CRISPR technologies for precise epigenome editing

![[Pasted image 20240923144712.png]] From: [CRISPR technologies for precise epigenome editing](https://www.nature.com/articles/s41556-020-00620-7) | **Panel** | **Process** | **Explanation** | **Analogy** | | -------------------------------------- | ---------------------------------------------------- | ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | **(a) Traditional CRISPR-Cas9** | **1. Guide RNA (gRNA) binding** | The [[CRISPR-Cas9]] system uses [[gRNA]] (grey) to bind a specific genomic DNA sequence (orange) adjacent to a [[PAM]] (blue). | [[gRNA]] acts like a GPS guiding [[Cas9]] "scissors" to the precise address in the genome. | | | **2. Double-strand break** | [[Cas9]] (light blue) cuts both strands of DNA, creating a double-strand break at the targeted site (red triangles). | [[Cas9]] works like scissors cutting a piece of string (DNA) at a precise location as directed by the GPS ([[gRNA]]). | | | **3. Repair through mutations or modifications** | The DNA break can be repaired either through random insertions or deletions (indels) or by using a template (green) for homology-directed repair. | It's like patching a hole in the road. The cell either patches it randomly (leading to bumps) or repairs it properly using a template matching the original design. | | **(b) CRISPR-dCas9 Epigenome Editing** | **1. [[dCas9]] binding to DNA** | [[dCas9]] (light blue) binds DNA without cutting it. The [[gRNA]] (grey) directs it to a specific DNA sequence (orange). | [[dCas9]] acts like a velcro patch, sticking to a specific DNA site without causing damage. | | **Subpanel (i)** | **dCas-mediated occlusion of transcription factors** | [[dCas9]] blocks the binding of transcription factors (TFs, brown) to their DNA-binding sites, preventing gene transcription. | [[dCas9]] is like someone blocking a doorway, preventing a transcription factor from entering and using the room (transcribing the gene). | | **Subpanel (ii)** | **Write or erase chromatin marks** | [[dCas9]] is fused with enzymes (colored dots) that add or remove chemical marks on [[chromatin]], regulating gene expression by acetylation or methylation. | [[dCas9]] acts like a painter adding colorful marks (acetyl groups) to make the DNA easier to "read," or an eraser removing marks (methyl groups), making it harder to "read." | | **Subpanel (iii)** | **Phase-separated condensates** | [[dCas9]] recruits proteins to form "phase-separated condensates" (green dots), specialized structures within the nucleus that regulate gene expression. | [[dCas9]] organizes a party at a specific DNA site, gathering proteins to create a microenvironment (the party) that influences the DNA's behavior. | | **Subpanel (iv)** | **Colocalizing [[chromatin]] in the nucleus** | [[dCas9]] brings together distant genomic regions (Locus A and Locus B) into specific areas in the nucleus (nuclear membrane), affecting gene expression through 3D genome organization. | [[dCas9]] acts like a mediator pulling two distant colleagues (Locus A and Locus B) into the same meeting room (nucleus), encouraging interactions that wouldn't happen otherwise. | # Outlook on [[Epigenetic Editing]] **The Frontier of [[CRISPR]] Epigenome Editing** - [[CRISPR]] technology, initially developed for genome editing, is now being adapted for epigenome editing, offering a highly versatile platform to target and modify epigenetic states. ### Timescales of Epigenetic State Induction - A critical aspect to explore is the ==**timescale** required to induce stable epigenetic changes.== - Recent advancements in **inducible [[CRISPR]] systems** using chemical, light, and protein-based controllers have shown that the duration of induction can result in either temporary or stable epigenetic modifications. - For example, **chemically inducible systems** revealed that longer induction periods can lead to stable changes, highlighting the need for further study on time-dependent effects in epigenome editing. ### Measurement of Epigenetic State - A==ccurate measurement of epigenetic modifications remains a significant challenge.== - **Sequencing technologies** have enhanced our understanding of the relationship between genomics, transcriptomics, and epigenomics. - **[[CRISPR]]-based tools** are being used to improve epigenetic measurement techniques: - **[[Cas]] cleavage** has been employed to enrich DNA inputs for **[[nanopore sequencing]]**, providing more sensitive detection of DNA methylation. - **Live-cell [[CRISPR]] imaging** has been applied to track dynamic processes like **DNA repair**, **phase separation**, and **enhancer-promoter interactions**. ### dCas-based Tools and Epigenetic Mapping - **[[dCas9]]**-based [[CRISPR]] systems are being utilized in **chromatin immunoprecipitation ([[ChIP]])** to capture locus-specific **protein-DNA interactions**. - This strategy has been extended to label specific **histones**, allowing the study of **histone inheritance** in active vs. repressed chromatin regions. - How would that work? ### Future Outlook - [[CRISPR]] technologies will continue to advance epigenome editing by combining tools for both perturbation and measurement. - The integration of **inducible systems** and advanced **imaging** or **sequencing techniques** will enable deeper insights into the timescales and stability of epigenetic modifications. # Table 1 Summary of CRISPR-based epigenetic engineering strategies and their effects From: [CRISPR technologies for precise epigenome editing](https://www.nature.com/articles/s41556-020-00620-7) |Application|Domains|Cell and tissue types|Targeted gene regulation|Epigenetic changes|Persistence|Notes| |---|---|---|---|---|---|---| |**Chromatin editing**| | | | | | | |H3K27 acetylation[26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."),[33](https://www.nature.com/articles/s41556-020-00620-7#ref-CR33 "Chavez, A. et al. Comparison of Cas9 activators in multiple species. Nat. Methods 13, 563–567 (2016)."),[56](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."),[57](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR57 "Kuscu, C. et al. Temporal and spatial epigenome editing allows precise gene regulation in mammalian cells. J. Mol. Biol. 431, 111–121 (2019)."),[58](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR58 "Chiarella, A. M. et al. Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery. Nat. Biotechnol. 38, 50–55 (2020)."),[59](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR59 "Hilton, I. B. et al. Epigenome editing by a CRISPR–Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015)."),[60](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR60 "Cheng, A. W. et al. Casilio: a versatile CRISPR–Cas9-Pumilio hybrid for gene regulation and genomic labeling. Cell Res. 26, 254–257 (2016)."),[61](https://www.nature.com/articles/s41556-020-00620-7#ref-CR61 "Chen, T. et al. Chemically controlled epigenome editing through an inducible dCas9 system. J. Am. Chem. Soc. 139, 11337–11340 (2017)."),[63](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR63 "Okada, M., Kanamori, M., Someya, K., Nakatsukasa, H. & Yoshimura, A. Stabilization of Foxp3 expression by CRISPR–dCas9-based epigenome editing in mouse primary T cells. Epigenetics Chromatin 10, 24 (2017)."),[64](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR64 "Shrimp, J. H. et al. Chemical control of a CRISPR–Cas9 acetyltransferase. ACS Chem. Biol. 13, 455–460 (2018)."),[65](https://www.nature.com/articles/s41556-020-00620-7#ref-CR65 "Zhang, X. et al. Gene activation in human cells using CRISPR/Cpf1-p300 and CRISPR/Cpf1-SunTag systems. Protein Cell 9, 380–383 (2018).")|p300 (refs. [26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."),[33](https://www.nature.com/articles/s41556-020-00620-7#ref-CR33 "Chavez, A. et al. Comparison of Cas9 activators in multiple species. Nat. Methods 13, 563–567 (2016)."),[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."),[57](https://www.nature.com/articles/s41556-020-00620-7#ref-CR57 "Kuscu, C. et al. Temporal and spatial epigenome editing allows precise gene regulation in mammalian cells. J. Mol. Biol. 431, 111–121 (2019)."),[59](https://www.nature.com/articles/s41556-020-00620-7#ref-CR59 "Hilton, I. B. et al. Epigenome editing by a CRISPR–Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015)."),[61](https://www.nature.com/articles/s41556-020-00620-7#ref-CR61 "Chen, T. et al. Chemically controlled epigenome editing through an inducible dCas9 system. J. Am. Chem. Soc. 139, 11337–11340 (2017)."),[63](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR63 "Okada, M., Kanamori, M., Someya, K., Nakatsukasa, H. & Yoshimura, A. Stabilization of Foxp3 expression by CRISPR–dCas9-based epigenome editing in mouse primary T cells. Epigenetics Chromatin 10, 24 (2017)."),[64](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR64 "Shrimp, J. H. et al. Chemical control of a CRISPR–Cas9 acetyltransferase. ACS Chem. Biol. 13, 455–460 (2018)."),[65](https://www.nature.com/articles/s41556-020-00620-7#ref-CR65 "Zhang, X. et al. Gene activation in human cells using CRISPR/Cpf1-p300 and CRISPR/Cpf1-SunTag systems. Protein Cell 9, 380–383 (2018).")), CBP[60](https://www.nature.com/articles/s41556-020-00620-7#ref-CR60 "Cheng, A. W. et al. Casilio: a versatile CRISPR–Cas9-Pumilio hybrid for gene regulation and genomic labeling. Cell Res. 26, 254–257 (2016)."), p300 and/or CBP[58](https://www.nature.com/articles/s41556-020-00620-7#ref-CR58 "Chiarella, A. M. et al. Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery. Nat. Biotechnol. 38, 50–55 (2020)."), VP64 + p300 (ref. [56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."))|HEK293 (ref. [64](https://www.nature.com/articles/s41556-020-00620-7#ref-CR64 "Shrimp, J. H. et al. Chemical control of a CRISPR–Cas9 acetyltransferase. ACS Chem. Biol. 13, 455–460 (2018).")), HEK293T[26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."),[33](https://www.nature.com/articles/s41556-020-00620-7#ref-CR33 "Chavez, A. et al. Comparison of Cas9 activators in multiple species. Nat. Methods 13, 563–567 (2016)."),[56](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."),[57](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR57 "Kuscu, C. et al. Temporal and spatial epigenome editing allows precise gene regulation in mammalian cells. J. Mol. Biol. 431, 111–121 (2019)."),[58](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR58 "Chiarella, A. M. et al. Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery. Nat. Biotechnol. 38, 50–55 (2020)."),[59](https://www.nature.com/articles/s41556-020-00620-7#ref-CR59 "Hilton, I. B. et al. Epigenome editing by a CRISPR–Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015)."),[61](https://www.nature.com/articles/s41556-020-00620-7#ref-CR61 "Chen, T. et al. Chemically controlled epigenome editing through an inducible dCas9 system. J. Am. Chem. Soc. 139, 11337–11340 (2017)."),[65](https://www.nature.com/articles/s41556-020-00620-7#ref-CR65 "Zhang, X. et al. Gene activation in human cells using CRISPR/Cpf1-p300 and CRISPR/Cpf1-SunTag systems. Protein Cell 9, 380–383 (2018)."), MCF7 (ref. [65](https://www.nature.com/articles/s41556-020-00620-7#ref-CR65 "Zhang, X. et al. Gene activation in human cells using CRISPR/Cpf1-p300 and CRISPR/Cpf1-SunTag systems. Protein Cell 9, 380–383 (2018).")), U2OS[65](https://www.nature.com/articles/s41556-020-00620-7#ref-CR65 "Zhang, X. et al. Gene activation in human cells using CRISPR/Cpf1-p300 and CRISPR/Cpf1-SunTag systems. Protein Cell 9, 380–383 (2018)."), K562 (refs. [26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."),[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."),[57](https://www.nature.com/articles/s41556-020-00620-7#ref-CR57 "Kuscu, C. et al. Temporal and spatial epigenome editing allows precise gene regulation in mammalian cells. J. Mol. Biol. 431, 111–121 (2019).")), HCT116 (ref. [58](https://www.nature.com/articles/s41556-020-00620-7#ref-CR58 "Chiarella, A. M. et al. Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery. Nat. Biotechnol. 38, 50–55 (2020).")), Jurkat[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."), 68-41 T cells[63](https://www.nature.com/articles/s41556-020-00620-7#ref-CR63 "Okada, M., Kanamori, M., Someya, K., Nakatsukasa, H. & Yoshimura, A. Stabilization of Foxp3 expression by CRISPR–dCas9-based epigenome editing in mouse primary T cells. Epigenetics Chromatin 10, 24 (2017)."), primary mouse T cells[63](https://www.nature.com/articles/s41556-020-00620-7#ref-CR63 "Okada, M., Kanamori, M., Someya, K., Nakatsukasa, H. & Yoshimura, A. Stabilization of Foxp3 expression by CRISPR–dCas9-based epigenome editing in mouse primary T cells. Epigenetics Chromatin 10, 24 (2017).")|_IL1RN_ (+++ (refs. [57](https://www.nature.com/articles/s41556-020-00620-7#ref-CR57 "Kuscu, C. et al. Temporal and spatial epigenome editing allows precise gene regulation in mammalian cells. J. Mol. Biol. 431, 111–121 (2019)."),[59](https://www.nature.com/articles/s41556-020-00620-7#ref-CR59 "Hilton, I. B. et al. Epigenome editing by a CRISPR–Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015)."),[64](https://www.nature.com/articles/s41556-020-00620-7#ref-CR64 "Shrimp, J. H. et al. Chemical control of a CRISPR–Cas9 acetyltransferase. ACS Chem. Biol. 13, 455–460 (2018)."),[65](https://www.nature.com/articles/s41556-020-00620-7#ref-CR65 "Zhang, X. et al. Gene activation in human cells using CRISPR/Cpf1-p300 and CRISPR/Cpf1-SunTag systems. Protein Cell 9, 380–383 (2018).")); ++ (ref. [61](https://www.nature.com/articles/s41556-020-00620-7#ref-CR61 "Chen, T. et al. Chemically controlled epigenome editing through an inducible dCas9 system. J. Am. Chem. Soc. 139, 11337–11340 (2017)."))), _MYOD1_ (+++ (refs. [26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."),[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."),[59](https://www.nature.com/articles/s41556-020-00620-7#ref-CR59 "Hilton, I. B. et al. Epigenome editing by a CRISPR–Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015)."),[65](https://www.nature.com/articles/s41556-020-00620-7#ref-CR65 "Zhang, X. et al. Gene activation in human cells using CRISPR/Cpf1-p300 and CRISPR/Cpf1-SunTag systems. Protein Cell 9, 380–383 (2018).")) ++ (refs. [56](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."),[57](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR57 "Kuscu, C. et al. Temporal and spatial epigenome editing allows precise gene regulation in mammalian cells. J. Mol. Biol. 431, 111–121 (2019)."),[58](https://www.nature.com/articles/s41556-020-00620-7#ref-CR58 "Chiarella, A. M. et al. Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery. Nat. Biotechnol. 38, 50–55 (2020).")); 0 (ref. [61](https://www.nature.com/articles/s41556-020-00620-7#ref-CR61 "Chen, T. et al. Chemically controlled epigenome editing through an inducible dCas9 system. J. Am. Chem. Soc. 139, 11337–11340 (2017)."))), _HBB_ (++ (ref. [56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020).")); + (ref. [56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020).")); 0 (ref. [59](https://www.nature.com/articles/s41556-020-00620-7#ref-CR59 "Hilton, I. B. et al. Epigenome editing by a CRISPR–Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015)."))), _HBE1_ (++ (refs. [56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."),[59](https://www.nature.com/articles/s41556-020-00620-7#ref-CR59 "Hilton, I. B. et al. Epigenome editing by a CRISPR–Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015).")); + (ref. [56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."))); (+++)**:** _OCT4_ (refs. [57](https://www.nature.com/articles/s41556-020-00620-7#ref-CR57 "Kuscu, C. et al. Temporal and spatial epigenome editing allows precise gene regulation in mammalian cells. J. Mol. Biol. 431, 111–121 (2019)."),[59](https://www.nature.com/articles/s41556-020-00620-7#ref-CR59 "Hilton, I. B. et al. Epigenome editing by a CRISPR–Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015)."),[60](https://www.nature.com/articles/s41556-020-00620-7#ref-CR60 "Cheng, A. W. et al. Casilio: a versatile CRISPR–Cas9-Pumilio hybrid for gene regulation and genomic labeling. Cell Res. 26, 254–257 (2016)."),[65](https://www.nature.com/articles/s41556-020-00620-7#ref-CR65 "Zhang, X. et al. Gene activation in human cells using CRISPR/Cpf1-p300 and CRISPR/Cpf1-SunTag systems. Protein Cell 9, 380–383 (2018).")), _HBG1/2_ (refs. [56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."),[59](https://www.nature.com/articles/s41556-020-00620-7#ref-CR59 "Hilton, I. B. et al. Epigenome editing by a CRISPR–Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015)."),[65](https://www.nature.com/articles/s41556-020-00620-7#ref-CR65 "Zhang, X. et al. Gene activation in human cells using CRISPR/Cpf1-p300 and CRISPR/Cpf1-SunTag systems. Protein Cell 9, 380–383 (2018).")), _ASCL1_ (ref. [33](https://www.nature.com/articles/s41556-020-00620-7#ref-CR33 "Chavez, A. et al. Comparison of Cas9 activators in multiple species. Nat. Methods 13, 563–567 (2016).")), _RHOXF2_ (ref. [64](https://www.nature.com/articles/s41556-020-00620-7#ref-CR64 "Shrimp, J. H. et al. Chemical control of a CRISPR–Cas9 acetyltransferase. ACS Chem. Biol. 13, 455–460 (2018).")), _TTN_[64](https://www.nature.com/articles/s41556-020-00620-7#ref-CR64 "Shrimp, J. H. et al. Chemical control of a CRISPR–Cas9 acetyltransferase. ACS Chem. Biol. 13, 455–460 (2018)."), FP[58](https://www.nature.com/articles/s41556-020-00620-7#ref-CR58 "Chiarella, A. M. et al. Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery. Nat. Biotechnol. 38, 50–55 (2020)."); (++): _NEUROD1_ (ref. [33](https://www.nature.com/articles/s41556-020-00620-7#ref-CR33 "Chavez, A. et al. Comparison of Cas9 activators in multiple species. Nat. Methods 13, 563–567 (2016).")), _HBA_[61](https://www.nature.com/articles/s41556-020-00620-7#ref-CR61 "Chen, T. et al. Chemically controlled epigenome editing through an inducible dCas9 system. J. Am. Chem. Soc. 139, 11337–11340 (2017)."), (+): _HBD_[59](https://www.nature.com/articles/s41556-020-00620-7#ref-CR59 "Hilton, I. B. et al. Epigenome editing by a CRISPR–Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015)."), _GRM2_ (ref. [61](https://www.nature.com/articles/s41556-020-00620-7#ref-CR61 "Chen, T. et al. Chemically controlled epigenome editing through an inducible dCas9 system. J. Am. Chem. Soc. 139, 11337–11340 (2017).")), _CXCR4_ (ref. [58](https://www.nature.com/articles/s41556-020-00620-7#ref-CR58 "Chiarella, A. M. et al. Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery. Nat. Biotechnol. 38, 50–55 (2020).")),_TAL1_ (ref. [56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020).")), _B2M_[26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."), _Foxp3_ (ref. [63](https://www.nature.com/articles/s41556-020-00620-7#ref-CR63 "Okada, M., Kanamori, M., Someya, K., Nakatsukasa, H. & Yoshimura, A. Stabilization of Foxp3 expression by CRISPR–dCas9-based epigenome editing in mouse primary T cells. Epigenetics Chromatin 10, 24 (2017)."))|H3K27ac[57](https://www.nature.com/articles/s41556-020-00620-7#ref-CR57 "Kuscu, C. et al. Temporal and spatial epigenome editing allows precise gene regulation in mammalian cells. J. Mol. Biol. 431, 111–121 (2019)."),[59](https://www.nature.com/articles/s41556-020-00620-7#ref-CR59 "Hilton, I. B. et al. Epigenome editing by a CRISPR–Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015)."),[61](https://www.nature.com/articles/s41556-020-00620-7#ref-CR61 "Chen, T. et al. Chemically controlled epigenome editing through an inducible dCas9 system. J. Am. Chem. Soc. 139, 11337–11340 (2017)."), H3ac[63](https://www.nature.com/articles/s41556-020-00620-7#ref-CR63 "Okada, M., Kanamori, M., Someya, K., Nakatsukasa, H. & Yoshimura, A. Stabilization of Foxp3 expression by CRISPR–dCas9-based epigenome editing in mouse primary T cells. Epigenetics Chromatin 10, 24 (2017)."), DNA interactions[57](https://www.nature.com/articles/s41556-020-00620-7#ref-CR57 "Kuscu, C. et al. Temporal and spatial epigenome editing allows precise gene regulation in mammalian cells. J. Mol. Biol. 431, 111–121 (2019).")|Transient[26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."), 1 day[57](https://www.nature.com/articles/s41556-020-00620-7#ref-CR57 "Kuscu, C. et al. Temporal and spatial epigenome editing allows precise gene regulation in mammalian cells. J. Mol. Biol. 431, 111–121 (2019)."),[61](https://www.nature.com/articles/s41556-020-00620-7#ref-CR61 "Chen, T. et al. Chemically controlled epigenome editing through an inducible dCas9 system. J. Am. Chem. Soc. 139, 11337–11340 (2017)."), 4 days[58](https://www.nature.com/articles/s41556-020-00620-7#ref-CR58 "Chiarella, A. M. et al. Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery. Nat. Biotechnol. 38, 50–55 (2020).")|Gene expression off-target effects assessed[59](https://www.nature.com/articles/s41556-020-00620-7#ref-CR59 "Hilton, I. B. et al. Epigenome editing by a CRISPR–Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015)."); dCas12a[65](https://www.nature.com/articles/s41556-020-00620-7#ref-CR65 "Zhang, X. et al. Gene activation in human cells using CRISPR/Cpf1-p300 and CRISPR/Cpf1-SunTag systems. Protein Cell 9, 380–383 (2018).")| |H3K27 deacetylation[62](https://www.nature.com/articles/s41556-020-00620-7#ref-CR62 "Kwon, D. Y., Zhao, Y.-T., Lamonica, J. M. & Zhou, Z. Locus-specific histone deacetylation using a synthetic CRISPR–Cas9-based HDAC. Nat. Commun. 8, 15315 (2017).")|HDAC3|N2a, MC3T3-e1|(0): _Isl1_, _Mecp2_, _Smn1_|**H3K27ac**||| |H3K4 methylation[40](https://www.nature.com/articles/s41556-020-00620-7#ref-CR40 "Cano-Rodriguez, D. et al. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner. Nat. Commun. 7, 12284 (2016)."),[53](https://www.nature.com/articles/s41556-020-00620-7#ref-CR53 "Kim, J.-M. et al. Cooperation between SMYD3 and PC4 drives a distinct transcriptional program in cancer cells. Nucleic Acids Res. 43, 8868–8883 (2015)."),[55](https://www.nature.com/articles/s41556-020-00620-7#ref-CR55 "Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017).")|SMYD3 (ref. [53](https://www.nature.com/articles/s41556-020-00620-7#ref-CR53 "Kim, J.-M. et al. Cooperation between SMYD3 and PC4 drives a distinct transcriptional program in cancer cells. Nucleic Acids Res. 43, 8868–8883 (2015).")), PRDM9 (ref. [40](https://www.nature.com/articles/s41556-020-00620-7#ref-CR40 "Cano-Rodriguez, D. et al. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner. Nat. Commun. 7, 12284 (2016).")), DOT1L[40](https://www.nature.com/articles/s41556-020-00620-7#ref-CR40 "Cano-Rodriguez, D. et al. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner. Nat. Commun. 7, 12284 (2016)."), UBE2A[40](https://www.nature.com/articles/s41556-020-00620-7#ref-CR40 "Cano-Rodriguez, D. et al. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner. Nat. Commun. 7, 12284 (2016)."), BAF (SS18 subunit)[55](https://www.nature.com/articles/s41556-020-00620-7#ref-CR55 "Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017).")|HEK293 (ref. [53](https://www.nature.com/articles/s41556-020-00620-7#ref-CR53 "Kim, J.-M. et al. Cooperation between SMYD3 and PC4 drives a distinct transcriptional program in cancer cells. Nucleic Acids Res. 43, 8868–8883 (2015).")), HEK293T[40](https://www.nature.com/articles/s41556-020-00620-7#ref-CR40 "Cano-Rodriguez, D. et al. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner. Nat. Commun. 7, 12284 (2016)."), A549 (ref. [40](https://www.nature.com/articles/s41556-020-00620-7#ref-CR40 "Cano-Rodriguez, D. et al. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner. Nat. Commun. 7, 12284 (2016).")), C33a[40](https://www.nature.com/articles/s41556-020-00620-7#ref-CR40 "Cano-Rodriguez, D. et al. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner. Nat. Commun. 7, 12284 (2016)."), HeLa[40](https://www.nature.com/articles/s41556-020-00620-7#ref-CR40 "Cano-Rodriguez, D. et al. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner. Nat. Commun. 7, 12284 (2016)."), mESCs[55](https://www.nature.com/articles/s41556-020-00620-7#ref-CR55 "Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017).")|_PLOD2_ (ref. [40](https://www.nature.com/articles/s41556-020-00620-7#ref-CR40 "Cano-Rodriguez, D. et al. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner. Nat. Commun. 7, 12284 (2016).")) (+ (DOT1L, PRDM9), 0 (UBE2A)) _EPCAM_[40](https://www.nature.com/articles/s41556-020-00620-7#ref-CR40 "Cano-Rodriguez, D. et al. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner. Nat. Commun. 7, 12284 (2016).") (+ (PRDM9), 0 (DOT1L) (++): _Nkx2.9_ (ref. [55](https://www.nature.com/articles/s41556-020-00620-7#ref-CR55 "Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017).")) (+): _FNBP1_ (ref. [53](https://www.nature.com/articles/s41556-020-00620-7#ref-CR53 "Kim, J.-M. et al. Cooperation between SMYD3 and PC4 drives a distinct transcriptional program in cancer cells. Nucleic Acids Res. 43, 8868–8883 (2015).")), _MFGE8_ (ref. [53](https://www.nature.com/articles/s41556-020-00620-7#ref-CR53 "Kim, J.-M. et al. Cooperation between SMYD3 and PC4 drives a distinct transcriptional program in cancer cells. Nucleic Acids Res. 43, 8868–8883 (2015).")), _Gata4_ (ref. [55](https://www.nature.com/articles/s41556-020-00620-7#ref-CR55 "Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017).")), _Nfatc1_ (ref. [55](https://www.nature.com/articles/s41556-020-00620-7#ref-CR55 "Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017).")), _Pcdh7_ (ref. [55](https://www.nature.com/articles/s41556-020-00620-7#ref-CR55 "Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017).")), _Prom1_ (ref. [55](https://www.nature.com/articles/s41556-020-00620-7#ref-CR55 "Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017).")) (0): _ICAM1_ (ref. [40](https://www.nature.com/articles/s41556-020-00620-7#ref-CR40 "Cano-Rodriguez, D. et al. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner. Nat. Commun. 7, 12284 (2016).")), _RASSF1A_[40](https://www.nature.com/articles/s41556-020-00620-7#ref-CR40 "Cano-Rodriguez, D. et al. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner. Nat. Commun. 7, 12284 (2016)."), _Ascl1_ (ref. [55](https://www.nature.com/articles/s41556-020-00620-7#ref-CR55 "Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017)."))|H3K4me (SMYD3 (ref. [53](https://www.nature.com/articles/s41556-020-00620-7#ref-CR53 "Kim, J.-M. et al. Cooperation between SMYD3 and PC4 drives a distinct transcriptional program in cancer cells. Nucleic Acids Res. 43, 8868–8883 (2015)."))), H3K4me3 (PRDM9 (ref. [40](https://www.nature.com/articles/s41556-020-00620-7#ref-CR40 "Cano-Rodriguez, D. et al. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner. Nat. Commun. 7, 12284 (2016).")), BAF[55](https://www.nature.com/articles/s41556-020-00620-7#ref-CR55 "Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017).")), H3K79me3 (DOT1L[40](https://www.nature.com/articles/s41556-020-00620-7#ref-CR40 "Cano-Rodriguez, D. et al. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner. Nat. Commun. 7, 12284 (2016).")), **H3K27me3** (BAF[55](https://www.nature.com/articles/s41556-020-00620-7#ref-CR55 "Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017)."))|PRDM9, DOT1L[40](https://www.nature.com/articles/s41556-020-00620-7#ref-CR40 "Cano-Rodriguez, D. et al. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner. Nat. Commun. 7, 12284 (2016)."): transient, 20 days; BAF[55](https://www.nature.com/articles/s41556-020-00620-7#ref-CR55 "Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017)."): transient|Larger expression changes and duration with small molecule[40](https://www.nature.com/articles/s41556-020-00620-7#ref-CR40 "Cano-Rodriguez, D. et al. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner. Nat. Commun. 7, 12284 (2016).")| |H3K9 and H3K27 methylation[45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015)."),[46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."),[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."),[54](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018)."),[55](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR55 "Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017)."),[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020).")|LSD1 (refs. [45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015)."),[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020).")), KRAB[45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015)."),[46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."),[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."),[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018)."),[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."), G9A[46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."), SUV39H1 (ref. [46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017).")) EZH2 (refs. [46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."),[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."),[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018).")), FOG1 (refs. [46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."),[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019).")), LSD1 + KRAB[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."), HP1 (ref. [55](https://www.nature.com/articles/s41556-020-00620-7#ref-CR55 "Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017)."))|mESCs[45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015)."),[55](https://www.nature.com/articles/s41556-020-00620-7#ref-CR55 "Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017)."), HCT116 (refs. [46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."),[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019).")), HEK293 (ref. [55](https://www.nature.com/articles/s41556-020-00620-7#ref-CR55 "Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017).")), HEK293T[46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."), K562 (ref. [56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020).")), mHSCs[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."), Hep3B[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018)."), LNCaP[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."), N2a[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."), 3T3 (ref. [49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."))|LSD1, KRAB: _Oct4_ (−, qualitative)[45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015)."), _Tbx3_ (−−)[45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015)."), _HBB_ (−)[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."), _HBE1_ (−)[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."), _HBG1/2_ (−)[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."); G9A, SUV39H1, EZH2 (ref. [46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017).")): _HER2_ (−), _MYC1_ (0), _EPCAM_ (0) KRAB: FP (−−)[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018)."), _HER2_ (−)[46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."), _MYC1_ (−)[46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."), _GRN_ (−)[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018)."), _Snurf_ (−)[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019).")_, EPCAM_ (0)[46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017).") FOG1 (ref. [46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017).")): _Snurf_ (−−)[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."), _HER2_ (−), _MYC1_ (−), _EPCAM_ (−); LSD1 + KRAB[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."): _HBB_ (−−), _HBE1_ (−−), _HBG1/2_ (−−), _TAL1_ (−), _Cebpa_ (−), _Spi1_ (−), _Gata1_ (−), _Gata2_ (−), _Runx1_ (−); HP1 (ref. [55](https://www.nature.com/articles/s41556-020-00620-7#ref-CR55 "Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017).")): _CXCR4_ (−−), _ASCL1_ (−), _NEUROD1_ (−), _Oct4_ (−); EZH2: FP (−−)[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018)."), _Snurf_ (−)[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."), _GRN_ (0)[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018).")|LSD1, KRAB: **H3K27ac**[45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015)."), H3K27ac[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."), DNA interactions[45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015)."), H3 occupancy[45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015)."); LSD1: **H3K4me2** (refs. [45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015)."),[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020).")), H3K9me3 (refs. [45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015)."),[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020).")), H3K27me3 (ref. [45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015).")); KRAB: H3K9me3 (refs. [45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015)."),[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018)."),[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020).")), H3K27me3 (ref. [45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015).")), DNA 5-mC[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018)."), HP1A binding[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018)."), **H3K4me2** (ref. [45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015).")) and **H3K4me3** (ref. [54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018).")), H3K27me3 (ref. [54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018).")); G9A[46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017).")**:** H3K9me3; SUV39H1 (ref. [46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017).")): H3K9me2, H3K9me3, H3K27me3; EZH2: H3K27me3 (refs. [46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."),[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018).")), **H3K4me3** (ref. [54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018).")), **H3K9me3** (ref. [54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018).")), H3K9me2 and H3K9me3 (ref. [46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017).")); DNA 5-mC[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018)."), HP1A binding[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018)."); FOG1 (ref. [46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017).")): H3K27me3, **H3K27ac**; LSD1, KRAB, LSD1 + KRAB[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."): **H3K4me1** and **H3K4me2**, **GATA1**/**TAL1** **binding**, H3K27me3, CTCF binding LSD1 + KRAB[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."): H3K9me3, **H3K27ac** HP1 (ref. [55](https://www.nature.com/articles/s41556-020-00620-7#ref-CR55 "Braun, S. M. G. et al. Rapid and reversible epigenome editing by endogenous chromatin regulators. Nat. Commun. 8, 560 (2017).")): H3K9me3|KRAB, FOG1: transient[46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."),[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."); EZH2: transient[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."), 14 days[46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017).")|dCas12a[46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."), gene expression off-target effects assessed[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020).")| |DNA methylation[26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."),[27](https://www.nature.com/articles/s41556-020-00620-7#ref-CR27 "Yamazaki, T. et al. Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase. PLoS ONE 12, e0177764 (2017)."),[46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."),[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."),[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018)."),[66](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR66 "Saunderson, E. A. et al. Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors. Nat. Commun. 8, 1450 (2017)."),[67](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR67 "Xiong, T. et al. Targeted DNA methylation in human cells using engineered dCas9-methyltransferases. Sci. Rep. 7, 6732 (2017)."),[68](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR68 "Galonska, C. et al. Genome-wide tracking of dCas9-methyltransferase footprints. Nat. Commun. 9, 597 (2018)."),[69](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR69 "Pflueger, C. et al. A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9–DNMT3A constructs. Genome Res. 28, 1193–1206 (2018)."),[70](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR70 "Lin, L. et al. Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases. Gigascience 7, 1–19 (2018)."),[71](https://www.nature.com/articles/s41556-020-00620-7#ref-CR71 "Hofacker, D. et al. Engineering of effector domains for targeted DNA methylation with reduced off-target effects. Int. J. Mol. Sci. 21, 502 (2020)."),[76](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR76 "Kantor, B. et al. Downregulation of SNCA expression by targeted editing of DNA methylation: a potential strategy for precision therapy in PD. Mol. Ther. 26, 2638–2649 (2018)."),[77](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR77 "Marx, N. et al. CRISPR-based targeted epigenetic editing enables gene expression modulation of the silenced beta-galactoside alpha-2,6-sialyltransferase 1 in CHO cells. Biotechnol. J. 13, 1700217 (2018)."),[78](https://www.nature.com/articles/s41556-020-00620-7#ref-CR78 "Mkannez, G. et al. DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease. Cardiovasc. Res. 114, 1525–1535 (2018)."),[80](https://www.nature.com/articles/s41556-020-00620-7#ref-CR80 "Ziller, M. J. et al. Dissecting the functional consequences of de novo DNA methylation dynamics in human motor neuron differentiation and physiology. Cell Stem Cell 22, 559–574.e9 (2018)."),[82](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR82 "McDonald, J. I. et al. Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation. Biol. Open 5, 866–874 (2016)."),[83](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR83 "Vojta, A. et al. Repurposing the CRISPR–Cas9 system for targeted DNA methylation. Nucleic Acids Res. 44, 5615–5628 (2016)."),[84](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016)."),[85](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR85 "Huang, Y.-H. et al. DNA epigenome editing using CRISPR–Cas SunTag-directed DNMT3A. Genome Biol. 18, 176 (2017)."),[86](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR86 "Stepper, P. et al. Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase. Nucleic Acids Res. 45, 1703–1713 (2017)."),[87](https://www.nature.com/articles/s41556-020-00620-7#ref-CR87 "Lei, Y. et al. Targeted DNA methylation in vivo using an engineered dCas9–MQ1 fusion protein. Nat. Commun. 8, 16026 (2017).")|DNMT3A[46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."),[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018)."),[68](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR68 "Galonska, C. et al. Genome-wide tracking of dCas9-methyltransferase footprints. Nat. Commun. 9, 597 (2018)."),[69](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR69 "Pflueger, C. et al. A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9–DNMT3A constructs. Genome Res. 28, 1193–1206 (2018)."),[70](https://www.nature.com/articles/s41556-020-00620-7#ref-CR70 "Lin, L. et al. Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases. Gigascience 7, 1–19 (2018)."),[76](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR76 "Kantor, B. et al. Downregulation of SNCA expression by targeted editing of DNA methylation: a potential strategy for precision therapy in PD. Mol. Ther. 26, 2638–2649 (2018)."),[77](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR77 "Marx, N. et al. CRISPR-based targeted epigenetic editing enables gene expression modulation of the silenced beta-galactoside alpha-2,6-sialyltransferase 1 in CHO cells. Biotechnol. J. 13, 1700217 (2018)."),[78](https://www.nature.com/articles/s41556-020-00620-7#ref-CR78 "Mkannez, G. et al. DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease. Cardiovasc. Res. 114, 1525–1535 (2018)."),[80](https://www.nature.com/articles/s41556-020-00620-7#ref-CR80 "Ziller, M. J. et al. Dissecting the functional consequences of de novo DNA methylation dynamics in human motor neuron differentiation and physiology. Cell Stem Cell 22, 559–574.e9 (2018)."),[82](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR82 "McDonald, J. I. et al. Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation. Biol. Open 5, 866–874 (2016)."),[83](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR83 "Vojta, A. et al. Repurposing the CRISPR–Cas9 system for targeted DNA methylation. Nucleic Acids Res. 44, 5615–5628 (2016)."),[84](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016)."),[85](https://www.nature.com/articles/s41556-020-00620-7#ref-CR85 "Huang, Y.-H. et al. DNA epigenome editing using CRISPR–Cas SunTag-directed DNMT3A. Genome Biol. 18, 176 (2017)."),[87](https://www.nature.com/articles/s41556-020-00620-7#ref-CR87 "Lei, Y. et al. Targeted DNA methylation in vivo using an engineered dCas9–MQ1 fusion protein. Nat. Commun. 8, 16026 (2017)."), DNMT3A + DNMT3L[66](https://www.nature.com/articles/s41556-020-00620-7#ref-CR66 "Saunderson, E. A. et al. Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors. Nat. Commun. 8, 1450 (2017)."),[71](https://www.nature.com/articles/s41556-020-00620-7#ref-CR71 "Hofacker, D. et al. Engineering of effector domains for targeted DNA methylation with reduced off-target effects. Int. J. Mol. Sci. 21, 502 (2020)."),[86](https://www.nature.com/articles/s41556-020-00620-7#ref-CR86 "Stepper, P. et al. Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase. Nucleic Acids Res. 45, 1703–1713 (2017)."), KRAB/EZH2/FOG1 + DNMT3A[46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."),[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."), KRAB + DNMT3A (+ DNMT3L)[26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."), _M.Sss1_ MQ1 (refs. [27](https://www.nature.com/articles/s41556-020-00620-7#ref-CR27 "Yamazaki, T. et al. Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase. PLoS ONE 12, e0177764 (2017)."),[67](https://www.nature.com/articles/s41556-020-00620-7#ref-CR67 "Xiong, T. et al. Targeted DNA methylation in human cells using engineered dCas9-methyltransferases. Sci. Rep. 7, 6732 (2017)."),[87](https://www.nature.com/articles/s41556-020-00620-7#ref-CR87 "Lei, Y. et al. Targeted DNA methylation in vivo using an engineered dCas9–MQ1 fusion protein. Nat. Commun. 8, 16026 (2017).")), DNMT1 (ref. [70](https://www.nature.com/articles/s41556-020-00620-7#ref-CR70 "Lin, L. et al. Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases. Gigascience 7, 1–19 (2018).")), DNMT3B[70](https://www.nature.com/articles/s41556-020-00620-7#ref-CR70 "Lin, L. et al. Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases. Gigascience 7, 1–19 (2018).")|K562 (refs. [26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."),[87](https://www.nature.com/articles/s41556-020-00620-7#ref-CR87 "Lei, Y. et al. Targeted DNA methylation in vivo using an engineered dCas9–MQ1 fusion protein. Nat. Commun. 8, 16026 (2017).")), HEK293 (refs. [71](https://www.nature.com/articles/s41556-020-00620-7#ref-CR71 "Hofacker, D. et al. Engineering of effector domains for targeted DNA methylation with reduced off-target effects. Int. J. Mol. Sci. 21, 502 (2020)."),[83](https://www.nature.com/articles/s41556-020-00620-7#ref-CR83 "Vojta, A. et al. Repurposing the CRISPR–Cas9 system for targeted DNA methylation. Nucleic Acids Res. 44, 5615–5628 (2016)."),[86](https://www.nature.com/articles/s41556-020-00620-7#ref-CR86 "Stepper, P. et al. Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase. Nucleic Acids Res. 45, 1703–1713 (2017).")), HEK293T[26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."),[46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."),[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018)."),[67](https://www.nature.com/articles/s41556-020-00620-7#ref-CR67 "Xiong, T. et al. Targeted DNA methylation in human cells using engineered dCas9-methyltransferases. Sci. Rep. 7, 6732 (2017)."),[68](https://www.nature.com/articles/s41556-020-00620-7#ref-CR68 "Galonska, C. et al. Genome-wide tracking of dCas9-methyltransferase footprints. Nat. Commun. 9, 597 (2018)."),[70](https://www.nature.com/articles/s41556-020-00620-7#ref-CR70 "Lin, L. et al. Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases. Gigascience 7, 1–19 (2018)."),[78](https://www.nature.com/articles/s41556-020-00620-7#ref-CR78 "Mkannez, G. et al. DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease. Cardiovasc. Res. 114, 1525–1535 (2018)."),[82](https://www.nature.com/articles/s41556-020-00620-7#ref-CR82 "McDonald, J. I. et al. Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation. Biol. Open 5, 866–874 (2016)."),[85](https://www.nature.com/articles/s41556-020-00620-7#ref-CR85 "Huang, Y.-H. et al. DNA epigenome editing using CRISPR–Cas SunTag-directed DNMT3A. Genome Biol. 18, 176 (2017)."),[87](https://www.nature.com/articles/s41556-020-00620-7#ref-CR87 "Lei, Y. et al. Targeted DNA methylation in vivo using an engineered dCas9–MQ1 fusion protein. Nat. Commun. 8, 16026 (2017)."), HeLa[69](https://www.nature.com/articles/s41556-020-00620-7#ref-CR69 "Pflueger, C. et al. A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9–DNMT3A constructs. Genome Res. 28, 1193–1206 (2018)."),[84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016)."), 1089 (ref. [66](https://www.nature.com/articles/s41556-020-00620-7#ref-CR66 "Saunderson, E. A. et al. Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors. Nat. Commun. 8, 1450 (2017).")), SKOV-3 (ref. [86](https://www.nature.com/articles/s41556-020-00620-7#ref-CR86 "Stepper, P. et al. Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase. Nucleic Acids Res. 45, 1703–1713 (2017).")), MCF7 (refs. [68](https://www.nature.com/articles/s41556-020-00620-7#ref-CR68 "Galonska, C. et al. Genome-wide tracking of dCas9-methyltransferase footprints. Nat. Commun. 9, 597 (2018)."),[69](https://www.nature.com/articles/s41556-020-00620-7#ref-CR69 "Pflueger, C. et al. A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9–DNMT3A constructs. Genome Res. 28, 1193–1206 (2018).")), HCT116 (refs. [46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."),[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019).")), LNCaP[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."), iPSC-derived neurons[76](https://www.nature.com/articles/s41556-020-00620-7#ref-CR76 "Kantor, B. et al. Downregulation of SNCA expression by targeted editing of DNA methylation: a potential strategy for precision therapy in PD. Mol. Ther. 26, 2638–2649 (2018)."), ESCs[80](https://www.nature.com/articles/s41556-020-00620-7#ref-CR80 "Ziller, M. J. et al. Dissecting the functional consequences of de novo DNA methylation dynamics in human motor neuron differentiation and physiology. Cell Stem Cell 22, 559–574.e9 (2018)."), myoepithelial cells[66](https://www.nature.com/articles/s41556-020-00620-7#ref-CR66 "Saunderson, E. A. et al. Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors. Nat. Commun. 8, 1450 (2017)."), VIC[78](https://www.nature.com/articles/s41556-020-00620-7#ref-CR78 "Mkannez, G. et al. DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease. Cardiovasc. Res. 114, 1525–1535 (2018)."), N2a[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."), 3T3 (ref. [49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019).")), mESCs[68](https://www.nature.com/articles/s41556-020-00620-7#ref-CR68 "Galonska, C. et al. Genome-wide tracking of dCas9-methyltransferase footprints. Nat. Commun. 9, 597 (2018)."),[84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016)."), 32D[82](https://www.nature.com/articles/s41556-020-00620-7#ref-CR82 "McDonald, J. I. et al. Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation. Biol. Open 5, 866–874 (2016)."), mouse tail[87](https://www.nature.com/articles/s41556-020-00620-7#ref-CR87 "Lei, Y. et al. Targeted DNA methylation in vivo using an engineered dCas9–MQ1 fusion protein. Nat. Commun. 8, 16026 (2017)."), mouse embryo[27](https://www.nature.com/articles/s41556-020-00620-7#ref-CR27 "Yamazaki, T. et al. Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase. PLoS ONE 12, e0177764 (2017)."), CHO[77](https://www.nature.com/articles/s41556-020-00620-7#ref-CR77 "Marx, N. et al. CRISPR-based targeted epigenetic editing enables gene expression modulation of the silenced beta-galactoside alpha-2,6-sialyltransferase 1 in CHO cells. Biotechnol. J. 13, 1700217 (2018).")|(+): _RUNX1_ (ref. [87](https://www.nature.com/articles/s41556-020-00620-7#ref-CR87 "Lei, Y. et al. Targeted DNA methylation in vivo using an engineered dCas9–MQ1 fusion protein. Nat. Commun. 8, 16026 (2017).")), _PAX6_ (ref. [80](https://www.nature.com/articles/s41556-020-00620-7#ref-CR80 "Ziller, M. J. et al. Dissecting the functional consequences of de novo DNA methylation dynamics in human motor neuron differentiation and physiology. Cell Stem Cell 22, 559–574.e9 (2018).")), _Nlrp12_ (ref. [84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016).")), _H2-Q10_ (ref. [84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016).")); (0): _VEGFA_[26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."), _CDKN2A_[82](https://www.nature.com/articles/s41556-020-00620-7#ref-CR82 "McDonald, J. I. et al. Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation. Biol. Open 5, 866–874 (2016)."), _ARF_[82](https://www.nature.com/articles/s41556-020-00620-7#ref-CR82 "McDonald, J. I. et al. Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation. Biol. Open 5, 866–874 (2016)."), _BACH2_ (ref. [83](https://www.nature.com/articles/s41556-020-00620-7#ref-CR83 "Vojta, A. et al. Repurposing the CRISPR–Cas9 system for targeted DNA methylation. Nucleic Acids Res. 44, 5615–5628 (2016).")), _KLF4_ (ref. [85](https://www.nature.com/articles/s41556-020-00620-7#ref-CR85 "Huang, Y.-H. et al. DNA epigenome editing using CRISPR–Cas SunTag-directed DNMT3A. Genome Biol. 18, 176 (2017).")), _HOXA5/11/13_ (ref. [87](https://www.nature.com/articles/s41556-020-00620-7#ref-CR87 "Lei, Y. et al. Targeted DNA methylation in vivo using an engineered dCas9–MQ1 fusion protein. Nat. Commun. 8, 16026 (2017).")), _HIC1_ (ref. [66](https://www.nature.com/articles/s41556-020-00620-7#ref-CR66 "Saunderson, E. A. et al. Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors. Nat. Commun. 8, 1450 (2017).")), _RASSF1B/C_[66](https://www.nature.com/articles/s41556-020-00620-7#ref-CR66 "Saunderson, E. A. et al. Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors. Nat. Commun. 8, 1450 (2017)."), _PTEN_[66](https://www.nature.com/articles/s41556-020-00620-7#ref-CR66 "Saunderson, E. A. et al. Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors. Nat. Commun. 8, 1450 (2017)."), _CXCR4_ (ref. [86](https://www.nature.com/articles/s41556-020-00620-7#ref-CR86 "Stepper, P. et al. Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase. Nucleic Acids Res. 45, 1703–1713 (2017).")), _SNCA_[76](https://www.nature.com/articles/s41556-020-00620-7#ref-CR76 "Kantor, B. et al. Downregulation of SNCA expression by targeted editing of DNA methylation: a potential strategy for precision therapy in PD. Mol. Ther. 26, 2638–2649 (2018)."), _PLPP3_ (ref. [78](https://www.nature.com/articles/s41556-020-00620-7#ref-CR78 "Mkannez, G. et al. DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease. Cardiovasc. Res. 114, 1525–1535 (2018).")) _TMEM206_ (ref. [69](https://www.nature.com/articles/s41556-020-00620-7#ref-CR69 "Pflueger, C. et al. A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9–DNMT3A constructs. Genome Res. 28, 1193–1206 (2018).")) _EPCAM_[46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."), _GRN_[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018)."), FP[78](https://www.nature.com/articles/s41556-020-00620-7#ref-CR78 "Mkannez, G. et al. DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease. Cardiovasc. Res. 114, 1525–1535 (2018)."); (−): _B2M_[26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."), _IFNAR1_ (ref. [26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016).")), _IL6ST_[83](https://www.nature.com/articles/s41556-020-00620-7#ref-CR83 "Vojta, A. et al. Repurposing the CRISPR–Cas9 system for targeted DNA methylation. Nucleic Acids Res. 44, 5615–5628 (2016)."), _HOXA5_ (ref. [85](https://www.nature.com/articles/s41556-020-00620-7#ref-CR85 "Huang, Y.-H. et al. DNA epigenome editing using CRISPR–Cas SunTag-directed DNMT3A. Genome Biol. 18, 176 (2017).")), _RASSF1A_[66](https://www.nature.com/articles/s41556-020-00620-7#ref-CR66 "Saunderson, E. A. et al. Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors. Nat. Commun. 8, 1450 (2017)."), _CDKN2A/p16INK4a/p14ARF_[66](https://www.nature.com/articles/s41556-020-00620-7#ref-CR66 "Saunderson, E. A. et al. Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors. Nat. Commun. 8, 1450 (2017)."), _TFRC_[86](https://www.nature.com/articles/s41556-020-00620-7#ref-CR86 "Stepper, P. et al. Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase. Nucleic Acids Res. 45, 1703–1713 (2017).")_, EPCAM_[86](https://www.nature.com/articles/s41556-020-00620-7#ref-CR86 "Stepper, P. et al. Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase. Nucleic Acids Res. 45, 1703–1713 (2017).")_, TGFBR3_ (ref. [70](https://www.nature.com/articles/s41556-020-00620-7#ref-CR70 "Lin, L. et al. Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases. Gigascience 7, 1–19 (2018).")), _HER2_ (refs. [46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."),[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019).")), _MYC_[46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."), _SNURF_[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."), _Cdkn1a_[82](https://www.nature.com/articles/s41556-020-00620-7#ref-CR82 "McDonald, J. I. et al. Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation. Biol. Open 5, 866–874 (2016)."), _ST6GAL1_ (ref. [77](https://www.nature.com/articles/s41556-020-00620-7#ref-CR77 "Marx, N. et al. CRISPR-based targeted epigenetic editing enables gene expression modulation of the silenced beta-galactoside alpha-2,6-sialyltransferase 1 in CHO cells. Biotechnol. J. 13, 1700217 (2018).")), FP[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018)."),[84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016)."); (−−): _UPA_[70](https://www.nature.com/articles/s41556-020-00620-7#ref-CR70 "Lin, L. et al. Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases. Gigascience 7, 1–19 (2018)."), _Snurf_[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019).");|DNA 5-mC[26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."),[27](https://www.nature.com/articles/s41556-020-00620-7#ref-CR27 "Yamazaki, T. et al. Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase. PLoS ONE 12, e0177764 (2017)."),[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."),[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018)."),[66](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR66 "Saunderson, E. A. et al. Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors. Nat. Commun. 8, 1450 (2017)."),[67](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR67 "Xiong, T. et al. Targeted DNA methylation in human cells using engineered dCas9-methyltransferases. Sci. Rep. 7, 6732 (2017)."),[68](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR68 "Galonska, C. et al. Genome-wide tracking of dCas9-methyltransferase footprints. Nat. Commun. 9, 597 (2018)."),[69](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR69 "Pflueger, C. et al. A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9–DNMT3A constructs. Genome Res. 28, 1193–1206 (2018)."),[70](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR70 "Lin, L. et al. Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases. Gigascience 7, 1–19 (2018)."),[71](https://www.nature.com/articles/s41556-020-00620-7#ref-CR71 "Hofacker, D. et al. Engineering of effector domains for targeted DNA methylation with reduced off-target effects. Int. J. Mol. Sci. 21, 502 (2020)."),[76](https://www.nature.com/articles/s41556-020-00620-7#ref-CR76 "Kantor, B. et al. Downregulation of SNCA expression by targeted editing of DNA methylation: a potential strategy for precision therapy in PD. Mol. Ther. 26, 2638–2649 (2018)."),[78](https://www.nature.com/articles/s41556-020-00620-7#ref-CR78 "Mkannez, G. et al. DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease. Cardiovasc. Res. 114, 1525–1535 (2018)."),[80](https://www.nature.com/articles/s41556-020-00620-7#ref-CR80 "Ziller, M. J. et al. Dissecting the functional consequences of de novo DNA methylation dynamics in human motor neuron differentiation and physiology. Cell Stem Cell 22, 559–574.e9 (2018)."),[82](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR82 "McDonald, J. I. et al. Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation. Biol. Open 5, 866–874 (2016)."),[83](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR83 "Vojta, A. et al. Repurposing the CRISPR–Cas9 system for targeted DNA methylation. Nucleic Acids Res. 44, 5615–5628 (2016)."),[84](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016)."),[85](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR85 "Huang, Y.-H. et al. DNA epigenome editing using CRISPR–Cas SunTag-directed DNMT3A. Genome Biol. 18, 176 (2017)."),[86](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR86 "Stepper, P. et al. Efficient targeted DNA methylation with chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase. Nucleic Acids Res. 45, 1703–1713 (2017)."),[87](https://www.nature.com/articles/s41556-020-00620-7#ref-CR87 "Lei, Y. et al. Targeted DNA methylation in vivo using an engineered dCas9–MQ1 fusion protein. Nat. Commun. 8, 16026 (2017)."), other DNA methylations[85](https://www.nature.com/articles/s41556-020-00620-7#ref-CR85 "Huang, Y.-H. et al. DNA epigenome editing using CRISPR–Cas SunTag-directed DNMT3A. Genome Biol. 18, 176 (2017)."), DNA interactions[84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016)."), heterochromatin formation[27](https://www.nature.com/articles/s41556-020-00620-7#ref-CR27 "Yamazaki, T. et al. Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase. PLoS ONE 12, e0177764 (2017)."), **CTCF binding**[69](https://www.nature.com/articles/s41556-020-00620-7#ref-CR69 "Pflueger, C. et al. A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9–DNMT3A constructs. Genome Res. 28, 1193–1206 (2018)."),[84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016)."),[87](https://www.nature.com/articles/s41556-020-00620-7#ref-CR87 "Lei, Y. et al. Targeted DNA methylation in vivo using an engineered dCas9–MQ1 fusion protein. Nat. Commun. 8, 16026 (2017)."), **TF binding**[69](https://www.nature.com/articles/s41556-020-00620-7#ref-CR69 "Pflueger, C. et al. A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9–DNMT3A constructs. Genome Res. 28, 1193–1206 (2018)."),[87](https://www.nature.com/articles/s41556-020-00620-7#ref-CR87 "Lei, Y. et al. Targeted DNA methylation in vivo using an engineered dCas9–MQ1 fusion protein. Nat. Commun. 8, 16026 (2017)."), H3K4me3 (ref. [54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018).")), H3K9me3 (ref. [54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018).")), H3K27me3 (ref. [54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018).")), HP1A binding[54](https://www.nature.com/articles/s41556-020-00620-7#ref-CR54 "Wang, H. et al. Epigenetic targeting of Granulin in hepatoma cells by synthetic CRISPR dCas9 Epi-suppressors. Mol. Ther. Nucleic Acids 11, 23–33 (2018)."); H3K27me3, **H3K27ac** (with EZH2 (ref. [49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."))); H3K9me3 (with KRAB[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019).")); DNA 5-mC (DNMT1 (ref. [70](https://www.nature.com/articles/s41556-020-00620-7#ref-CR70 "Lin, L. et al. Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases. Gigascience 7, 1–19 (2018).")))|Transient[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."), 3 days (for off-target methylation)[68](https://www.nature.com/articles/s41556-020-00620-7#ref-CR68 "Galonska, C. et al. Genome-wide tracking of dCas9-methyltransferase footprints. Nat. Commun. 9, 597 (2018)."), 7 days (21 d in vivo)[87](https://www.nature.com/articles/s41556-020-00620-7#ref-CR87 "Lei, Y. et al. Targeted DNA methylation in vivo using an engineered dCas9–MQ1 fusion protein. Nat. Commun. 8, 16026 (2017)."), 8 days[70](https://www.nature.com/articles/s41556-020-00620-7#ref-CR70 "Lin, L. et al. Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases. Gigascience 7, 1–19 (2018)."), 10 days[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."),[82](https://www.nature.com/articles/s41556-020-00620-7#ref-CR82 "McDonald, J. I. et al. Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation. Biol. Open 5, 866–874 (2016)."),[83](https://www.nature.com/articles/s41556-020-00620-7#ref-CR83 "Vojta, A. et al. Repurposing the CRISPR–Cas9 system for targeted DNA methylation. Nucleic Acids Res. 44, 5615–5628 (2016)."), 14 days[46](https://www.nature.com/articles/s41556-020-00620-7#ref-CR46 "O’Geen, H. et al. dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression. Nucleic Acids Res. 45, 9901–9916 (2017)."), 24 days[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."), 30 days[26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."), 40 days[66](https://www.nature.com/articles/s41556-020-00620-7#ref-CR66 "Saunderson, E. A. et al. Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors. Nat. Commun. 8, 1450 (2017)."), 50 days[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019).")|Gene expression[26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."),[70](https://www.nature.com/articles/s41556-020-00620-7#ref-CR70 "Lin, L. et al. Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases. Gigascience 7, 1–19 (2018)."),[76](https://www.nature.com/articles/s41556-020-00620-7#ref-CR76 "Kantor, B. et al. Downregulation of SNCA expression by targeted editing of DNA methylation: a potential strategy for precision therapy in PD. Mol. Ther. 26, 2638–2649 (2018).") and epigenetic[26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."),[49](https://www.nature.com/articles/s41556-020-00620-7#ref-CR49 "O’Geen, H. et al. Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner. Epigenetics Chromatin 12, 26 (2019)."),[66](https://www.nature.com/articles/s41556-020-00620-7#ref-CR66 "Saunderson, E. A. et al. Hit-and-run epigenetic editing prevents senescence entry in primary breast cells from healthy donors. Nat. Commun. 8, 1450 (2017)."),[68](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR68 "Galonska, C. et al. Genome-wide tracking of dCas9-methyltransferase footprints. Nat. Commun. 9, 597 (2018)."),[69](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR69 "Pflueger, C. et al. A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9–DNMT3A constructs. Genome Res. 28, 1193–1206 (2018)."),[70](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR70 "Lin, L. et al. Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases. Gigascience 7, 1–19 (2018)."),[71](https://www.nature.com/articles/s41556-020-00620-7#ref-CR71 "Hofacker, D. et al. Engineering of effector domains for targeted DNA methylation with reduced off-target effects. Int. J. Mol. Sci. 21, 502 (2020)."),[76](https://www.nature.com/articles/s41556-020-00620-7#ref-CR76 "Kantor, B. et al. Downregulation of SNCA expression by targeted editing of DNA methylation: a potential strategy for precision therapy in PD. Mol. Ther. 26, 2638–2649 (2018)."),[80](https://www.nature.com/articles/s41556-020-00620-7#ref-CR80 "Ziller, M. J. et al. Dissecting the functional consequences of de novo DNA methylation dynamics in human motor neuron differentiation and physiology. Cell Stem Cell 22, 559–574.e9 (2018)."),[83](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR83 "Vojta, A. et al. Repurposing the CRISPR–Cas9 system for targeted DNA methylation. Nucleic Acids Res. 44, 5615–5628 (2016)."),[84](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016)."),[85](https://www.nature.com/articles/s41556-020-00620-7#ref-CR85 "Huang, Y.-H. et al. DNA epigenome editing using CRISPR–Cas SunTag-directed DNMT3A. Genome Biol. 18, 176 (2017)."),[87](https://www.nature.com/articles/s41556-020-00620-7#ref-CR87 "Lei, Y. et al. Targeted DNA methylation in vivo using an engineered dCas9–MQ1 fusion protein. Nat. Commun. 8, 16026 (2017).") off-target effects assessed; compared DNMT3A isoforms[85](https://www.nature.com/articles/s41556-020-00620-7#ref-CR85 "Huang, Y.-H. et al. DNA epigenome editing using CRISPR–Cas SunTag-directed DNMT3A. Genome Biol. 18, 176 (2017).")| |DNA demethylation[26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."),[63](https://www.nature.com/articles/s41556-020-00620-7#ref-CR63 "Okada, M., Kanamori, M., Someya, K., Nakatsukasa, H. & Yoshimura, A. Stabilization of Foxp3 expression by CRISPR–dCas9-based epigenome editing in mouse primary T cells. Epigenetics Chromatin 10, 24 (2017)."),[69](https://www.nature.com/articles/s41556-020-00620-7#ref-CR69 "Pflueger, C. et al. A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9–DNMT3A constructs. Genome Res. 28, 1193–1206 (2018)."),[72](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR72 "Xu, X. et al. A CRISPR-based approach for targeted DNA demethylation. Cell Discov. 2, 16009 (2016)."),[73](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR73 "Choudhury, S. R., Cui, Y., Lubecka, K., Stefanska, B. & Irudayaraj, J. CRISPR–dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter. Oncotarget 7, 46545–46556 (2016)."),[74](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR74 "Morita, S. et al. Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat. Biotechnol. 34, 1060–1065 (2016)."),[75](https://www.nature.com/articles/s41556-020-00620-7#ref-CR75 "Liu, X. S. et al. Rescue of fragile X syndrome neurons by DNA methylation editing of the FMR1 gene. Cell 172, 979–992.e6 (2018)."),[77](https://www.nature.com/articles/s41556-020-00620-7#ref-CR77 "Marx, N. et al. CRISPR-based targeted epigenetic editing enables gene expression modulation of the silenced beta-galactoside alpha-2,6-sialyltransferase 1 in CHO cells. Biotechnol. J. 13, 1700217 (2018)."),[79](https://www.nature.com/articles/s41556-020-00620-7#ref-CR79 "Xu, X. et al. High-fidelity CRISPR/Cas9-based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis. Nat. Commun. 9, 3509 (2018)."),[81](https://www.nature.com/articles/s41556-020-00620-7#ref-CR81 "Baumann, V. et al. Targeted removal of epigenetic barriers during transcriptional reprogramming. Nat. Commun. 10, 2119 (2019)."),[84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016).")|TET1 (refs. [26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."),[63](https://www.nature.com/articles/s41556-020-00620-7#ref-CR63 "Okada, M., Kanamori, M., Someya, K., Nakatsukasa, H. & Yoshimura, A. Stabilization of Foxp3 expression by CRISPR–dCas9-based epigenome editing in mouse primary T cells. Epigenetics Chromatin 10, 24 (2017)."),[69](https://www.nature.com/articles/s41556-020-00620-7#ref-CR69 "Pflueger, C. et al. A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9–DNMT3A constructs. Genome Res. 28, 1193–1206 (2018)."),[72](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR72 "Xu, X. et al. A CRISPR-based approach for targeted DNA demethylation. Cell Discov. 2, 16009 (2016)."),[73](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR73 "Choudhury, S. R., Cui, Y., Lubecka, K., Stefanska, B. & Irudayaraj, J. CRISPR–dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter. Oncotarget 7, 46545–46556 (2016)."),[74](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR74 "Morita, S. et al. Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat. Biotechnol. 34, 1060–1065 (2016)."),[75](https://www.nature.com/articles/s41556-020-00620-7#ref-CR75 "Liu, X. S. et al. Rescue of fragile X syndrome neurons by DNA methylation editing of the FMR1 gene. Cell 172, 979–992.e6 (2018)."),[77](https://www.nature.com/articles/s41556-020-00620-7#ref-CR77 "Marx, N. et al. CRISPR-based targeted epigenetic editing enables gene expression modulation of the silenced beta-galactoside alpha-2,6-sialyltransferase 1 in CHO cells. Biotechnol. J. 13, 1700217 (2018)."),[84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016).")), TET3 (ref. [79](https://www.nature.com/articles/s41556-020-00620-7#ref-CR79 "Xu, X. et al. High-fidelity CRISPR/Cas9-based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis. Nat. Commun. 9, 3509 (2018).")), CRISPRa + TET1 (ref. [81](https://www.nature.com/articles/s41556-020-00620-7#ref-CR81 "Baumann, V. et al. Targeted removal of epigenetic barriers during transcriptional reprogramming. Nat. Commun. 10, 2119 (2019)."))|K562 (refs. [26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016).")), HeLa[69](https://www.nature.com/articles/s41556-020-00620-7#ref-CR69 "Pflueger, C. et al. A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9–DNMT3A constructs. Genome Res. 28, 1193–1206 (2018)."),[72](https://www.nature.com/articles/s41556-020-00620-7#ref-CR72 "Xu, X. et al. A CRISPR-based approach for targeted DNA demethylation. Cell Discov. 2, 16009 (2016)."),[73](https://www.nature.com/articles/s41556-020-00620-7#ref-CR73 "Choudhury, S. R., Cui, Y., Lubecka, K., Stefanska, B. & Irudayaraj, J. CRISPR–dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter. Oncotarget 7, 46545–46556 (2016)."), MCF7 (ref. [73](https://www.nature.com/articles/s41556-020-00620-7#ref-CR73 "Choudhury, S. R., Cui, Y., Lubecka, K., Stefanska, B. & Irudayaraj, J. CRISPR–dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter. Oncotarget 7, 46545–46556 (2016).")), HEK293T[74](https://www.nature.com/articles/s41556-020-00620-7#ref-CR74 "Morita, S. et al. Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat. Biotechnol. 34, 1060–1065 (2016)."),[84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016)."), HEK293FT[72](https://www.nature.com/articles/s41556-020-00620-7#ref-CR72 "Xu, X. et al. A CRISPR-based approach for targeted DNA demethylation. Cell Discov. 2, 16009 (2016)."), SH-SY5Y[72](https://www.nature.com/articles/s41556-020-00620-7#ref-CR72 "Xu, X. et al. A CRISPR-based approach for targeted DNA demethylation. Cell Discov. 2, 16009 (2016)."), TK188 (ref. [79](https://www.nature.com/articles/s41556-020-00620-7#ref-CR79 "Xu, X. et al. High-fidelity CRISPR/Cas9-based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis. Nat. Commun. 9, 3509 (2018).")), HK2 (ref. [79](https://www.nature.com/articles/s41556-020-00620-7#ref-CR79 "Xu, X. et al. High-fidelity CRISPR/Cas9-based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis. Nat. Commun. 9, 3509 (2018).")), iPSCs (and derived neurons)[75](https://www.nature.com/articles/s41556-020-00620-7#ref-CR75 "Liu, X. S. et al. Rescue of fragile X syndrome neurons by DNA methylation editing of the FMR1 gene. Cell 172, 979–992.e6 (2018)."), 68-41 T cells[63](https://www.nature.com/articles/s41556-020-00620-7#ref-CR63 "Okada, M., Kanamori, M., Someya, K., Nakatsukasa, H. & Yoshimura, A. Stabilization of Foxp3 expression by CRISPR–dCas9-based epigenome editing in mouse primary T cells. Epigenetics Chromatin 10, 24 (2017)."), MCT[79](https://www.nature.com/articles/s41556-020-00620-7#ref-CR79 "Xu, X. et al. High-fidelity CRISPR/Cas9-based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis. Nat. Commun. 9, 3509 (2018)."), primary mouse T cells[63](https://www.nature.com/articles/s41556-020-00620-7#ref-CR63 "Okada, M., Kanamori, M., Someya, K., Nakatsukasa, H. & Yoshimura, A. Stabilization of Foxp3 expression by CRISPR–dCas9-based epigenome editing in mouse primary T cells. Epigenetics Chromatin 10, 24 (2017)."), mESCs[74](https://www.nature.com/articles/s41556-020-00620-7#ref-CR74 "Morita, S. et al. Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat. Biotechnol. 34, 1060–1065 (2016)."),[84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016)."), mouse neurons/fibroblasts/epidermis[84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016)."), C3H10T1/2[84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016)."), mNPC[74](https://www.nature.com/articles/s41556-020-00620-7#ref-CR74 "Morita, S. et al. Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat. Biotechnol. 34, 1060–1065 (2016)."),[81](https://www.nature.com/articles/s41556-020-00620-7#ref-CR81 "Baumann, V. et al. Targeted removal of epigenetic barriers during transcriptional reprogramming. Nat. Commun. 10, 2119 (2019)."), mouse brain[74](https://www.nature.com/articles/s41556-020-00620-7#ref-CR74 "Morita, S. et al. Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat. Biotechnol. 34, 1060–1065 (2016)."),[84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016).")/kidney fibroblasts[79](https://www.nature.com/articles/s41556-020-00620-7#ref-CR79 "Xu, X. et al. High-fidelity CRISPR/Cas9-based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis. Nat. Commun. 9, 3509 (2018)."), CHO[77](https://www.nature.com/articles/s41556-020-00620-7#ref-CR77 "Marx, N. et al. CRISPR-based targeted epigenetic editing enables gene expression modulation of the silenced beta-galactoside alpha-2,6-sialyltransferase 1 in CHO cells. Biotechnol. J. 13, 1700217 (2018).")|(+++): _SH3BP2_ (ref. [74](https://www.nature.com/articles/s41556-020-00620-7#ref-CR74 "Morita, S. et al. Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat. Biotechnol. 34, 1060–1065 (2016).")), _MAGEB2_ (ref. [72](https://www.nature.com/articles/s41556-020-00620-7#ref-CR72 "Xu, X. et al. A CRISPR-based approach for targeted DNA demethylation. Cell Discov. 2, 16009 (2016).")), _FMR1_ (ref. [75](https://www.nature.com/articles/s41556-020-00620-7#ref-CR75 "Liu, X. S. et al. Rescue of fragile X syndrome neurons by DNA methylation editing of the FMR1 gene. Cell 172, 979–992.e6 (2018).")), _ST6GAL1_ (ref. [77](https://www.nature.com/articles/s41556-020-00620-7#ref-CR77 "Marx, N. et al. CRISPR-based targeted epigenetic editing enables gene expression modulation of the silenced beta-galactoside alpha-2,6-sialyltransferase 1 in CHO cells. Biotechnol. J. 13, 1700217 (2018).")); (++): _RHOXF2B_[74](https://www.nature.com/articles/s41556-020-00620-7#ref-CR74 "Morita, S. et al. Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat. Biotechnol. 34, 1060–1065 (2016)."), _CARD9_ (ref. [74](https://www.nature.com/articles/s41556-020-00620-7#ref-CR74 "Morita, S. et al. Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat. Biotechnol. 34, 1060–1065 (2016).")), _CNKSR1_ (ref. [74](https://www.nature.com/articles/s41556-020-00620-7#ref-CR74 "Morita, S. et al. Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat. Biotechnol. 34, 1060–1065 (2016).")), _RANKL_[72](https://www.nature.com/articles/s41556-020-00620-7#ref-CR72 "Xu, X. et al. A CRISPR-based approach for targeted DNA demethylation. Cell Discov. 2, 16009 (2016)."); (+): _B2M_[26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."), _BRCA1_ (ref. [73](https://www.nature.com/articles/s41556-020-00620-7#ref-CR73 "Choudhury, S. R., Cui, Y., Lubecka, K., Stefanska, B. & Irudayaraj, J. CRISPR–dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter. Oncotarget 7, 46545–46556 (2016).")), _MMP2_ (ref. [72](https://www.nature.com/articles/s41556-020-00620-7#ref-CR72 "Xu, X. et al. A CRISPR-based approach for targeted DNA demethylation. Cell Discov. 2, 16009 (2016).")), _RASAL1_ (ref. [79](https://www.nature.com/articles/s41556-020-00620-7#ref-CR79 "Xu, X. et al. High-fidelity CRISPR/Cas9-based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis. Nat. Commun. 9, 3509 (2018).")), _EYA1_ (ref. [79](https://www.nature.com/articles/s41556-020-00620-7#ref-CR79 "Xu, X. et al. High-fidelity CRISPR/Cas9-based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis. Nat. Commun. 9, 3509 (2018).")), _LRFN2_ (ref. [79](https://www.nature.com/articles/s41556-020-00620-7#ref-CR79 "Xu, X. et al. High-fidelity CRISPR/Cas9-based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis. Nat. Commun. 9, 3509 (2018).")), _KL_[79](https://www.nature.com/articles/s41556-020-00620-7#ref-CR79 "Xu, X. et al. High-fidelity CRISPR/Cas9-based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis. Nat. Commun. 9, 3509 (2018)."), _Rasal1_ (ref. [79](https://www.nature.com/articles/s41556-020-00620-7#ref-CR79 "Xu, X. et al. High-fidelity CRISPR/Cas9-based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis. Nat. Commun. 9, 3509 (2018).")), _Klotho_[79](https://www.nature.com/articles/s41556-020-00620-7#ref-CR79 "Xu, X. et al. High-fidelity CRISPR/Cas9-based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis. Nat. Commun. 9, 3509 (2018)."), _Bdnf_[84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016)."), _Myod1_ (ref. [84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016).")), _Gfap_[74](https://www.nature.com/articles/s41556-020-00620-7#ref-CR74 "Morita, S. et al. Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat. Biotechnol. 34, 1060–1065 (2016)."), _Sox1_ (ref. [81](https://www.nature.com/articles/s41556-020-00620-7#ref-CR81 "Baumann, V. et al. Targeted removal of epigenetic barriers during transcriptional reprogramming. Nat. Commun. 10, 2119 (2019).")), FP[84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016)."); (0): _Foxp3_ (ref. [63](https://www.nature.com/articles/s41556-020-00620-7#ref-CR63 "Okada, M., Kanamori, M., Someya, K., Nakatsukasa, H. & Yoshimura, A. Stabilization of Foxp3 expression by CRISPR–dCas9-based epigenome editing in mouse primary T cells. Epigenetics Chromatin 10, 24 (2017).")), _H19_ (ref. [74](https://www.nature.com/articles/s41556-020-00620-7#ref-CR74 "Morita, S. et al. Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat. Biotechnol. 34, 1060–1065 (2016)."))|**DNA 5-mC**[26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."),[63](https://www.nature.com/articles/s41556-020-00620-7#ref-CR63 "Okada, M., Kanamori, M., Someya, K., Nakatsukasa, H. & Yoshimura, A. Stabilization of Foxp3 expression by CRISPR–dCas9-based epigenome editing in mouse primary T cells. Epigenetics Chromatin 10, 24 (2017)."),[69](https://www.nature.com/articles/s41556-020-00620-7#ref-CR69 "Pflueger, C. et al. A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9–DNMT3A constructs. Genome Res. 28, 1193–1206 (2018)."),[72](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR72 "Xu, X. et al. A CRISPR-based approach for targeted DNA demethylation. Cell Discov. 2, 16009 (2016)."),[73](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR73 "Choudhury, S. R., Cui, Y., Lubecka, K., Stefanska, B. & Irudayaraj, J. CRISPR–dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter. Oncotarget 7, 46545–46556 (2016)."),[74](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR74 "Morita, S. et al. Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat. Biotechnol. 34, 1060–1065 (2016)."),[75](https://www.nature.com/articles/s41556-020-00620-7#ref-CR75 "Liu, X. S. et al. Rescue of fragile X syndrome neurons by DNA methylation editing of the FMR1 gene. Cell 172, 979–992.e6 (2018)."),[79](https://www.nature.com/articles/s41556-020-00620-7#ref-CR79 "Xu, X. et al. High-fidelity CRISPR/Cas9-based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis. Nat. Commun. 9, 3509 (2018)."),[81](https://www.nature.com/articles/s41556-020-00620-7#ref-CR81 "Baumann, V. et al. Targeted removal of epigenetic barriers during transcriptional reprogramming. Nat. Commun. 10, 2119 (2019)."),[84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016)."), **H3K9me3** (ref. [75](https://www.nature.com/articles/s41556-020-00620-7#ref-CR75 "Liu, X. S. et al. Rescue of fragile X syndrome neurons by DNA methylation editing of the FMR1 gene. Cell 172, 979–992.e6 (2018).")), DNA 5-hmC[73](https://www.nature.com/articles/s41556-020-00620-7#ref-CR73 "Choudhury, S. R., Cui, Y., Lubecka, K., Stefanska, B. & Irudayaraj, J. CRISPR–dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter. Oncotarget 7, 46545–46556 (2016)."),[79](https://www.nature.com/articles/s41556-020-00620-7#ref-CR79 "Xu, X. et al. High-fidelity CRISPR/Cas9-based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis. Nat. Commun. 9, 3509 (2018)."),[84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016)."), H3K4me3 (ref. [75](https://www.nature.com/articles/s41556-020-00620-7#ref-CR75 "Liu, X. S. et al. Rescue of fragile X syndrome neurons by DNA methylation editing of the FMR1 gene. Cell 172, 979–992.e6 (2018).")), H3K27ac[75](https://www.nature.com/articles/s41556-020-00620-7#ref-CR75 "Liu, X. S. et al. Rescue of fragile X syndrome neurons by DNA methylation editing of the FMR1 gene. Cell 172, 979–992.e6 (2018).")|4 days[72](https://www.nature.com/articles/s41556-020-00620-7#ref-CR72 "Xu, X. et al. A CRISPR-based approach for targeted DNA demethylation. Cell Discov. 2, 16009 (2016)."), 13 days[75](https://www.nature.com/articles/s41556-020-00620-7#ref-CR75 "Liu, X. S. et al. Rescue of fragile X syndrome neurons by DNA methylation editing of the FMR1 gene. Cell 172, 979–992.e6 (2018)."), 26 days[26](https://www.nature.com/articles/s41556-020-00620-7#ref-CR26 "Amabile, A. et al. Inheritable silencing of endogenous genes by hit-and-run targeted epigenetic editing. Cell 167, 219–232.e14 (2016)."), 100 days[77](https://www.nature.com/articles/s41556-020-00620-7#ref-CR77 "Marx, N. et al. CRISPR-based targeted epigenetic editing enables gene expression modulation of the silenced beta-galactoside alpha-2,6-sialyltransferase 1 in CHO cells. Biotechnol. J. 13, 1700217 (2018).")|Gene expression[72](https://www.nature.com/articles/s41556-020-00620-7#ref-CR72 "Xu, X. et al. A CRISPR-based approach for targeted DNA demethylation. Cell Discov. 2, 16009 (2016)."),[74](https://www.nature.com/articles/s41556-020-00620-7#ref-CR74 "Morita, S. et al. Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat. Biotechnol. 34, 1060–1065 (2016).") and demethylation[69](https://www.nature.com/articles/s41556-020-00620-7#ref-CR69 "Pflueger, C. et al. A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9–DNMT3A constructs. Genome Res. 28, 1193–1206 (2018)."),[74](https://www.nature.com/articles/s41556-020-00620-7#ref-CR74 "Morita, S. et al. Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat. Biotechnol. 34, 1060–1065 (2016)."),[84](https://www.nature.com/articles/s41556-020-00620-7#ref-CR84 "Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016).") off-target effects assessed| |Other effector characterization[31](https://www.nature.com/articles/s41556-020-00620-7#ref-CR31 "Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR–Cas9 complex. Nature 517, 583–588 (2015)."),[38](https://www.nature.com/articles/s41556-020-00620-7#ref-CR38 "Polstein, L. R. et al. Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators. Genome Res. 25, 1158–1169 (2015)."),[58](https://www.nature.com/articles/s41556-020-00620-7#ref-CR58 "Chiarella, A. M. et al. Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery. Nat. Biotechnol. 38, 50–55 (2020)."),[93](https://www.nature.com/articles/s41556-020-00620-7#ref-CR93 "Barkal, A. A., Srinivasan, S., Hashimoto, T., Gifford, D. K. & Sherwood, R. I. Cas9 functionally opens chromatin. PLoS ONE 11, e0152683 (2016).")|BRD4 (ref. [58](https://www.nature.com/articles/s41556-020-00620-7#ref-CR58 "Chiarella, A. M. et al. Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery. Nat. Biotechnol. 38, 50–55 (2020).")), BRPF1 (ref. [58](https://www.nature.com/articles/s41556-020-00620-7#ref-CR58 "Chiarella, A. M. et al. Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery. Nat. Biotechnol. 38, 50–55 (2020).")), CRISPRa[31](https://www.nature.com/articles/s41556-020-00620-7#ref-CR31 "Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR–Cas9 complex. Nature 517, 583–588 (2015)."),[38](https://www.nature.com/articles/s41556-020-00620-7#ref-CR38 "Polstein, L. R. et al. Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators. Genome Res. 25, 1158–1169 (2015)."), dCas[93](https://www.nature.com/articles/s41556-020-00620-7#ref-CR93 "Barkal, A. A., Srinivasan, S., Hashimoto, T., Gifford, D. K. & Sherwood, R. I. Cas9 functionally opens chromatin. PLoS ONE 11, e0152683 (2016).")|HEK293T[38](https://www.nature.com/articles/s41556-020-00620-7#ref-CR38 "Polstein, L. R. et al. Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators. Genome Res. 25, 1158–1169 (2015)."),[58](https://www.nature.com/articles/s41556-020-00620-7#ref-CR58 "Chiarella, A. M. et al. Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery. Nat. Biotechnol. 38, 50–55 (2020)."), HEK293FT[31](https://www.nature.com/articles/s41556-020-00620-7#ref-CR31 "Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR–Cas9 complex. Nature 517, 583–588 (2015)."), HCT116 (ref. [58](https://www.nature.com/articles/s41556-020-00620-7#ref-CR58 "Chiarella, A. M. et al. Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery. Nat. Biotechnol. 38, 50–55 (2020).")), mESCs[93](https://www.nature.com/articles/s41556-020-00620-7#ref-CR93 "Barkal, A. A., Srinivasan, S., Hashimoto, T., Gifford, D. K. & Sherwood, R. I. Cas9 functionally opens chromatin. PLoS ONE 11, e0152683 (2016).")|BRD4 (ref. [58](https://www.nature.com/articles/s41556-020-00620-7#ref-CR58 "Chiarella, A. M. et al. Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery. Nat. Biotechnol. 38, 50–55 (2020).")): _MYOD1_ (++), _CXCR4_ (+), _IL1RN_ (+++), _OCT4_ (+), _MYC1_ (0), FP (++); BRPF1 (ref. [58](https://www.nature.com/articles/s41556-020-00620-7#ref-CR58 "Chiarella, A. M. et al. Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery. Nat. Biotechnol. 38, 50–55 (2020).")): _MYOD1_ (0), FP (+)|BRD4 relocalization[58](https://www.nature.com/articles/s41556-020-00620-7#ref-CR58 "Chiarella, A. M. et al. Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery. Nat. Biotechnol. 38, 50–55 (2020)."), DNA accessibility[38](https://www.nature.com/articles/s41556-020-00620-7#ref-CR38 "Polstein, L. R. et al. Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators. Genome Res. 25, 1158–1169 (2015)."),[93](https://www.nature.com/articles/s41556-020-00620-7#ref-CR93 "Barkal, A. A., Srinivasan, S., Hashimoto, T., Gifford, D. K. & Sherwood, R. I. Cas9 functionally opens chromatin. PLoS ONE 11, e0152683 (2016)."), long ncRNA expression[31](https://www.nature.com/articles/s41556-020-00620-7#ref-CR31 "Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR–Cas9 complex. Nature 517, 583–588 (2015)."), RAR binding[93](https://www.nature.com/articles/s41556-020-00620-7#ref-CR93 "Barkal, A. A., Srinivasan, S., Hashimoto, T., Gifford, D. K. & Sherwood, R. I. Cas9 functionally opens chromatin. PLoS ONE 11, e0152683 (2016).")|4 days (BRD4)[58](https://www.nature.com/articles/s41556-020-00620-7#ref-CR58 "Chiarella, A. M. et al. Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery. Nat. Biotechnol. 38, 50–55 (2020).")|DNA accessibility changes with dCas binding alone[38](https://www.nature.com/articles/s41556-020-00620-7#ref-CR38 "Polstein, L. R. et al. Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators. Genome Res. 25, 1158–1169 (2015)."),[93](https://www.nature.com/articles/s41556-020-00620-7#ref-CR93 "Barkal, A. A., Srinivasan, S., Hashimoto, T., Gifford, D. K. & Sherwood, R. I. Cas9 functionally opens chromatin. PLoS ONE 11, e0152683 (2016).")| |**Noncoding elements**| | | | | | | |ncRNA characterization[36](https://www.nature.com/articles/s41556-020-00620-7#ref-CR36 "Raffeiner, P. et al. An MXD1-derived repressor peptide identifies noncoding mediators of MYC-driven cell proliferation. Proc. Natl Acad. Sci. USA 117, 6571–6579 (2020)."),[96](https://www.nature.com/articles/s41556-020-00620-7#ref-CR96 "Aparicio-Prat, E. et al. DECKO: single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs. BMC Genomics 16, 846 (2015)."),[98](https://www.nature.com/articles/s41556-020-00620-7#ref-CR98 "Zhu, S. et al. Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR–Cas9 library. Nat. Biotechnol. 34, 1279–1286 (2016)."),[123](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR123 "Liu, Y. et al. Genome-wide screening for functional long noncoding RNAs in human cells by Cas9 targeting of splice sites. Nat. Biotechnol. 36, 1203–1210 (2018)."),[124](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR124 "Joung, J. et al. Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood. Nature 548, 343–346 (2017)."),[125](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR125 "Liu, S. J. et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science 355, eaah7111 (2017)."),[126](https://www.nature.com/articles/s41556-020-00620-7#ref-CR126 "Bester, A. C. et al. An integrated genome-wide CRISPRa approach to functionalize lncRNAs in drug resistance. Cell 173, 649–664.e20 (2018).")|Cas editing[96](https://www.nature.com/articles/s41556-020-00620-7#ref-CR96 "Aparicio-Prat, E. et al. DECKO: single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs. BMC Genomics 16, 846 (2015)."),[98](https://www.nature.com/articles/s41556-020-00620-7#ref-CR98 "Zhu, S. et al. Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR–Cas9 library. Nat. Biotechnol. 34, 1279–1286 (2016)."),[123](https://www.nature.com/articles/s41556-020-00620-7#ref-CR123 "Liu, Y. et al. Genome-wide screening for functional long noncoding RNAs in human cells by Cas9 targeting of splice sites. Nat. Biotechnol. 36, 1203–1210 (2018)."), KRAB[36](https://www.nature.com/articles/s41556-020-00620-7#ref-CR36 "Raffeiner, P. et al. An MXD1-derived repressor peptide identifies noncoding mediators of MYC-driven cell proliferation. Proc. Natl Acad. Sci. USA 117, 6571–6579 (2020)."),[98](https://www.nature.com/articles/s41556-020-00620-7#ref-CR98 "Zhu, S. et al. Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR–Cas9 library. Nat. Biotechnol. 34, 1279–1286 (2016)."),[125](https://www.nature.com/articles/s41556-020-00620-7#ref-CR125 "Liu, S. J. et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science 355, eaah7111 (2017)."), SID[36](https://www.nature.com/articles/s41556-020-00620-7#ref-CR36 "Raffeiner, P. et al. An MXD1-derived repressor peptide identifies noncoding mediators of MYC-driven cell proliferation. Proc. Natl Acad. Sci. USA 117, 6571–6579 (2020)."), CRISPRa[98](https://www.nature.com/articles/s41556-020-00620-7#ref-CR98 "Zhu, S. et al. Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR–Cas9 library. Nat. Biotechnol. 34, 1279–1286 (2016)."),[124](https://www.nature.com/articles/s41556-020-00620-7#ref-CR124 "Joung, J. et al. Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood. Nature 548, 343–346 (2017)."),[126](https://www.nature.com/articles/s41556-020-00620-7#ref-CR126 "Bester, A. C. et al. An integrated genome-wide CRISPRa approach to functionalize lncRNAs in drug resistance. Cell 173, 649–664.e20 (2018).")|IMR90 (ref. [96](https://www.nature.com/articles/s41556-020-00620-7#ref-CR96 "Aparicio-Prat, E. et al. DECKO: single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs. BMC Genomics 16, 846 (2015).")), HCT116 (ref. [96](https://www.nature.com/articles/s41556-020-00620-7#ref-CR96 "Aparicio-Prat, E. et al. DECKO: single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs. BMC Genomics 16, 846 (2015).")), HEK293T[96](https://www.nature.com/articles/s41556-020-00620-7#ref-CR96 "Aparicio-Prat, E. et al. DECKO: single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs. BMC Genomics 16, 846 (2015)."),[126](https://www.nature.com/articles/s41556-020-00620-7#ref-CR126 "Bester, A. C. et al. An integrated genome-wide CRISPRa approach to functionalize lncRNAs in drug resistance. Cell 173, 649–664.e20 (2018)."), Huh-7.5 (ref. [98](https://www.nature.com/articles/s41556-020-00620-7#ref-CR98 "Zhu, S. et al. Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR–Cas9 library. Nat. Biotechnol. 34, 1279–1286 (2016).")), HeLa[98](https://www.nature.com/articles/s41556-020-00620-7#ref-CR98 "Zhu, S. et al. Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR–Cas9 library. Nat. Biotechnol. 34, 1279–1286 (2016)."),[123](https://www.nature.com/articles/s41556-020-00620-7#ref-CR123 "Liu, Y. et al. Genome-wide screening for functional long noncoding RNAs in human cells by Cas9 targeting of splice sites. Nat. Biotechnol. 36, 1203–1210 (2018)."),[125](https://www.nature.com/articles/s41556-020-00620-7#ref-CR125 "Liu, S. J. et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science 355, eaah7111 (2017)."), A375 (ref. [124](https://www.nature.com/articles/s41556-020-00620-7#ref-CR124 "Joung, J. et al. Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood. Nature 548, 343–346 (2017).")), K562 (refs. [123](https://www.nature.com/articles/s41556-020-00620-7#ref-CR123 "Liu, Y. et al. Genome-wide screening for functional long noncoding RNAs in human cells by Cas9 targeting of splice sites. Nat. Biotechnol. 36, 1203–1210 (2018)."),[125](https://www.nature.com/articles/s41556-020-00620-7#ref-CR125 "Liu, S. J. et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science 355, eaah7111 (2017)."),[126](https://www.nature.com/articles/s41556-020-00620-7#ref-CR126 "Bester, A. C. et al. An integrated genome-wide CRISPRa approach to functionalize lncRNAs in drug resistance. Cell 173, 649–664.e20 (2018).")), U87 (ref. [125](https://www.nature.com/articles/s41556-020-00620-7#ref-CR125 "Liu, S. J. et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science 355, eaah7111 (2017).")), MCF7 (ref. [125](https://www.nature.com/articles/s41556-020-00620-7#ref-CR125 "Liu, S. J. et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science 355, eaah7111 (2017).")), MDA-MB231 (ref. [125](https://www.nature.com/articles/s41556-020-00620-7#ref-CR125 "Liu, S. J. et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science 355, eaah7111 (2017).")), iPSCs[125](https://www.nature.com/articles/s41556-020-00620-7#ref-CR125 "Liu, S. J. et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science 355, eaah7111 (2017)."), MOLM14 (ref. [126](https://www.nature.com/articles/s41556-020-00620-7#ref-CR126 "Bester, A. C. et al. An integrated genome-wide CRISPRa approach to functionalize lncRNAs in drug resistance. Cell 173, 649–664.e20 (2018).")), HL60 (ref. [126](https://www.nature.com/articles/s41556-020-00620-7#ref-CR126 "Bester, A. C. et al. An integrated genome-wide CRISPRa approach to functionalize lncRNAs in drug resistance. Cell 173, 649–664.e20 (2018).")), RAMOS[36](https://www.nature.com/articles/s41556-020-00620-7#ref-CR36 "Raffeiner, P. et al. An MXD1-derived repressor peptide identifies noncoding mediators of MYC-driven cell proliferation. Proc. Natl Acad. Sci. USA 117, 6571–6579 (2020)."), P493-6 (ref. [36](https://www.nature.com/articles/s41556-020-00620-7#ref-CR36 "Raffeiner, P. et al. An MXD1-derived repressor peptide identifies noncoding mediators of MYC-driven cell proliferation. Proc. Natl Acad. Sci. USA 117, 6571–6579 (2020).")), GM12878 (ref. [123](https://www.nature.com/articles/s41556-020-00620-7#ref-CR123 "Liu, Y. et al. Genome-wide screening for functional long noncoding RNAs in human cells by Cas9 targeting of splice sites. Nat. Biotechnol. 36, 1203–1210 (2018)."))||||| |CTCF site characterization[91](https://www.nature.com/articles/s41556-020-00620-7#ref-CR91 "Tarjan, D. R., Flavahan, W. A. & Bernstein, B. E. Epigenome editing strategies for the functional annotation of CTCF insulators. Nat. Commun. 10, 4258 (2019)."),[132](https://www.nature.com/articles/s41556-020-00620-7#ref-CR132 "Guo, Y. et al. CRISPR inversion of CTCF sites alters genome topology and enhancer/promoter function. Cell 162, 900–910 (2015)."),[133](https://www.nature.com/articles/s41556-020-00620-7#ref-CR133 "Guo, Y. et al. CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops. Genome Biol. 19, 160 (2018).")|Cas editing[91](https://www.nature.com/articles/s41556-020-00620-7#ref-CR91 "Tarjan, D. R., Flavahan, W. A. & Bernstein, B. E. Epigenome editing strategies for the functional annotation of CTCF insulators. Nat. Commun. 10, 4258 (2019)."),[132](https://www.nature.com/articles/s41556-020-00620-7#ref-CR132 "Guo, Y. et al. CRISPR inversion of CTCF sites alters genome topology and enhancer/promoter function. Cell 162, 900–910 (2015)."),[133](https://www.nature.com/articles/s41556-020-00620-7#ref-CR133 "Guo, Y. et al. CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops. Genome Biol. 19, 160 (2018)."), dCas binding[91](https://www.nature.com/articles/s41556-020-00620-7#ref-CR91 "Tarjan, D. R., Flavahan, W. A. & Bernstein, B. E. Epigenome editing strategies for the functional annotation of CTCF insulators. Nat. Commun. 10, 4258 (2019)."), KRAB (+ DNMT3A/DNMT3L)[91](https://www.nature.com/articles/s41556-020-00620-7#ref-CR91 "Tarjan, D. R., Flavahan, W. A. & Bernstein, B. E. Epigenome editing strategies for the functional annotation of CTCF insulators. Nat. Commun. 10, 4258 (2019).")|HEC-1B[132](https://www.nature.com/articles/s41556-020-00620-7#ref-CR132 "Guo, Y. et al. CRISPR inversion of CTCF sites alters genome topology and enhancer/promoter function. Cell 162, 900–910 (2015)."), K562 (ref. [132](https://www.nature.com/articles/s41556-020-00620-7#ref-CR132 "Guo, Y. et al. CRISPR inversion of CTCF sites alters genome topology and enhancer/promoter function. Cell 162, 900–910 (2015).")), HEK293 (ref. [91](https://www.nature.com/articles/s41556-020-00620-7#ref-CR91 "Tarjan, D. R., Flavahan, W. A. & Bernstein, B. E. Epigenome editing strategies for the functional annotation of CTCF insulators. Nat. Commun. 10, 4258 (2019).")), HEK293T[132](https://www.nature.com/articles/s41556-020-00620-7#ref-CR132 "Guo, Y. et al. CRISPR inversion of CTCF sites alters genome topology and enhancer/promoter function. Cell 162, 900–910 (2015)."),[133](https://www.nature.com/articles/s41556-020-00620-7#ref-CR133 "Guo, Y. et al. CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops. Genome Biol. 19, 160 (2018)."), 22RV1 (ref. [133](https://www.nature.com/articles/s41556-020-00620-7#ref-CR133 "Guo, Y. et al. CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops. Genome Biol. 19, 160 (2018).")), HAP1 (ref. [133](https://www.nature.com/articles/s41556-020-00620-7#ref-CR133 "Guo, Y. et al. CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops. Genome Biol. 19, 160 (2018).")), GSC6 (ref. [91](https://www.nature.com/articles/s41556-020-00620-7#ref-CR91 "Tarjan, D. R., Flavahan, W. A. & Bernstein, B. E. Epigenome editing strategies for the functional annotation of CTCF insulators. Nat. Commun. 10, 4258 (2019)."))|(+++): _KCNN3_ (ref. [133](https://www.nature.com/articles/s41556-020-00620-7#ref-CR133 "Guo, Y. et al. CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops. Genome Biol. 19, 160 (2018).")), _KRT78_ (ref. [133](https://www.nature.com/articles/s41556-020-00620-7#ref-CR133 "Guo, Y. et al. CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops. Genome Biol. 19, 160 (2018).")), _KRT4_ (ref. [133](https://www.nature.com/articles/s41556-020-00620-7#ref-CR133 "Guo, Y. et al. CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops. Genome Biol. 19, 160 (2018).")), _KRT79_ (ref. [133](https://www.nature.com/articles/s41556-020-00620-7#ref-CR133 "Guo, Y. et al. CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops. Genome Biol. 19, 160 (2018).")); (++): _KRT80_ (ref. [133](https://www.nature.com/articles/s41556-020-00620-7#ref-CR133 "Guo, Y. et al. CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops. Genome Biol. 19, 160 (2018).")); (0): _KRT8_ (ref. [133](https://www.nature.com/articles/s41556-020-00620-7#ref-CR133 "Guo, Y. et al. CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops. Genome Biol. 19, 160 (2018).")), _PDGFRA_[91](https://www.nature.com/articles/s41556-020-00620-7#ref-CR91 "Tarjan, D. R., Flavahan, W. A. & Bernstein, B. E. Epigenome editing strategies for the functional annotation of CTCF insulators. Nat. Commun. 10, 4258 (2019)."), _LRP1_ (ref. [91](https://www.nature.com/articles/s41556-020-00620-7#ref-CR91 "Tarjan, D. R., Flavahan, W. A. & Bernstein, B. E. Epigenome editing strategies for the functional annotation of CTCF insulators. Nat. Commun. 10, 4258 (2019).")), _HIP1_ (ref. [91](https://www.nature.com/articles/s41556-020-00620-7#ref-CR91 "Tarjan, D. R., Flavahan, W. A. & Bernstein, B. E. Epigenome editing strategies for the functional annotation of CTCF insulators. Nat. Commun. 10, 4258 (2019).")); (−): _Pcdh_[132](https://www.nature.com/articles/s41556-020-00620-7#ref-CR132 "Guo, Y. et al. CRISPR inversion of CTCF sites alters genome topology and enhancer/promoter function. Cell 162, 900–910 (2015).")|DNA interactions[132](https://www.nature.com/articles/s41556-020-00620-7#ref-CR132 "Guo, Y. et al. CRISPR inversion of CTCF sites alters genome topology and enhancer/promoter function. Cell 162, 900–910 (2015)."), CTCF binding[132](https://www.nature.com/articles/s41556-020-00620-7#ref-CR132 "Guo, Y. et al. CRISPR inversion of CTCF sites alters genome topology and enhancer/promoter function. Cell 162, 900–910 (2015)."), H3K27ac[133](https://www.nature.com/articles/s41556-020-00620-7#ref-CR133 "Guo, Y. et al. CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop anchors identifies repressive chromatin loops. Genome Biol. 19, 160 (2018)."), **Rad21 binding**[132](https://www.nature.com/articles/s41556-020-00620-7#ref-CR132 "Guo, Y. et al. CRISPR inversion of CTCF sites alters genome topology and enhancer/promoter function. Cell 162, 900–910 (2015)."), **CTCF binding**[91](https://www.nature.com/articles/s41556-020-00620-7#ref-CR91 "Tarjan, D. R., Flavahan, W. A. & Bernstein, B. E. Epigenome editing strategies for the functional annotation of CTCF insulators. Nat. Commun. 10, 4258 (2019)."); H3K9me3 (KRAB[91](https://www.nature.com/articles/s41556-020-00620-7#ref-CR91 "Tarjan, D. R., Flavahan, W. A. & Bernstein, B. E. Epigenome editing strategies for the functional annotation of CTCF insulators. Nat. Commun. 10, 4258 (2019).")); H3K9me3, DNA 5-mC (DNMT3A + DNMT3L[91](https://www.nature.com/articles/s41556-020-00620-7#ref-CR91 "Tarjan, D. R., Flavahan, W. A. & Bernstein, B. E. Epigenome editing strategies for the functional annotation of CTCF insulators. Nat. Commun. 10, 4258 (2019).")); H3K9me3, DNA 5-mC, DNA interactions, **H3K27ac** (KRAB + DNMT3A + DNMT3L[91](https://www.nature.com/articles/s41556-020-00620-7#ref-CR91 "Tarjan, D. R., Flavahan, W. A. & Bernstein, B. E. Epigenome editing strategies for the functional annotation of CTCF insulators. Nat. Commun. 10, 4258 (2019)."))|12 days (KRAB), 20 days (DNMT3A / 3L)[91](https://www.nature.com/articles/s41556-020-00620-7#ref-CR91 "Tarjan, D. R., Flavahan, W. A. & Bernstein, B. E. Epigenome editing strategies for the functional annotation of CTCF insulators. Nat. Commun. 10, 4258 (2019).")|Profiled off-target effects on CTCF binding[91](https://www.nature.com/articles/s41556-020-00620-7#ref-CR91 "Tarjan, D. R., Flavahan, W. A. & Bernstein, B. E. Epigenome editing strategies for the functional annotation of CTCF insulators. Nat. Commun. 10, 4258 (2019).")| |DNA element characterization[37](https://www.nature.com/articles/s41556-020-00620-7#ref-CR37 "Thakore, P. I. et al. Highly specific epigenome editing by CRISPR–Cas9 repressors for silencing of distal regulatory elements. Nat. Methods 12, 1143–1149 (2015)."),[39](https://www.nature.com/articles/s41556-020-00620-7#ref-CR39 "Fulco, C. P. et al. Systematic mapping of functional enhancer–promoter connections with CRISPR interference. Science 354, 769–773 (2016)."),[43](https://www.nature.com/articles/s41556-020-00620-7#ref-CR43 "Xie, S., Duan, J., Li, B., Zhou, P. & Hon, G. C. Multiplexed engineering and analysis of combinatorial enhancer activity in single cells. Mol. Cell 66, 285–299.e5 (2017)."),[45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015)."),[47](https://www.nature.com/articles/s41556-020-00620-7#ref-CR47 "Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017)."),[48](https://www.nature.com/articles/s41556-020-00620-7#ref-CR48 "Simeonov, D. R. et al. Discovery of stimulation-responsive immune enhancers with CRISPR activation. Nature 549, 111–115 (2017)."),[50](https://www.nature.com/articles/s41556-020-00620-7#ref-CR50 "Morton, A. R. et al. Functional enhancers shape extrachromosomal oncogene amplifications. Cell 179, 1330–1341.e13 (2019)."),[51](https://www.nature.com/articles/s41556-020-00620-7#ref-CR51 "Fulco, C. P. et al. Activity-by-contact model of enhancer–promoter regulation from thousands of CRISPR perturbations. Nat. Genet. 51, 1664–1669 (2019)."),[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."),[57](https://www.nature.com/articles/s41556-020-00620-7#ref-CR57 "Kuscu, C. et al. Temporal and spatial epigenome editing allows precise gene regulation in mammalian cells. J. Mol. Biol. 431, 111–121 (2019)."),[78](https://www.nature.com/articles/s41556-020-00620-7#ref-CR78 "Mkannez, G. et al. DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease. Cardiovasc. Res. 114, 1525–1535 (2018)."),[88](https://www.nature.com/articles/s41556-020-00620-7#ref-CR88 "Gasperini, M. et al. A genome-wide framework for mapping gene regulation via cellular genetic screens. Cell 176, 377–390.e19 (2019)."),[89](https://www.nature.com/articles/s41556-020-00620-7#ref-CR89 "D’Ippolito, A. M. et al. Pre-established chromatin interactions mediate the genomic response to glucocorticoids. Cell Syst. 7, 146–160.e7 (2018)."),[105](https://www.nature.com/articles/s41556-020-00620-7#ref-CR105 "Pradeepa, M. M. et al. Histone H3 globular domain acetylation identifies a new class of enhancers. Nat. Genet. 48, 681–686 (2016)."),[110](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR110 "Diao, Y. et al. A tiling-deletion-based genetic screen for cis-regulatory element identification in mammalian cells. Nat. Methods 14, 629–635 (2017)."),[111](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR111 "Gasperini, M. et al. CRISPR/Cas9-mediated scanning for regulatory elements required for HPRT1 expression via thousands of large, programmed genomic deletions. Am. J. Hum. Genet. 101, 192–205 (2017)."),[112](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR112 "Canver, M. C. et al. Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci. Nat. Genet. 49, 625–634 (2017)."),[113](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR113 "Dickel, D. E. et al. Ultraconserved enhancers are required for normal development. Cell 172, 491–499.e15 (2018)."),[114](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR114 "Osterwalder, M. et al. Enhancer redundancy provides phenotypic robustness in mammalian development. Nature 554, 239–243 (2018)."),[115](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR115 "Canver, M. C. et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature 527, 192–197 (2015)."),[116](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR116 "Lupiáñez, D. G. et al. Disruptions of topological chromatin domains cause pathogenic rewiring of gene–enhancer interactions. Cell 161, 1012–1025 (2015)."),[117](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR117 "Diao, Y. et al. A new class of temporarily phenotypic enhancers identified by CRISPR/Cas9-mediated genetic screening. Genome Res. 26, 397–405 (2016)."),[118](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR118 "Korkmaz, G. et al. Functional genetic screens for enhancer elements in the human genome using CRISPR–Cas9. Nat. Biotechnol. 34, 192–198 (2016)."),[119](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR119 "Sanjana, N. E. et al. High-resolution interrogation of functional elements in the noncoding genome. Science 353, 1545–1549 (2016)."),[120](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR120 "Rajagopal, N. et al. High-throughput mapping of regulatory DNA. Nat. Biotechnol. 34, 167–174 (2016)."),[121](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR121 "Sen, D. R. et al. The epigenetic landscape of T cell exhaustion. Science 354, 1165–1169 (2016)."),[122](https://www.nature.com/articles/s41556-020-00620-7#ref-CR122 "Shin, H. Y. et al. Hierarchy within the mammary STAT5-driven Wap super-enhancer. Nat. Genet. 48, 904–911 (2016)."),[134](https://www.nature.com/articles/s41556-020-00620-7#ref-CR134 "Flavahan, W. A. et al. Altered chromosomal topology drives oncogenic programs in SDH-deficient GISTs. Nature 575, 229–233 (2019).")|CRISPRa[48](https://www.nature.com/articles/s41556-020-00620-7#ref-CR48 "Simeonov, D. R. et al. Discovery of stimulation-responsive immune enhancers with CRISPR activation. Nature 549, 111–115 (2017)."),[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."), p300 (refs. [45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015)."),[47](https://www.nature.com/articles/s41556-020-00620-7#ref-CR47 "Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017)."),[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."),[57](https://www.nature.com/articles/s41556-020-00620-7#ref-CR57 "Kuscu, C. et al. Temporal and spatial epigenome editing allows precise gene regulation in mammalian cells. J. Mol. Biol. 431, 111–121 (2019).")), VP64 + p300 (ref. [56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020).")), LSD1 (refs. [45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015)."),[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020).")), KRAB[37](https://www.nature.com/articles/s41556-020-00620-7#ref-CR37 "Thakore, P. I. et al. Highly specific epigenome editing by CRISPR–Cas9 repressors for silencing of distal regulatory elements. Nat. Methods 12, 1143–1149 (2015)."),[39](https://www.nature.com/articles/s41556-020-00620-7#ref-CR39 "Fulco, C. P. et al. Systematic mapping of functional enhancer–promoter connections with CRISPR interference. Science 354, 769–773 (2016)."),[43](https://www.nature.com/articles/s41556-020-00620-7#ref-CR43 "Xie, S., Duan, J., Li, B., Zhou, P. & Hon, G. C. Multiplexed engineering and analysis of combinatorial enhancer activity in single cells. Mol. Cell 66, 285–299.e5 (2017)."),[45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015)."),[47](https://www.nature.com/articles/s41556-020-00620-7#ref-CR47 "Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017)."),[50](https://www.nature.com/articles/s41556-020-00620-7#ref-CR50 "Morton, A. R. et al. Functional enhancers shape extrachromosomal oncogene amplifications. Cell 179, 1330–1341.e13 (2019)."),[51](https://www.nature.com/articles/s41556-020-00620-7#ref-CR51 "Fulco, C. P. et al. Activity-by-contact model of enhancer–promoter regulation from thousands of CRISPR perturbations. Nat. Genet. 51, 1664–1669 (2019)."),[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."),[88](https://www.nature.com/articles/s41556-020-00620-7#ref-CR88 "Gasperini, M. et al. A genome-wide framework for mapping gene regulation via cellular genetic screens. Cell 176, 377–390.e19 (2019)."),[89](https://www.nature.com/articles/s41556-020-00620-7#ref-CR89 "D’Ippolito, A. M. et al. Pre-established chromatin interactions mediate the genomic response to glucocorticoids. Cell Syst. 7, 146–160.e7 (2018)."), LSD1 + KRAB[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020)."), SID[105](https://www.nature.com/articles/s41556-020-00620-7#ref-CR105 "Pradeepa, M. M. et al. Histone H3 globular domain acetylation identifies a new class of enhancers. Nat. Genet. 48, 681–686 (2016)."), DNMT3A[78](https://www.nature.com/articles/s41556-020-00620-7#ref-CR78 "Mkannez, G. et al. DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease. Cardiovasc. Res. 114, 1525–1535 (2018)."), Cas editing[105](https://www.nature.com/articles/s41556-020-00620-7#ref-CR105 "Pradeepa, M. M. et al. Histone H3 globular domain acetylation identifies a new class of enhancers. Nat. Genet. 48, 681–686 (2016)."),[110](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR110 "Diao, Y. et al. A tiling-deletion-based genetic screen for cis-regulatory element identification in mammalian cells. Nat. Methods 14, 629–635 (2017)."),[111](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR111 "Gasperini, M. et al. CRISPR/Cas9-mediated scanning for regulatory elements required for HPRT1 expression via thousands of large, programmed genomic deletions. Am. J. Hum. Genet. 101, 192–205 (2017)."),[112](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR112 "Canver, M. C. et al. Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci. Nat. Genet. 49, 625–634 (2017)."),[113](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR113 "Dickel, D. E. et al. Ultraconserved enhancers are required for normal development. Cell 172, 491–499.e15 (2018)."),[114](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR114 "Osterwalder, M. et al. Enhancer redundancy provides phenotypic robustness in mammalian development. Nature 554, 239–243 (2018)."),[115](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR115 "Canver, M. C. et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature 527, 192–197 (2015)."),[116](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR116 "Lupiáñez, D. G. et al. Disruptions of topological chromatin domains cause pathogenic rewiring of gene–enhancer interactions. Cell 161, 1012–1025 (2015)."),[117](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR117 "Diao, Y. et al. A new class of temporarily phenotypic enhancers identified by CRISPR/Cas9-mediated genetic screening. Genome Res. 26, 397–405 (2016)."),[118](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR118 "Korkmaz, G. et al. Functional genetic screens for enhancer elements in the human genome using CRISPR–Cas9. Nat. Biotechnol. 34, 192–198 (2016)."),[119](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR119 "Sanjana, N. E. et al. High-resolution interrogation of functional elements in the noncoding genome. Science 353, 1545–1549 (2016)."),[120](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR120 "Rajagopal, N. et al. High-throughput mapping of regulatory DNA. Nat. Biotechnol. 34, 167–174 (2016)."),[121](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR121 "Sen, D. R. et al. The epigenetic landscape of T cell exhaustion. Science 354, 1165–1169 (2016)."),[122](https://www.nature.com/articles/s41556-020-00620-7#ref-CR122 "Shin, H. Y. et al. Hierarchy within the mammary STAT5-driven Wap super-enhancer. Nat. Genet. 48, 904–911 (2016)."),[134](https://www.nature.com/articles/s41556-020-00620-7#ref-CR134 "Flavahan, W. A. et al. Altered chromosomal topology drives oncogenic programs in SDH-deficient GISTs. Nature 575, 229–233 (2019).")|HEK293T[47](https://www.nature.com/articles/s41556-020-00620-7#ref-CR47 "Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017)."),[57](https://www.nature.com/articles/s41556-020-00620-7#ref-CR57 "Kuscu, C. et al. Temporal and spatial epigenome editing allows precise gene regulation in mammalian cells. J. Mol. Biol. 431, 111–121 (2019)."),[78](https://www.nature.com/articles/s41556-020-00620-7#ref-CR78 "Mkannez, G. et al. DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease. Cardiovasc. Res. 114, 1525–1535 (2018)."), K562 (refs. [37](https://www.nature.com/articles/s41556-020-00620-7#ref-CR37 "Thakore, P. I. et al. Highly specific epigenome editing by CRISPR–Cas9 repressors for silencing of distal regulatory elements. Nat. Methods 12, 1143–1149 (2015)."),[39](https://www.nature.com/articles/s41556-020-00620-7#ref-CR39 "Fulco, C. P. et al. Systematic mapping of functional enhancer–promoter connections with CRISPR interference. Science 354, 769–773 (2016)."),[43](https://www.nature.com/articles/s41556-020-00620-7#ref-CR43 "Xie, S., Duan, J., Li, B., Zhou, P. & Hon, G. C. Multiplexed engineering and analysis of combinatorial enhancer activity in single cells. Mol. Cell 66, 285–299.e5 (2017)."),[47](https://www.nature.com/articles/s41556-020-00620-7#ref-CR47 "Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017)."),[51](https://www.nature.com/articles/s41556-020-00620-7#ref-CR51 "Fulco, C. P. et al. 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Nature 527, 192–197 (2015).")), BJ[118](https://www.nature.com/articles/s41556-020-00620-7#ref-CR118 "Korkmaz, G. et al. Functional genetic screens for enhancer elements in the human genome using CRISPR–Cas9. Nat. Biotechnol. 34, 192–198 (2016)."), MCF7 (ref. [118](https://www.nature.com/articles/s41556-020-00620-7#ref-CR118 "Korkmaz, G. et al. Functional genetic screens for enhancer elements in the human genome using CRISPR–Cas9. Nat. Biotechnol. 34, 192–198 (2016).")), T47D[118](https://www.nature.com/articles/s41556-020-00620-7#ref-CR118 "Korkmaz, G. et al. Functional genetic screens for enhancer elements in the human genome using CRISPR–Cas9. Nat. Biotechnol. 34, 192–198 (2016)."), MDA-MB-231 (ref. [118](https://www.nature.com/articles/s41556-020-00620-7#ref-CR118 "Korkmaz, G. et al. Functional genetic screens for enhancer elements in the human genome using CRISPR–Cas9. Nat. Biotechnol. 34, 192–198 (2016).")), A375 (ref. [119](https://www.nature.com/articles/s41556-020-00620-7#ref-CR119 "Sanjana, N. E. et al. High-resolution interrogation of functional elements in the noncoding genome. Science 353, 1545–1549 (2016).")), A431 (ref. [47](https://www.nature.com/articles/s41556-020-00620-7#ref-CR47 "Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017).")), A549 (ref. [89](https://www.nature.com/articles/s41556-020-00620-7#ref-CR89 "D’Ippolito, A. M. et al. Pre-established chromatin interactions mediate the genomic response to glucocorticoids. Cell Syst. 7, 146–160.e7 (2018).")), HAP1 (ref. [111](https://www.nature.com/articles/s41556-020-00620-7#ref-CR111 "Gasperini, M. et al. CRISPR/Cas9-mediated scanning for regulatory elements required for HPRT1 expression via thousands of large, programmed genomic deletions. Am. J. Hum. 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Genome Res. 26, 397–405 (2016)."), erythroblasts[112](https://www.nature.com/articles/s41556-020-00620-7#ref-CR112 "Canver, M. C. et al. Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci. Nat. Genet. 49, 625–634 (2017)."), MEL-745A[115](https://www.nature.com/articles/s41556-020-00620-7#ref-CR115 "Canver, M. C. et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature 527, 192–197 (2015)."), EL4 (ref. [121](https://www.nature.com/articles/s41556-020-00620-7#ref-CR121 "Sen, D. R. et al. The epigenetic landscape of T cell exhaustion. Science 354, 1165–1169 (2016).")), mESCs[45](https://www.nature.com/articles/s41556-020-00620-7#ref-CR45 "Kearns, N. A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015)."),[105](https://www.nature.com/articles/s41556-020-00620-7#ref-CR105 "Pradeepa, M. M. et al. 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Disruptions of topological chromatin domains cause pathogenic rewiring of gene–enhancer interactions. Cell 161, 1012–1025 (2015).")/mammary[122](https://www.nature.com/articles/s41556-020-00620-7#ref-CR122 "Shin, H. Y. et al. Hierarchy within the mammary STAT5-driven Wap super-enhancer. Nat. Genet. 48, 904–911 (2016).")/brain tissues[113](https://www.nature.com/articles/s41556-020-00620-7#ref-CR113 "Dickel, D. E. et al. Ultraconserved enhancers are required for normal development. Cell 172, 491–499.e15 (2018).")||DNA interactions[116](https://www.nature.com/articles/s41556-020-00620-7#ref-CR116 "Lupiáñez, D. G. et al. Disruptions of topological chromatin domains cause pathogenic rewiring of gene–enhancer interactions. Cell 161, 1012–1025 (2015)."),[134](https://www.nature.com/articles/s41556-020-00620-7#ref-CR134 "Flavahan, W. A. et al. Altered chromosomal topology drives oncogenic programs in SDH-deficient GISTs. Nature 575, 229–233 (2019)."), **eRNA expression**[118](https://www.nature.com/articles/s41556-020-00620-7#ref-CR118 "Korkmaz, G. et al. Functional genetic screens for enhancer elements in the human genome using CRISPR–Cas9. Nat. Biotechnol. 34, 192–198 (2016)."), **TF binding**[37](https://www.nature.com/articles/s41556-020-00620-7#ref-CR37 "Thakore, P. I. et al. Highly specific epigenome editing by CRISPR–Cas9 repressors for silencing of distal regulatory elements. Nat. Methods 12, 1143–1149 (2015)."),[112](https://www.nature.com/articles/s41556-020-00620-7#ref-CR112 "Canver, M. C. et al. Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci. Nat. Genet. 49, 625–634 (2017)."),[118](https://www.nature.com/articles/s41556-020-00620-7#ref-CR118 "Korkmaz, G. et al. Functional genetic screens for enhancer elements in the human genome using CRISPR–Cas9. Nat. Biotechnol. 34, 192–198 (2016)."),[119](https://www.nature.com/articles/s41556-020-00620-7#ref-CR119 "Sanjana, N. E. et al. High-resolution interrogation of functional elements in the noncoding genome. Science 353, 1545–1549 (2016)."),[122](https://www.nature.com/articles/s41556-020-00620-7#ref-CR122 "Shin, H. Y. et al. Hierarchy within the mammary STAT5-driven Wap super-enhancer. Nat. Genet. 48, 904–911 (2016)."), **H3K4me**[119](https://www.nature.com/articles/s41556-020-00620-7#ref-CR119 "Sanjana, N. E. et al. High-resolution interrogation of functional elements in the noncoding genome. Science 353, 1545–1549 (2016)."), **H3K27ac**[39](https://www.nature.com/articles/s41556-020-00620-7#ref-CR39 "Fulco, C. P. et al. Systematic mapping of functional enhancer–promoter connections with CRISPR interference. Science 354, 769–773 (2016)."),[119](https://www.nature.com/articles/s41556-020-00620-7#ref-CR119 "Sanjana, N. E. et al. High-resolution interrogation of functional elements in the noncoding genome. Science 353, 1545–1549 (2016)."),[122](https://www.nature.com/articles/s41556-020-00620-7#ref-CR122 "Shin, H. Y. et al. Hierarchy within the mammary STAT5-driven Wap super-enhancer. Nat. Genet. 48, 904–911 (2016)."), **DNA accessibility**[37](https://www.nature.com/articles/s41556-020-00620-7#ref-CR37 "Thakore, P. I. et al. Highly specific epigenome editing by CRISPR–Cas9 repressors for silencing of distal regulatory elements. Nat. Methods 12, 1143–1149 (2015)."),[122](https://www.nature.com/articles/s41556-020-00620-7#ref-CR122 "Shin, H. Y. et al. Hierarchy within the mammary STAT5-driven Wap super-enhancer. Nat. Genet. 48, 904–911 (2016)."), H3K9me3 (KRAB[37](https://www.nature.com/articles/s41556-020-00620-7#ref-CR37 "Thakore, P. I. et al. Highly specific epigenome editing by CRISPR–Cas9 repressors for silencing of distal regulatory elements. Nat. Methods 12, 1143–1149 (2015)."),[43](https://www.nature.com/articles/s41556-020-00620-7#ref-CR43 "Xie, S., Duan, J., Li, B., Zhou, P. & Hon, G. C. Multiplexed engineering and analysis of combinatorial enhancer activity in single cells. Mol. Cell 66, 285–299.e5 (2017)."),[47](https://www.nature.com/articles/s41556-020-00620-7#ref-CR47 "Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017).")), **H3K122ac** (SID[105](https://www.nature.com/articles/s41556-020-00620-7#ref-CR105 "Pradeepa, M. M. et al. Histone H3 globular domain acetylation identifies a new class of enhancers. Nat. Genet. 48, 681–686 (2016).")), H3K27ac (p300 (ref. [47](https://www.nature.com/articles/s41556-020-00620-7#ref-CR47 "Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017).")))|Various durations[110](https://www.nature.com/articles/s41556-020-00620-7#ref-CR110 "Diao, Y. et al. A tiling-deletion-based genetic screen for cis-regulatory element identification in mammalian cells. Nat. Methods 14, 629–635 (2017)."),[117](https://www.nature.com/articles/s41556-020-00620-7#ref-CR117 "Diao, Y. et al. A new class of temporarily phenotypic enhancers identified by CRISPR/Cas9-mediated genetic screening. Genome Res. 26, 397–405 (2016).")|Induced de novo enhancer activity[57](https://www.nature.com/articles/s41556-020-00620-7#ref-CR57 "Kuscu, C. et al. Temporal and spatial epigenome editing allows precise gene regulation in mammalian cells. J. Mol. Biol. 431, 111–121 (2019)."); in vivo screen[56](https://www.nature.com/articles/s41556-020-00620-7#ref-CR56 "Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 11, 485 (2020).")| |DNA interaction control[47](https://www.nature.com/articles/s41556-020-00620-7#ref-CR47 "Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017)."),[129](https://www.nature.com/articles/s41556-020-00620-7#ref-CR129 "Kim, J. H. et al. LADL: light-activated dynamic looping for endogenous gene expression control. Nat. Methods 16, 633–639 (2019).")|Heterodimerization domains[47](https://www.nature.com/articles/s41556-020-00620-7#ref-CR47 "Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017)."),[129](https://www.nature.com/articles/s41556-020-00620-7#ref-CR129 "Kim, J. H. et al. LADL: light-activated dynamic looping for endogenous gene expression control. Nat. Methods 16, 633–639 (2019).")|K562 (ref. [47](https://www.nature.com/articles/s41556-020-00620-7#ref-CR47 "Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017).")), HEK293T[47](https://www.nature.com/articles/s41556-020-00620-7#ref-CR47 "Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017)."), mESCs[129](https://www.nature.com/articles/s41556-020-00620-7#ref-CR129 "Kim, J. H. et al. LADL: light-activated dynamic looping for endogenous gene expression control. Nat. Methods 16, 633–639 (2019).")|(+++): _HBB_[47](https://www.nature.com/articles/s41556-020-00620-7#ref-CR47 "Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017)."); (0): _OCT4_ (ref. [47](https://www.nature.com/articles/s41556-020-00620-7#ref-CR47 "Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017).")), _Zfp462_ (ref. [129](https://www.nature.com/articles/s41556-020-00620-7#ref-CR129 "Kim, J. H. et al. LADL: light-activated dynamic looping for endogenous gene expression control. Nat. Methods 16, 633–639 (2019).")), _Klf4_ (ref. [129](https://www.nature.com/articles/s41556-020-00620-7#ref-CR129 "Kim, J. H. et al. LADL: light-activated dynamic looping for endogenous gene expression control. Nat. Methods 16, 633–639 (2019)."))|DNA interactions[47](https://www.nature.com/articles/s41556-020-00620-7#ref-CR47 "Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017)."),[129](https://www.nature.com/articles/s41556-020-00620-7#ref-CR129 "Kim, J. H. et al. LADL: light-activated dynamic looping for endogenous gene expression control. Nat. Methods 16, 633–639 (2019)."), H3K4me3 (ref. [47](https://www.nature.com/articles/s41556-020-00620-7#ref-CR47 "Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017).")), protein binding[47](https://www.nature.com/articles/s41556-020-00620-7#ref-CR47 "Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017).")|<1 day for short interactions, 10 days for longer interactions[47](https://www.nature.com/articles/s41556-020-00620-7#ref-CR47 "Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017).")|| |**Other epigenetic studies**| | | | | | | |Cellular reprogramming[25](https://www.nature.com/articles/s41556-020-00620-7#ref-CR25 "Gao, X. et al. Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers. Nucleic Acids Res. 42, e155 (2014)."),[41](https://www.nature.com/articles/s41556-020-00620-7#ref-CR41 "Black, J. B. et al. Targeted epigenetic remodeling of endogenous loci by CRISPR/Cas9-based transcriptional activators directly converts fibroblasts to neuronal cells. Cell Stem Cell 19, 406–414 (2016)."),[42](https://www.nature.com/articles/s41556-020-00620-7#ref-CR42 "Liao, H.-K. et al. In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation. Cell 171, 1495–1507.e15 (2017)."),[44](https://www.nature.com/articles/s41556-020-00620-7#ref-CR44 "Liu, P., Chen, M., Liu, Y., Qi, L. S. & Ding, S. CRISPR-based chromatin remodeling of the endogenous Oct4 or Sox2 locus enables reprogramming to pluripotency. Cell Stem Cell 22, 252–261.e4 (2018).")|CRISPRa[25](https://www.nature.com/articles/s41556-020-00620-7#ref-CR25 "Gao, X. et al. Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers. Nucleic Acids Res. 42, e155 (2014)."),[41](https://www.nature.com/articles/s41556-020-00620-7#ref-CR41 "Black, J. B. et al. Targeted epigenetic remodeling of endogenous loci by CRISPR/Cas9-based transcriptional activators directly converts fibroblasts to neuronal cells. Cell Stem Cell 19, 406–414 (2016)."),[42](https://www.nature.com/articles/s41556-020-00620-7#ref-CR42 "Liao, H.-K. et al. In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation. Cell 171, 1495–1507.e15 (2017)."),[44](https://www.nature.com/articles/s41556-020-00620-7#ref-CR44 "Liu, P., Chen, M., Liu, Y., Qi, L. S. & Ding, S. CRISPR-based chromatin remodeling of the endogenous Oct4 or Sox2 locus enables reprogramming to pluripotency. Cell Stem Cell 22, 252–261.e4 (2018)."), p300 (ref. [44](https://www.nature.com/articles/s41556-020-00620-7#ref-CR44 "Liu, P., Chen, M., Liu, Y., Qi, L. S. & Ding, S. CRISPR-based chromatin remodeling of the endogenous Oct4 or Sox2 locus enables reprogramming to pluripotency. Cell Stem Cell 22, 252–261.e4 (2018).")), KRAB[25](https://www.nature.com/articles/s41556-020-00620-7#ref-CR25 "Gao, X. et al. Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers. Nucleic Acids Res. 42, e155 (2014).")|HEK293 (ref. [42](https://www.nature.com/articles/s41556-020-00620-7#ref-CR42 "Liao, H.-K. et al. In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation. Cell 171, 1495–1507.e15 (2017).")), HEK293T[41](https://www.nature.com/articles/s41556-020-00620-7#ref-CR41 "Black, J. B. et al. Targeted epigenetic remodeling of endogenous loci by CRISPR/Cas9-based transcriptional activators directly converts fibroblasts to neuronal cells. Cell Stem Cell 19, 406–414 (2016)."), N2a[42](https://www.nature.com/articles/s41556-020-00620-7#ref-CR42 "Liao, H.-K. et al. In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation. Cell 171, 1495–1507.e15 (2017)."), mEFs[25](https://www.nature.com/articles/s41556-020-00620-7#ref-CR25 "Gao, X. et al. Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers. Nucleic Acids Res. 42, e155 (2014)."),[41](https://www.nature.com/articles/s41556-020-00620-7#ref-CR41 "Black, J. B. et al. Targeted epigenetic remodeling of endogenous loci by CRISPR/Cas9-based transcriptional activators directly converts fibroblasts to neuronal cells. Cell Stem Cell 19, 406–414 (2016)."),[44](https://www.nature.com/articles/s41556-020-00620-7#ref-CR44 "Liu, P., Chen, M., Liu, Y., Qi, L. S. & Ding, S. CRISPR-based chromatin remodeling of the endogenous Oct4 or Sox2 locus enables reprogramming to pluripotency. Cell Stem Cell 22, 252–261.e4 (2018)."), mESCs/epiSCs[25](https://www.nature.com/articles/s41556-020-00620-7#ref-CR25 "Gao, X. et al. Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers. Nucleic Acids Res. 42, e155 (2014)."),[44](https://www.nature.com/articles/s41556-020-00620-7#ref-CR44 "Liu, P., Chen, M., Liu, Y., Qi, L. S. & Ding, S. CRISPR-based chromatin remodeling of the endogenous Oct4 or Sox2 locus enables reprogramming to pluripotency. Cell Stem Cell 22, 252–261.e4 (2018)."), mouse tissues[42](https://www.nature.com/articles/s41556-020-00620-7#ref-CR42 "Liao, H.-K. et al. In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation. Cell 171, 1495–1507.e15 (2017).")|(+++): _ASCL1_ (ref. [41](https://www.nature.com/articles/s41556-020-00620-7#ref-CR41 "Black, J. B. et al. Targeted epigenetic remodeling of endogenous loci by CRISPR/Cas9-based transcriptional activators directly converts fibroblasts to neuronal cells. Cell Stem Cell 19, 406–414 (2016).")), _BRN2_ (ref. [41](https://www.nature.com/articles/s41556-020-00620-7#ref-CR41 "Black, J. B. et al. Targeted epigenetic remodeling of endogenous loci by CRISPR/Cas9-based transcriptional activators directly converts fibroblasts to neuronal cells. Cell Stem Cell 19, 406–414 (2016).")), FP[42](https://www.nature.com/articles/s41556-020-00620-7#ref-CR42 "Liao, H.-K. et al. In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation. Cell 171, 1495–1507.e15 (2017)."); _Fst_[42](https://www.nature.com/articles/s41556-020-00620-7#ref-CR42 "Liao, H.-K. et al. In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation. Cell 171, 1495–1507.e15 (2017)."), _Klotho_[42](https://www.nature.com/articles/s41556-020-00620-7#ref-CR42 "Liao, H.-K. et al. In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation. Cell 171, 1495–1507.e15 (2017)."), _Il10_ (ref. [42](https://www.nature.com/articles/s41556-020-00620-7#ref-CR42 "Liao, H.-K. et al. In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation. Cell 171, 1495–1507.e15 (2017).")), _Six2_ (ref. [42](https://www.nature.com/articles/s41556-020-00620-7#ref-CR42 "Liao, H.-K. et al. In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation. Cell 171, 1495–1507.e15 (2017).")), _Cebpa_[44](https://www.nature.com/articles/s41556-020-00620-7#ref-CR44 "Liu, P., Chen, M., Liu, Y., Qi, L. S. & Ding, S. CRISPR-based chromatin remodeling of the endogenous Oct4 or Sox2 locus enables reprogramming to pluripotency. Cell Stem Cell 22, 252–261.e4 (2018)."); (++): _Sox2_ (ref. [44](https://www.nature.com/articles/s41556-020-00620-7#ref-CR44 "Liu, P., Chen, M., Liu, Y., Qi, L. S. & Ding, S. CRISPR-based chromatin remodeling of the endogenous Oct4 or Sox2 locus enables reprogramming to pluripotency. Cell Stem Cell 22, 252–261.e4 (2018).")), _Klf4_ (ref. [44](https://www.nature.com/articles/s41556-020-00620-7#ref-CR44 "Liu, P., Chen, M., Liu, Y., Qi, L. S. & Ding, S. CRISPR-based chromatin remodeling of the endogenous Oct4 or Sox2 locus enables reprogramming to pluripotency. Cell Stem Cell 22, 252–261.e4 (2018).")); (+): _Pdx1_ (ref. [42](https://www.nature.com/articles/s41556-020-00620-7#ref-CR42 "Liao, H.-K. et al. In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation. Cell 171, 1495–1507.e15 (2017).")), _utrophin_[42](https://www.nature.com/articles/s41556-020-00620-7#ref-CR42 "Liao, H.-K. et al. In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation. Cell 171, 1495–1507.e15 (2017)."), _MYT1L_[41](https://www.nature.com/articles/s41556-020-00620-7#ref-CR41 "Black, J. B. et al. Targeted epigenetic remodeling of endogenous loci by CRISPR/Cas9-based transcriptional activators directly converts fibroblasts to neuronal cells. Cell Stem Cell 19, 406–414 (2016)."), _c-Myc_[44](https://www.nature.com/articles/s41556-020-00620-7#ref-CR44 "Liu, P., Chen, M., Liu, Y., Qi, L. S. & Ding, S. CRISPR-based chromatin remodeling of the endogenous Oct4 or Sox2 locus enables reprogramming to pluripotency. Cell Stem Cell 22, 252–261.e4 (2018)."), _Nr5a2_ (ref. [44](https://www.nature.com/articles/s41556-020-00620-7#ref-CR44 "Liu, P., Chen, M., Liu, Y., Qi, L. S. & Ding, S. CRISPR-based chromatin remodeling of the endogenous Oct4 or Sox2 locus enables reprogramming to pluripotency. Cell Stem Cell 22, 252–261.e4 (2018).")), _Glis1_ (ref. [44](https://www.nature.com/articles/s41556-020-00620-7#ref-CR44 "Liu, P., Chen, M., Liu, Y., Qi, L. S. & Ding, S. CRISPR-based chromatin remodeling of the endogenous Oct4 or Sox2 locus enables reprogramming to pluripotency. Cell Stem Cell 22, 252–261.e4 (2018).")); _Oct4_ (+++ (ref. [44](https://www.nature.com/articles/s41556-020-00620-7#ref-CR44 "Liu, P., Chen, M., Liu, Y., Qi, L. S. & Ding, S. CRISPR-based chromatin remodeling of the endogenous Oct4 or Sox2 locus enables reprogramming to pluripotency. Cell Stem Cell 22, 252–261.e4 (2018).")), +/− (ref. [25](https://www.nature.com/articles/s41556-020-00620-7#ref-CR25 "Gao, X. et al. Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers. Nucleic Acids Res. 42, e155 (2014)."))), _Nanog_ (+/−)[25](https://www.nature.com/articles/s41556-020-00620-7#ref-CR25 "Gao, X. et al. Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers. Nucleic Acids Res. 42, e155 (2014).")|p300 binding[25](https://www.nature.com/articles/s41556-020-00620-7#ref-CR25 "Gao, X. et al. Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers. Nucleic Acids Res. 42, e155 (2014)."), H3K4me3 (refs. [41](https://www.nature.com/articles/s41556-020-00620-7#ref-CR41 "Black, J. B. et al. Targeted epigenetic remodeling of endogenous loci by CRISPR/Cas9-based transcriptional activators directly converts fibroblasts to neuronal cells. Cell Stem Cell 19, 406–414 (2016)."),[42](https://www.nature.com/articles/s41556-020-00620-7#ref-CR42 "Liao, H.-K. et al. In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation. Cell 171, 1495–1507.e15 (2017).")), H3K27ac[25](https://www.nature.com/articles/s41556-020-00620-7#ref-CR25 "Gao, X. et al. Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers. Nucleic Acids Res. 42, e155 (2014)."),[41](https://www.nature.com/articles/s41556-020-00620-7#ref-CR41 "Black, J. B. et al. Targeted epigenetic remodeling of endogenous loci by CRISPR/Cas9-based transcriptional activators directly converts fibroblasts to neuronal cells. Cell Stem Cell 19, 406–414 (2016)."),[42](https://www.nature.com/articles/s41556-020-00620-7#ref-CR42 "Liao, H.-K. et al. In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation. Cell 171, 1495–1507.e15 (2017)."),[44](https://www.nature.com/articles/s41556-020-00620-7#ref-CR44 "Liu, P., Chen, M., Liu, Y., Qi, L. S. & Ding, S. CRISPR-based chromatin remodeling of the endogenous Oct4 or Sox2 locus enables reprogramming to pluripotency. Cell Stem Cell 22, 252–261.e4 (2018)."), **TF binding**[25](https://www.nature.com/articles/s41556-020-00620-7#ref-CR25 "Gao, X. et al. Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers. Nucleic Acids Res. 42, e155 (2014).")|10 days[25](https://www.nature.com/articles/s41556-020-00620-7#ref-CR25 "Gao, X. et al. Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers. Nucleic Acids Res. 42, e155 (2014)."), 18 days[41](https://www.nature.com/articles/s41556-020-00620-7#ref-CR41 "Black, J. B. et al. Targeted epigenetic remodeling of endogenous loci by CRISPR/Cas9-based transcriptional activators directly converts fibroblasts to neuronal cells. Cell Stem Cell 19, 406–414 (2016).")|dCas alone reduced TF binding[25](https://www.nature.com/articles/s41556-020-00620-7#ref-CR25 "Gao, X. et al. Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers. Nucleic Acids Res. 42, e155 (2014).")| |CRISPR–ChIP[147](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR147 "Fujita, T. & Fujii, H. Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochem. Biophys. Res. Commun. 439, 132–136 (2013)."),[148](https://www.nature.com/articles/s41556-020-00620-7/tables/1#ref-CR148 "Fujita, T., Yuno, M. & Fujii, H. Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins. Genes Cells 21, 370–377 (2016)."),[149](https://www.nature.com/articles/s41556-020-00620-7#ref-CR149 "Liu, X. et al. In situ capture of chromatin interactions by biotinylated dCas9. Cell 170, 1028–1043.e19 (2017)."),[151](https://www.nature.com/articles/s41556-020-00620-7#ref-CR151 "Tsui, C. et al. dCas9-targeted locus-specific protein isolation method identifies histone gene regulators. Proc. Natl Acad. Sci. USA 115, E2734–E2741 (2018).")|dCas (FLAG tag[147](https://www.nature.com/articles/s41556-020-00620-7#ref-CR147 "Fujita, T. & Fujii, H. Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochem. Biophys. Res. Commun. 439, 132–136 (2013)."),[148](https://www.nature.com/articles/s41556-020-00620-7#ref-CR148 "Fujita, T., Yuno, M. & Fujii, H. Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins. Genes Cells 21, 370–377 (2016)."),[151](https://www.nature.com/articles/s41556-020-00620-7#ref-CR151 "Tsui, C. et al. dCas9-targeted locus-specific protein isolation method identifies histone gene regulators. Proc. Natl Acad. Sci. USA 115, E2734–E2741 (2018)."), biotinylated[149](https://www.nature.com/articles/s41556-020-00620-7#ref-CR149 "Liu, X. et al. In situ capture of chromatin interactions by biotinylated dCas9. Cell 170, 1028–1043.e19 (2017)."))|HEK293T[147](https://www.nature.com/articles/s41556-020-00620-7#ref-CR147 "Fujita, T. & Fujii, H. Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochem. Biophys. Res. Commun. 439, 132–136 (2013)."),[148](https://www.nature.com/articles/s41556-020-00620-7#ref-CR148 "Fujita, T., Yuno, M. & Fujii, H. Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins. Genes Cells 21, 370–377 (2016)."), K562 (ref. [149](https://www.nature.com/articles/s41556-020-00620-7#ref-CR149 "Liu, X. et al. In situ capture of chromatin interactions by biotinylated dCas9. Cell 170, 1028–1043.e19 (2017).")), HeLa[151](https://www.nature.com/articles/s41556-020-00620-7#ref-CR151 "Tsui, C. et al. dCas9-targeted locus-specific protein isolation method identifies histone gene regulators. Proc. Natl Acad. Sci. USA 115, E2734–E2741 (2018)."), mESCs[149](https://www.nature.com/articles/s41556-020-00620-7#ref-CR149 "Liu, X. et al. In situ capture of chromatin interactions by biotinylated dCas9. Cell 170, 1028–1043.e19 (2017).")||||| |Protein labelling[141](https://www.nature.com/articles/s41556-020-00620-7#ref-CR141 "Han, S. et al. RNA–protein interaction mapping via MS2- or Cas13-based APEX targeting. Proc. Natl Acad. Sci. USA https://doi.org/10.1073/pnas.2006617117 (2020)."),[150](https://www.nature.com/articles/s41556-020-00620-7#ref-CR150 "Myers, S. A. et al. Discovery of proteins associated with a predefined genomic locus via dCas9–APEX-mediated proximity labeling. Nat. Methods 15, 1 (2018).")|APEX2 (refs. [141](https://www.nature.com/articles/s41556-020-00620-7#ref-CR141 "Han, S. et al. RNA–protein interaction mapping via MS2- or Cas13-based APEX targeting. Proc. Natl Acad. Sci. USA https://doi.org/10.1073/pnas.2006617117 (2020)."),[150](https://www.nature.com/articles/s41556-020-00620-7#ref-CR150 "Myers, S. A. et al. Discovery of proteins associated with a predefined genomic locus via dCas9–APEX-mediated proximity labeling. Nat. Methods 15, 1 (2018).")), BirA[150](https://www.nature.com/articles/s41556-020-00620-7#ref-CR150 "Myers, S. A. et al. Discovery of proteins associated with a predefined genomic locus via dCas9–APEX-mediated proximity labeling. Nat. Methods 15, 1 (2018).")|HEK293T[141](https://www.nature.com/articles/s41556-020-00620-7#ref-CR141 "Han, S. et al. RNA–protein interaction mapping via MS2- or Cas13-based APEX targeting. Proc. Natl Acad. Sci. USA https://doi.org/10.1073/pnas.2006617117 (2020)."),[150](https://www.nature.com/articles/s41556-020-00620-7#ref-CR150 "Myers, S. A. et al. Discovery of proteins associated with a predefined genomic locus via dCas9–APEX-mediated proximity labeling. Nat. Methods 15, 1 (2018)."), mESCs[150](https://www.nature.com/articles/s41556-020-00620-7#ref-CR150 "Myers, S. A. et al. Discovery of proteins associated with a predefined genomic locus via dCas9–APEX-mediated proximity labeling. Nat. Methods 15, 1 (2018).")||||Tracked histone dynamics[150](https://www.nature.com/articles/s41556-020-00620-7#ref-CR150 "Myers, S. A. et al. Discovery of proteins associated with a predefined genomic locus via dCas9–APEX-mediated proximity labeling. Nat. Methods 15, 1 (2018)."); RNA-protein interactions[141](https://www.nature.com/articles/s41556-020-00620-7#ref-CR141 "Han, S. et al. RNA–protein interaction mapping via MS2- or Cas13-based APEX targeting. Proc. Natl Acad. Sci. USA https://doi.org/10.1073/pnas.2006617117 (2020).")| |TF displacement[94](https://www.nature.com/articles/s41556-020-00620-7#ref-CR94 "Dall’Agnese, A. et al. Transcription factor-directed re-wiring of chromatin architecture for somatic cell nuclear reprogramming toward trans-differentiation. Mol. Cell 76, 453–472.e8 (2019)."),[95](https://www.nature.com/articles/s41556-020-00620-7#ref-CR95 "Shariati, S. A. et al. Reversible disruption of specific transcription factor–DNA interactions using CRISPR/Cas9. Mol. Cell 74, 622–633.e4 (2019).")|dCas[94](https://www.nature.com/articles/s41556-020-00620-7#ref-CR94 "Dall’Agnese, A. et al. Transcription factor-directed re-wiring of chromatin architecture for somatic cell nuclear reprogramming toward trans-differentiation. Mol. Cell 76, 453–472.e8 (2019)."),[95](https://www.nature.com/articles/s41556-020-00620-7#ref-CR95 "Shariati, S. A. et al. Reversible disruption of specific transcription factor–DNA interactions using CRISPR/Cas9. Mol. Cell 74, 622–633.e4 (2019).")|IMR90 (ref. [94](https://www.nature.com/articles/s41556-020-00620-7#ref-CR94 "Dall’Agnese, A. et al. Transcription factor-directed re-wiring of chromatin architecture for somatic cell nuclear reprogramming toward trans-differentiation. Mol. Cell 76, 453–472.e8 (2019).")), mESCs[95](https://www.nature.com/articles/s41556-020-00620-7#ref-CR95 "Shariati, S. A. et al. Reversible disruption of specific transcription factor–DNA interactions using CRISPR/Cas9. Mol. Cell 74, 622–633.e4 (2019).")|(+): _Pax6_ (ref. [95](https://www.nature.com/articles/s41556-020-00620-7#ref-CR95 "Shariati, S. A. et al. Reversible disruption of specific transcription factor–DNA interactions using CRISPR/Cas9. Mol. Cell 74, 622–633.e4 (2019).")), _Nanog_[95](https://www.nature.com/articles/s41556-020-00620-7#ref-CR95 "Shariati, S. A. et al. Reversible disruption of specific transcription factor–DNA interactions using CRISPR/Cas9. Mol. Cell 74, 622–633.e4 (2019).") (0): _TNNT2_ (ref. [94](https://www.nature.com/articles/s41556-020-00620-7#ref-CR94 "Dall’Agnese, A. et al. Transcription factor-directed re-wiring of chromatin architecture for somatic cell nuclear reprogramming toward trans-differentiation. Mol. Cell 76, 453–472.e8 (2019).")); (−): _ITGA7_ (ref. [94](https://www.nature.com/articles/s41556-020-00620-7#ref-CR94 "Dall’Agnese, A. et al. Transcription factor-directed re-wiring of chromatin architecture for somatic cell nuclear reprogramming toward trans-differentiation. Mol. Cell 76, 453–472.e8 (2019).")), _RDH5_ (ref. [94](https://www.nature.com/articles/s41556-020-00620-7#ref-CR94 "Dall’Agnese, A. et al. Transcription factor-directed re-wiring of chromatin architecture for somatic cell nuclear reprogramming toward trans-differentiation. Mol. Cell 76, 453–472.e8 (2019).")), _Utf1_ (ref. [95](https://www.nature.com/articles/s41556-020-00620-7#ref-CR95 "Shariati, S. A. et al. Reversible disruption of specific transcription factor–DNA interactions using CRISPR/Cas9. Mol. Cell 74, 622–633.e4 (2019).")), _Nanog_[95](https://www.nature.com/articles/s41556-020-00620-7#ref-CR95 "Shariati, S. A. et al. Reversible disruption of specific transcription factor–DNA interactions using CRISPR/Cas9. Mol. Cell 74, 622–633.e4 (2019).")|**TF binding**[94](https://www.nature.com/articles/s41556-020-00620-7#ref-CR94 "Dall’Agnese, A. et al. Transcription factor-directed re-wiring of chromatin architecture for somatic cell nuclear reprogramming toward trans-differentiation. Mol. Cell 76, 453–472.e8 (2019)."),[95](https://www.nature.com/articles/s41556-020-00620-7#ref-CR95 "Shariati, S. A. et al. Reversible disruption of specific transcription factor–DNA interactions using CRISPR/Cas9. Mol. Cell 74, 622–633.e4 (2019).")|<1 h[95](https://www.nature.com/articles/s41556-020-00620-7#ref-CR95 "Shariati, S. A. et al. Reversible disruption of specific transcription factor–DNA interactions using CRISPR/Cas9. Mol. Cell 74, 622–633.e4 (2019).")|| |Genome reorganization[130](https://www.nature.com/articles/s41556-020-00620-7#ref-CR130 "Wang, H. et al. CRISPR-mediated programmable 3D genome positioning and nuclear organization. Cell 175, 1405–1417.e14 (2018).")|Heterodimerization domains|U2OS, HeLa|(0): _CXCR4_, _XIST_; (−): _ACAP2_, _PPP1R2_, _TFRC_, FP||1 day|Target bound DNA to various cellular compartments| |Phase separation[135](https://www.nature.com/articles/s41556-020-00620-7#ref-CR135 "Shin, Y. et al. Liquid nuclear condensates mechanically sense and restructure the genome. Cell 175, 1481–1491.e13 (2018).")|BRD4, FUS, TAF15|HEK293, HEK293T, NIH3T3, U2OS||Formation of condensates, repositioning of chromatin|<1 min|| |RNA localization[131](https://www.nature.com/articles/s41556-020-00620-7#ref-CR131 "Xu, X. et al. Gene activation by a CRISPR-assisted trans enhancer. eLife 8, e45973 (2019)."),[140](https://www.nature.com/articles/s41556-020-00620-7#ref-CR140 "Shechner, D. M., Hacisuleyman, E., Younger, S. T. & Rinn, J. L. Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display. Nat. Methods 12, 664–670 (2015).")|dCas[131](https://www.nature.com/articles/s41556-020-00620-7#ref-CR131 "Xu, X. et al. Gene activation by a CRISPR-assisted trans enhancer. eLife 8, e45973 (2019)."),[140](https://www.nature.com/articles/s41556-020-00620-7#ref-CR140 "Shechner, D. M., Hacisuleyman, E., Younger, S. T. & Rinn, J. L. Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display. Nat. Methods 12, 664–670 (2015).")|HEK293FT[140](https://www.nature.com/articles/s41556-020-00620-7#ref-CR140 "Shechner, D. M., Hacisuleyman, E., Younger, S. T. & Rinn, J. L. Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display. Nat. Methods 12, 664–670 (2015)."), HEK293T[131](https://www.nature.com/articles/s41556-020-00620-7#ref-CR131 "Xu, X. et al. Gene activation by a CRISPR-assisted trans enhancer. eLife 8, e45973 (2019)."), HepG2 (ref. [131](https://www.nature.com/articles/s41556-020-00620-7#ref-CR131 "Xu, X. et al. Gene activation by a CRISPR-assisted trans enhancer. eLife 8, e45973 (2019).")), A549 (ref. [131](https://www.nature.com/articles/s41556-020-00620-7#ref-CR131 "Xu, X. et al. Gene activation by a CRISPR-assisted trans enhancer. eLife 8, e45973 (2019).")), HeLa[131](https://www.nature.com/articles/s41556-020-00620-7#ref-CR131 "Xu, X. et al. Gene activation by a CRISPR-assisted trans enhancer. eLife 8, e45973 (2019)."), SKOV3 (ref. [131](https://www.nature.com/articles/s41556-020-00620-7#ref-CR131 "Xu, X. et al. Gene activation by a CRISPR-assisted trans enhancer. eLife 8, e45973 (2019).")), PANC-1 (ref. [131](https://www.nature.com/articles/s41556-020-00620-7#ref-CR131 "Xu, X. et al. Gene activation by a CRISPR-assisted trans enhancer. eLife 8, e45973 (2019).")), HT29 (ref. [131](https://www.nature.com/articles/s41556-020-00620-7#ref-CR131 "Xu, X. et al. Gene activation by a CRISPR-assisted trans enhancer. eLife 8, e45973 (2019)."))|FP (0)[140](https://www.nature.com/articles/s41556-020-00620-7#ref-CR140 "Shechner, D. M., Hacisuleyman, E., Younger, S. T. & Rinn, J. L. Multiplexable, locus-specific targeting of long RNAs with CRISPR-Display. Nat. Methods 12, 664–670 (2015).")|||| |Epitranscriptomics[139](https://www.nature.com/articles/s41556-020-00620-7#ref-CR139 "Liu, X.-M., Zhou, J., Mao, Y., Ji, Q. & Qian, S.-B. Programmable RNA N6-methyladenosine editing by CRISPR–Cas9 conjugates. Nat. Chem. Biol. 15, 865–871 (2019).")|METTL3 + METTL14/ALKBH5/FTO|HeLa|FP (0)|RNA m6A, **mRNA stability**; (METTL3 + METTL14); **RNA m****6****A**, **HNRNPC binding** (ALKBH5, FTO)||| |Artificial chromatin[136](https://www.nature.com/articles/s41556-020-00620-7#ref-CR136 "Park, M., Patel, N., Keung, A. J. & Khalil, A. S. Engineering epigenetic regulation using synthetic read–write modules. Cell 176, 227–238.e20 (2019).")|Dam|HEK293FT||GATC m6A|Variable|Used reader effectors to activate or repress GFP reporter| 1. CRISPRa indicates the use of domains including VP16, VPR, and the SAM system or variants thereof. Certain cell types indicated as follows: ESCs, embryonic stem cells; epiSCs, epiblast-derived stem cells; HSCs, hematopoetic stem cells; EFs, embryonic fibroblasts; m preceding these cell types indicates cells of mouse origin (otherwise human). The degree of targeted gene regulation is indicated as follows: (0), <2-fold activation or repression; (+), 2–10-fold activation; (++), >10–30-fold activation; (+++), >30-fold activation; (−), 2–10-fold repression; (−−), 10-30-fold repression; (−−−), >30-fold repression. FP indicates a _trans_ gene, such as a fluorescent protein. Epigenetic changes are highlighted as follows: bold text, reduction of the specified mark at the locus; black text, increase of specified mark; underscored text: measured, but no change observed. Persistence means persistence of the effects post-transfection or after removal of effector. 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