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Editing DNA Methylation in the Mammalian Genome

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Editing DNA Methylation in the Mammalian Genome Resource Editing DNA Methylation in the Mammalian Genome Graphical Abstract Authors X. Shawn Liu, Hao Wu, Xiong Ji, ..., Daniel Dadon, Richard A. Young, Rudolf Jaenisch Correspondence [email protected] In Brief DNA methylation patterns can be specifically altered in mammalian cells using CRISPR/Cas9-based approaches. Highlights d dCas9-Tet1 and -Dnmt3a enable precise editing of CpG methylation in vitro and in vivo d Targeted demethylation of BDNF promoter IV activates BDNF in neurons d Targeted enhancer demethylation facilitates MyoD-induced muscle cell reprogramming d Targeted de novo methylation of CTCF motifs alters CTCF- mediated gene loops Liu et al., 2016, Cell 167, 233–247 September 22, 2016 ª 2016 Elsevier Inc. http://dx.doi.org/10.1016/j.cell.2016.08.056 Resource Editing DNA Methylation in the Mammalian Genome X. Shawn Liu,1,4 Hao Wu,1,4 Xiong Ji,1,5 Yonatan Stelzer,1 Xuebing Wu,1 Szymon Czauderna,1,3 Jian Shu,1 Daniel Dadon,1,2 Richard A. Young,1,2 and Rudolf Jaenisch1,2,6,* 1Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA 2Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA 3Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, 31-007 Krako´ w, Poland 4Co-first author 5Present address: School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China 6Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cell.2016.08.056 SUMMARY resolution during different stages of normal development as well as disease (De Jager et al., 2014; Landau et al., 2014). However, Mammalian DNA methylation is a critical epigenetic investigation of the functional significance of these DMRs re- mechanism orchestrating gene expression networks mains a challenge due to lack of appropriate molecular tools in many biological processes. However, investiga- that enable efficient editing of DNA methylation in a targeted tion of the functions of specific methylation events manner. remains challenging. Here, we demonstrate that We set out to establish such a toolbox by hybridization of the fusion of Tet1 or Dnmt3a with a catalytically inactive key enzymes in the DNA methylation pathway with reprogram- mable sequence-specific DNA-targeting molecular machinery. Cas9 (dCas9) enables targeted DNA methylation ed- DNA methylation is established by two de novo DNA methyl- iting. Targeting of the dCas9-Tet1 or -Dnmt3a fusion transferases (Dnmt3a/b) and is maintained by Dnmt1 (Smith protein to methylated or unmethylated promoter se- and Meissner, 2013). Gene activation during development is quences caused activation or silencing, respectively, associated with demethylation of promoter and enhancer se- of an endogenous reporter. Targeted demethylation quences with the best-understood mechanism being passive of the BDNF promoter IV or the MyoD distal enhancer demethylation by inhibition of Dnmt1. In addition, demethyla- by dCas9-Tet1 induced BDNF expression in post- tion can be achieved through oxidation of the methyl group by mitotic neurons or activated MyoD facilitating re- TET (ten-eleven translocation) dioxygenases to form 5-hydroxy- programming of fibroblasts into myoblasts, respec- methylcytosine (5-hmC), and then restoration into unmodified tively. Targeted de novo methylation of a CTCF cytosines by either DNA replication-dependent dilution or DNA loop anchor site by dCas9-Dnmt3a blocked CTCF glycosylase-initiated base excision repair (BER), a process termed as active demethylation and proposed to operate during binding and interfered with DNA looping, causing specific developmental stages such as preimplantation embryos altered gene expression in the neighboring loop. or in post-mitotic neurons (Wu and Zhang, 2014). Finally, we show that these tools can edit DNA Clustered regularly interspaced palindromic repeats (CRISPR), methylation in mice, demonstrating their wide utility a type II bacterial adaptive immune system, has been modified for functional studies of epigenetic regulation. to target the Cas9 nuclease to the desired genomic loci with seq- uence-specific guide RNAs for genome editing (Cong et al., INTRODUCTION 2013; Jinek et al., 2012; Mali et al., 2013). Importantly, a catalyt- ically inactive Cas9 (dCas9) was generated and engineered in Mammalian DNA methylation at 5-cytosine plays critical roles in several systems as a DNA targeting module to bring effector pro- many biological processes, including genomic imprinting, cell- teins such as transcriptional activator/suppressor, chromatin fate determination, chromatin architecture organization, and modifier, and green fluorescence protein to regulate gene regulation of gene expression (Bird, 2002; Jaenisch and Bird, expression, to modify chromatin, and to image genomic loci 2003; Smith and Meissner, 2013). Genetic studies have revealed respectively (Chen et al., 2013; Gilbert et al., 2013; Hilton et al., that DNA methylation is essential for mammalian development 2015; Jinek et al., 2012; Konermann et al., 2015; Qi et al., 2013). and adaptation to environmental signals (Jaenisch and Bird, In this study, we demonstrate that fusion of dCas9 with the 2003). Abnormal DNA methylation has been observed in cancer Tet1 enzymatic domain or Dnmt3a allows for targeted erasure and neurological disorders (Robertson, 2005). Owing to the or establishment of DNA methylation, respectively. As a proof advancement in sequencing technologies, single-nucleotide of principle, we first induced alterations to DNA methylation resolution methylation maps for many types of human and in two synthetic methylation reporters integrated in mouse mouse cells and tissues have been depicted (Lister et al., embryonic stem cells (mESCs). We further show that targeted 2009, 2013). Importantly, these maps have allowed for the iden- demethylation of BDNF promoter IV is sufficient to activate tification of differentially methylated regions (DMRs) at base pair its expression in mouse cortical neurons, and that targeted Cell 167, 233–247, September 22, 2016 ª 2016 Elsevier Inc. 233 A B gRNA (Cherry) + Tet dCas9 mC mC mC mC mC mC mC mC X Dazl locus Snrpn GFP Ub mC mC mC mC mC C C C dCas9 NLS effector Dazl locus Snrpn GFP U6 CMV gRNA puro T2A Cherry C D DIC GFP Cherry 40 40 31.3 26.3 ies Mock n 30 30 colo ecells 20 20 Ppositiv positive dC-T + dC-T sc gRNA 10 fGF 10 GFP f %o 0.5 1.2 1.2 0 %o 0 dC-T+ dC-T+ Mock dC-T+ dC-T+ sc gRNA target gRNA dC-T + dC-T sc gRNA target gRNA 250 um target gRNA E F Dazl locus Snrpn dC-T + dC-T + Mock 100 sc gRNA target gRNA 90 Mock 80 70 60 50 methylation of 40 dC-T + dC-T sc gRNA % 30 20 10 0 1 3 5 7 9 11 13 15 2 4 6 8 10 12 14 dC-T + dC-T target gRNA DazI Snrpn Figure 1. Activation of the Dazl-Snrpn-GFP Reporter by dCas9-Tet1 (A) Upper panel: schematic representation of a catalytically inactive mutant Cas9 (dCas9) fused with Tet1 for erasing DNA methylation, and with Dnmt3a for de novo methylation of specific sequences. Lower panel: an optimized dCas9-effector construct and a guide RNA (gRNA) construct with puro and Cherry cassettes. (B) Schematic representation of targeting the Snrpn promoter region by dCas9-Tet1 with specific gRNAs to erase methylation and activate GFP expression. (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. (legend continued on next page) 234 Cell 167, 233–247, September 22, 2016 demethylation of a MyoD distal enhancer promotes reprogram- ation-sensing promoter (conserved sequence elements from the ming of fibroblasts into myoblasts and facilitates myotube for- promoter of an imprinted gene, Snrpn) that controls the expres- mation. With dCas9-Dnmt3a, we demonstrate that targeted sion of a GFP. Insertion of this reporter construct into a genomic methylation at CTCF binding sites is able to block CTCF recruit- locus was shown to faithfully report on the methylation state of ment and to alter the expression of genes in the neighborhood the adjacent sequences (Stelzer et al., 2015). loop by increasing their interaction frequencies with the super- Demethylation of Specific CpGs enhancers insulated in the targeted loops. Furthermore, lentiviral To test whether defined sequences could be demethylated, we delivery of dCas9-Tet1 with target gRNAs into mice enabled introduced the dCas9-Tet1 construct in combination with gRNAs in vivo activation of a methylation reporter by demethylation of to target the Snrpn-GFP reporter inserted into the Dazl promoter its promoter. Thus, dCas9-Tet1 and dCas9-Dnmt3a provide (Figure 1B). Dazl is a germ cell specific gene, which is hyperme- powerful tools to investigate the functional significance of DNA thylated and not active in ES cells, and thus the GFP reporter is methylation in a locus-specific manner. not expressed. To activate GFP expression by dCas9-Tet1, we designed four gRNAs targeting all 14 CpGs in the Snrpn promoter RESULTS region (Figure S2A). After infection with lentiviral vectors co-ex- pressing dCas9-Tet1 and the four gRNAs for 3 days, some infec- A Modified CRISPR System to Edit DNA Methylation tion-positive cells as labeled by Cherry positive signal expressed To achieve targeted editing of DNA methylation, we fused dCas9 from gRNA construct began to turn on GFP (Figure S2B). To with enzymes in the methylation/demethylation pathway (Fig- assess the activation efficiency by dCas9-Tet1 with target ure 1A). Based on previous studies using the TALE system to gRNAs, we analyzed the cells infected by both viruses using target specific CpGs (Bernstein et al., 2015; Maeder et al., FACS.
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