University of Groningen Advances of epigenetic editing Gjaltema, Rutger A F; Rots, Marianne G Published in: Current Opinion in Chemical Biology DOI: 10.1016/j.cbpa.2020.04.020 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2020 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Gjaltema, R. A. F., & Rots, M. G. (2020). Advances of epigenetic editing. Current Opinion in Chemical Biology, 57, 75-81. https://doi.org/10.1016/j.cbpa.2020.04.020 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 25-09-2021 Available online at www.sciencedirect.com ScienceDirect Advances of epigenetic editing Rutger A. F. Gjaltema1 and Marianne G. Rots2 Abstract questions at the endogenous locus level, as well to Epigenetic editing refers to the locus-specific targeting of function as preclinical tools to engineer gene transcrip- epigenetic enzymes to rewrite the local epigenetic landscape tion. The foundation of epigenetic editing is formed by of an endogenous genomic site, often with the aim of tran- the ability to generate fusion proteins of epigenetic scriptional reprogramming. Implementing clustered regularly enzymes or their catalytic domains (CDs)with pro- interspaced short palindromic repeat–dCas9 greatly acceler- grammable DNA-binding platforms such as the clus- ated the advancement of epigenetic editing, yielding preclinical tered regularly interspaced short palindromic repeat therapeutic successes using a variety of epigenetic enzymes. (CRISPR) Cas9 to target these to an endogenous locus ,CRISPR/dCas9 Here, were review the current applications of of choice (Figure 1)[2,3]. The enzymatic fusion protein these epigenetic editing tools in mammalians and shed light on then dictates the initial deposited modification while biochemical improvements that facilitate versatile applications. subsequent cross-talk within the local chromatin envi- ronment likely influences epigenetic and transcriptional Addresses output. In this review, we discuss recent advances of 1 Otto-Warburg-Laboratory, Max Planck Institute for Molecular Ge- epigenetic editing in mammals based on the CRISPRe netics, Berlin, Germany 2 Department of Pathology and Medical Biology, University of Gronin- dCas9 platform, with emphasis on the latest chemical gen, University Medical Center Groningen, Groningen, the Netherlands and biotechnological developments to control temporal and on-target activity. Corresponding author: Rots, Marianne G ([email protected]) Epigenetic editing of DNA methylation Current Opinion in Chemical Biology 2020, 57:75–81 DNA methylation (5mC) at CpG islands in promoter This reviews comes from a themed issue on Chemical Genetics and regions is associated with transcriptional repressive Epigenetics states. Targeting DNA methyltransferases (DNMTs) to Edited by Akane Kawamura and Arasu Ganesan those regions would allow target gene repression through inducing de novo 5mC. Indeed, the full length or For a complete overview see the Issue and the Editorial the CD of human or mouse DNMT3A in fusion with dCas9 (dCas9eDNMT3A and dCas9eDNMT3ACD, https://doi.org/10.1016/j.cbpa.2020.04.020 respectively) introduced de novo 5mC up to w60% at 1367-5931/© 2020 The Author(s). Published by Elsevier Ltd. This is an targeted regions (mostly promoters) which was followed open access article under the CC BY license (http://creativecommons. by inhibition of transcription [4]. In a direct comparison org/licenses/by/4.0/). between full-length dCas9eDNMT3A and the dCas9e DNMT3ACD, the latter displayed more efficient 5mC Keywords activity, whereas dCas9eDNMT3A induced less off- CRISPR/dCas, Epigenome editing, Targeted DNA methylation, Targe- target 5mC [5]. ted histone modifications, Expression reprogramming. For enhanced targeted 5mC, various approaches have Introduction been tested: first, fusions of DNMT3ACD and DNMT3L, a stimulator of DNMT3A catalytic activity Epigenetic modifications of DNA and histones are [6] (dCas9eDNMT3A3 L), could induce w5-fold known for their multifaceted contributions to tran- more 5mC deposition at various target loci than those scriptional regulation. As these modifications are faith- of dCas9eDNMT3ACD [7], although not nearing a fully propagated throughout DNA replication [1], they fully methylated state of target regions. Another report are considered central players in cellular memory of confirmed these observations [8]. As a second transcriptional states. Many efforts in the last decade approach, full-length DNMT3A was applied to an have generated a vast understanding of individual adaptation of the SunTag system [5] to enable the epigenetic modifications and their contribution to recruitment of multiple copies of scFv-DNMT3ACD transcriptional regulation. However, standing questions fusion proteins (Figure 2B) [9]. Despite multimer remain regarding how and which modifications recruitment, de novo 5mC was lower than that of a contribute to a certain transcriptional output. Epige- dCas9eDNMT3ACD fusion. Alternatively, increasing netic editing offers powerful tools to dissect these the nuclear trafficking of dCas9eDNMT3ACD www.sciencedirect.com Current Opinion in Chemical Biology 2020, 57:75–81 76 Chemical Genetics and Epigenetics Figure 1 Principle of epigenetic editing. Epigenetic editing with the CRISPR/dCas9 platform involves targeting an effector domain (ED) fused to dCas9 (dCas9-ED) (a) Upon sgRNA-mediated recruitment to a target location (e.g. promoter) the dCas9-ED is able to rewrite the local epigenetic state such as histone tails or 5mC (depicted as lollipops) and thereby modify transcriptional activity (b). CRISPR, clustered regularly interspaced short palindromic repeat. through cloning a nucleoplasmin nuclear localization hydroxymethylcytosine and further intermediates. To signal C-terminally of DNMT3ACD improved targeted initiate targeted demethylation of 5mC, dCas9 fusions 5mC from w40 to w60% [10], suggesting that con- with TET1CD were targeted to methylated regions ventional dCas9eDNMT3ACD fusions experience [10,13e17] but with a varied degree of demethylation lower nuclear translocation. In addition, simultaneous efficiencies, likely depending on genetic and chromatin targeting of dCas9 fusions with DNMT3A, DNMT3L, context, as well as on delivery efficacy of the dCas9 and the Kru¨ppel associated box (KRAB) repressor has tools. Even despite partial DNA demethylation of been successfully applied to induce repressive tran- target regions, transcriptional reactivation of the scriptional memory [11] and effective epigenetic targeted genes was rather weak, likely caused by reprogramming at CCCTC-binding factor (CTCF) remaining repressive microenvironment (e.g. deacety- binding sites [8]. In addition to mammalian DNMTs, lated histones, H3K9me2) [15]. In addition to cultured the prokaryotic CpG methyltransferase (M.SssI) has cells, targeted DNA demethylation has also been been explored for targeted DNA methylation. Target- applied in preclinical mouse models. For example, ing a humanized M.SssI derivative (dCas9eMQ1) targeted demethylation of CGG-repeats within the introduced high levels of de novo 5mC (up to w70%) fragile X mental retardation 1 (FMR1) promoter that was widely spread alongside the target region [12]. through lentiviral expression of dCas9-TET1CD in However, due to extensive off-target 5mC, further post-mitotic neurons obtained from patient-derived modifications to M.SssI are required for it to be induced pluripotent stem cells (iPSCs) restored exploited for targeted DNA methylation ( ‘Precision FMR1 expression and neuronal function in culture and epigenetic editing’). was even maintained following engrafting into mouse brains [19]. In another report, in vivo lentiviral delivery As various disease-related genes are repressed by DNA of dCas9eTET3CD in a kidney fibrosis mouse model methylation, targeted demethylation would offer resulted in targeted promoter demethylation and sub- unique therapeutic possibilities. Active DNA deme- sequent reactivation of two antifibrotic genes, which thylation is initiated by ten-eleven translocation diox- attenuated kidney fibrosis and restored kidney function ygenases (TETs) that oxidize 5mC to 5- [20]. Current Opinion in Chemical Biology 2020, 57:75–81 www.sciencedirect.com Transcriptional reprogramming by epigenome engineering Gjaltema and Rots 77 Figure 2 Enhanced CRISPR–dCas9–based epigenetic editors. (a) Second-generation CRISPR systems contain RNA aptamers (MS2 or PP7) linked to the sgRNA handle, which recruit their corresponding aptamer coat protein (MCP or PCP, respectively) fused to (epigenetic) effector domains (EDs). (b) The SunTag system consists of a dCas9 fusion with GCN4 peptide repeats that enable the recruitment of multiple copies of