasmic ratio of dCas9-NLS-Tet1 and NLS-dCas9-NLS-Tet1 in HEK293T cells in the absence or presence of a gRNA, or an E-gNRA in a Box and Whiskers plot. Average dCas9 intensity of cytoplasmic and nuclear domain along a cross-sectional line as illustrated in C was used for the quantification. “+” denotes mean value of the 20 data points in each group; the boxes indicate the extreme data points (top and bottom bars), the 25%–75% interval (box), and the median (central line).

(C) Comparison of the cellular localization of dCas9-NLS-Tet1 and NLS-dCas9-NLS-Tet1 in HEK293T cells with or without co-expression of a gRNA or E-gNRA targeting the same position in the MyoD locus. In the absence of sgRNAs, dCas9-NLS-Tet1 is predominantly excluded from the nuclear compartment, and NLS-dCas9-NLS-Tet1 shows weak nuclear localization in transfected HEK293T cells. Co-expression of either gRNA or E-gRNA induced cytoplasm-to-nucleus translocation of these two proteins. Stained in green for dCas9, red for Cherry and blue for DAPI in the merged images. The red dashed lines in the first two panels indicate the cross section of the images for GFP intensity quantification. Scale bar: 10 um.

(B) Expression of dCas-NLS-Tet1 and NLS-dCas9-NLS-Tet1 was analyzed by immunoblotting with anti-Cas9 antibody after transfection with these constructs in HEK293T cells for 2 days. α-tubulin was used as a loading control.

(D) Left, representative fluorescence images of the sorted Cherry positive cells in (C) after culturing for 1 week. Scale bar, 250 μm. Right, percentages of GFP positive colonies were quantified and shown as the mean percentages of GFP positive colonies ±SD of two biological replicates.

(C) Dazl-Snrpn-GFP mESCs were infected with lentiviruses expressing dCas9-Tet1 (dC-T) with a scrambled gRNA (sc gRNA) or four gRNAs targeting the Snrpn promoter region (target gRNA). Percentages of GFP positive cells were calculated by flow cytometric analysis of these cells 3 days post-infection and shown as the mean percentages of GFP positive cells ±SD of two biological replicates. Note that the percentages of GFP-positive cells are expressed as the fraction of infected Cherry-positive cells.

Discussion

In this study, we have repurposed the CRISPR/Cas9 system to edit the methylation status of genomic sequences. The catalytically inactive Cas9 protein (dCas9) was fused either to the catalytic domain of Tet1 (dCas9-Tet1) or to Dnmt3a (dCas9-Dnmt3a) to predictably alter the epigenetic state of target sequences. A GFP reporter inserted into the promoter region of the methylated and silenced Dazl gene was demethylated and activated when targeted by dCas9-Tet1, whereas the GFP reporter inserted into the promoter region of the active and unmethylated Gapdh gene was de novo methylated and silenced when targeted by dCas9-Dnmt3a. When the dCas9-Tet1 was targeted to the inactive BDNF promoter IV in post-mitotic neurons, the promoter became demethylated and activated. Importantly, this tool predictably altered the methylation state and activity of regulatory regions: targeted demethylation of the inactive distal enhancer of MyoD activated the gene and facilitated muscle cell transdifferentiation and targeted methylation of CTCF anchor sites inhibited CTCF binding and interfered with its function as an insulator between gene loops. Finally, the editing tools can in vivo alter the methylation state of regulatory sequences as injection of the lentiviral vectors of dCas9-Tet1 with target gRNAs into the dermis or brain of transgenic mice demethylated the methylated Snrpn promoter in the Dlk1-Dio3 imprinted locus and activated the methylation-sensing GFP reporter.

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et al. Widespread plasticity in CTCF occupancy linked to DNA methylation. Recent studies of mammalian chromosome structures reveal that chromatin is organized in topologically associating domains and gene loops mediated by chromatin architecture proteins such as Cohesin and CTCF (). Emerging data suggest that higher-order chromatin structures confer epigenetic information during development and are frequently altered in cancer (). It has been reported that binding of CTCF is inhibited when its recognition sequence is methylated (). Targeting of dCas9-Dnmt3a to two CTCF binding sites induced de novo methylation of CpGs in these sites and interfered with the insulator function of the protein as evidenced by increased interaction frequencies between insulated super-enhancers in the targeted loop and genes in the neighboring loop causing upregulation of these genes. This suggests that the dCas9-Dnmt3a system is a useful tool to manipulate chromatin structure and to assess its functional significance during development and in disease context.