CRISPR/Cas9-Based Engineering of the Epigenome Julian Pulecio, Nipun Verma, Eva Mejía-Ramírez, Danwei Huangfu, Angel Raya  Cell Stem Cell  Volume 21, Issue 4, Pages 431-447 (October 2017) DOI: 10.1016/j.stem.2017.09.006 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 CRISPR/dCas9-Based Applications to Study and Manipulate the Epigenome For a Figure360 author presentation of Figure 1, see the figure legend at https://doi.org/10.1016/j.stem.2017.09.006#mmc2. Main applications of the CRISPR/dCas9 system to study and manipulate the epigenome are based on the feasibility to allocate chromatin modifiers and fluorescent molecules in a very precise way. This includes the targeted reposition of transcriptional regulators (A), histone modifiers (B), enzymes responsible for changes in the DNA methylation (C), chromatin-interacting ncRNAs (D), and fluorescent molecules (E). (Gray dotted-boxes can be replaced with the colored boxes corresponding to reported examples of chromatin modifiers and fluorescent molecules). HDM, histone demethylase; HMT, histone methyltransferase; HAT, histone acetyltransferase; HDAC, histone deacyetylase; HUbq, histone ubiquitin ligase; DNMT, DNA methyltransferase; TET, ten-eleven translocation enzymes. Ac, acetylation; Me, methylation; Ub, ubiquitination. Figure360: An Author Presentation of Figure 1 Cell Stem Cell 2017 21, 431-447DOI: (10.1016/j.stem.2017.09.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Technical Considerations to Improve CRISPR/dCas9 Versatility Several factors can improve the efficiency of the epigenetic targeted modifications by CRISPR/dCas9. (A) Using only the effector domain of the chromatin modifiers, as well as binding peptidic linkers and nuclear localization signals (NLS) to the dCas9 protein, significantly increases the precision and efficiency of the intended chromatin modifications. (B) The CRISPR/dCas9 system offers four different options to amplify the effect of single chromatin modifications, based on the possibility to multiplex the amount of effectors positioned in a specific locus. (C) The CRISPR/dCas9 system offers the possibility to simultaneously induce several targeted chromatin modifications, as the gRNA molecules can function as scaffolds to recruit different RNA-binding proteins linked to various effector domains. Another option to target multiple loci is to exploit the orthogonality of this system, using Cas9 enzymes from different species and their corresponding gRNAs. Sp (Streptococcus pyogenes), St (Streptococcus thermophiles), Nm (Neisseria meningitides), Sa (Staphylococcus aureus). Cell Stem Cell 2017 21, 431-447DOI: (10.1016/j.stem.2017.09.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Future Applications The use of the CRISPR/(d)Cas9 system to interrogate and manipulate the epigenome will pave the road for the study and modification of chromatin at different levels of complexity. This system will significantly help us comprehend and control a broad range of epigenetic events based on its capacity to modify single chromatin marks, control endogenous gene expression, dissect the establishment of epigenetic features, and trace chromatin events inside the nucleus. Cell Stem Cell 2017 21, 431-447DOI: (10.1016/j.stem.2017.09.006) Copyright © 2017 Elsevier Inc. Terms and Conditions