CRISPR 101: Epigenetics and Editing the Epigenome

Posted by Mary Gearing on Jun 24, 2020 1:45:00 PM


Originally published Feb 14, 2017 and updated Jun 24, 2020.

Epigenetic modifications are an additional layer of control over gene expression that go beyond genomic sequence. Dysregulation of the epigenome (the sum of epigenetic modifications across the genome) has been implicated in disease states, and targeting the epigenome may make certain processes, like cellular reprogramming of iPSCs, more efficient. In general, epigenetic chromatin modifications are correlated with alterations in gene expression, but causality and mechanisms remain unclear. Today, targeted epigenetic modification at specific genomic loci is possible using CRISPR, and Addgene has a number of tools for this purpose.

Epigenetics began as a correlative field in which covalent modifications to DNA or histones, the proteins that help package DNA, were associated with gene expression or silencing. To alter DNA modifications, researchers used blunt tools like histone deacetylases, but targeted epigenetic modification was impossible. With the genome engineering revolution came epigenome-engineering tools - zinc finger nucleases and TALENs fused to epigenetic modifiers enabled epigenetic modifications at a user-specified locus.

Researchers showed that TALE-TET1 constructs, which fused a TALEN to the Tet1 demethylase catalytic domain, could mediate demethylation and induce transcription at CpG regions of various promoters. Other researchers additionally fused a TAL effector to LSD1 histone demethylase to demethylate enhancer regions (Mendenhall et al., 2013). By comparing gene activation when enhancers were active or silent, they could identify the target genes of previously uncharacterized enhancers. The popular TALEN-based LITE system, which uses light to regulate transcription, also includes light-regulated histone methyltransferases and deacetylases.

CRISPR and epigenetics

As with many TALEN-based technologies, the advent of CRISPR has made targeting much easier! With the help of guide RNAs, fusions between Cas proteins and the epigenetic modifier could be targeted to specific DNA sequences. Non-editing CRISPR applications direct catalytically dead dCas9 fused to a variety of epigenetic modifiers to specific loci without inducing double strand breaks. Below you'll find some of the CRISPR-based epigenetic modifiers available from Addgene. For most of these constructs, catalytically dead modifiers are also available as controls. For an up-to-date list of CRISPR epigenetic tools, check out our CRISPR Epigenetics resource.

Transcriptional activation

p300 acetyltransferase

dCas9 fused to the catalytic domain of p300 acetyltransferase increases levels of H3K27ac histone modification at specified loci. Charles Gersbach’s lab has deposited mammalian expression constructs including pcDNA-dCas9-p300 Core and pcDNA3.3-Nm-dCas9-p300 Core, as well as pLV-dCas9-p300-P2A-PuroR for lentiviral expression.

CRISPR acetylation

Tet1 demethylase

Ronggui Hu’s lab has created pdCas9-Tet1-CD for targeted cytosine demethylation in mammalian cells. This plasmid is used with pcDNA3.1-MS2-Tet1-CD to decrease methylation and activate transcription. A lentiviral vector with the same modifier, Fuw-dCas9-Tet1CD, is available from Rudolf Jaenisch’s lab in plasmid form or as ready-to-use lentivirus.

CRISPR demethylation

Tet1 initiates cytosine demethylation of DNA. However, several proteins in the DNA oxidation and repair pathways work downstream of Tet1 to restore the DNA after cytosine removal. Recently, Albert Cheng’s lab developed Casilio-ME, which is based on their Casilio system. This system allows for targeted delivery of Tet1 alone, or coupled with DNA oxidation and repair factors that allow for increased gene activation at the targeted site compared to other Tet1 delivery systems.

Transcriptional repression

DNA Methyltransferase 3 Alpha (DNMT3A)

Vlatka Zoldoš’ lab has deposited pdCas9-DNMT3A-EGFP and pdCas9-DNMT3A-PuroR for targeted cytosine methylation in mammalian cells. Co-expression markers EGFP and PuroR enable sorting and selection of transduced cells. Grant Challen’s lab also created constitutive (pCMV-dCas9-D3A) and Tet-dependent (TetO-dCas9-D3A) constructs. For lentiviral expression, Fuw-dCas9-Dnmt3a and Fuw-dCas9-Dnmt3a-P2A-tagBFP are available from Rudolf Jaenisch’s lab, with the former also available as ready-to-use lentivirus.

CRISPR methylation

DNA Methyltransferase MQ1

Margaret Goodell's lab has deposited pcDNA3.1-dCas9-MQ1(Q147L)-EGFP, a fusion of dCas9 to a small DNA methyltransferase from the prokaryote Mollicutes spiroplasma (M. Sss1) (termed MQ1.) The Q147L mutation improves methylation kinetics such that cytosine methylation occurs within 24 hours rather than over a period of several days, as seen with other epigenome-editing tools. pLV hUbC-dCas9-MQ1(Q147L)-EGFP is also available for lentiviral transduction.

Lysine-specific Demethylase 1 (LSD1)

Tatjana Sauka-Spengler's lab has deposited pX330a dCas9-LSD1 for targeted removal of H3K4me1/2 and H3K9me2 histone modifications. Like the TALE-LSD1 system described above, dCas9-LSD1 inactivates targeted enhancers. Although the lab used this vector in chick embryos, it also functions in mammalian expression systems.
 
Christopher Newgard’s Lab has fused LSD1 to the smaller nuclease-dead Staphylococcus aureus Cas9 (Sa-Cas) and deposited plasmid IF311: pMAGIC (R4-R3) NLS-Sa dCas9-NLS-LSD1. LSD1 is a particularly large protein, and this fusion allows for packaging into viral vectors and targeted delivery into hard-to-manipulate cell types. They have also fused LSD1 to x-Cas9(3.7). x-Cas9(3.7) is a SpCas9 mutant that exhibits increased PAM flexibility, and this fusion protein allows for LSD1 targeting to a wider set of genomic targets. These two vectors are part of the larger pMVP/pMAGIC cloning system, which contains Cas9 fusions for several epigenetic modifiers, and is available as a kit from Addgene.
 

Find Ready-to-Use Viral Preps for Epigenetic Modification

Why use epigenetic modifiers?

Epigenetic modification is certainly not the only CRISPR-based technology designed to alter gene expression. Fusing dCas9 to a transcriptional activator like VP64 or VPR activates transcription, whereas dCas9-KRAB fusions repress transcription. Both of these methods also recruit epigenetic machinery - but is there an advantage to using direct epigenetic modifiers?

As with any experiment, your desired outcome will determine the tool that you should use. If you want to study the effects of one particular modification for which a targeted editor, like H3K27ac, is available, an epigenetic tool would be your best bet.

Another potential advantage of CRISPR epigenetic tools is their persistence and inheritance. CRISPR activators and repressors are thought to be reversible once the effector is inactivated/removed from the system. In contrast, epigenetic marks left by targeted epigenetic modifiers may be more frequently inherited by daughter cells. For example, KRAB induced silencing was transient and quickly reversed in culture. However, DNMT3A-induced methylation persisted throughout a 100 day experimental period, as this mark was faithfully propagated in culture and in vivo.

In certain cases, epigenetic modifiers may work better than activators/repressors - dCas9-p300 increased transcriptional activation more than dCas9-VP64, especially when targeting distal enhancers. As the effects of these tools are likely cell type- and context-dependent, it may make sense to try multiple CRISPR tools when setting up your experimental system. Let us know about your experience with these constructs in the comments!

Find Plasmids for Epigenetic Modification

Click to Download Addgene's CRISPR 101 eBook

 


Leah Schwiesow contributed to updating this article.

References

Haldeman JM, Conway AE, Arlotto ME, et al (2018) Creation of versatile cloning platforms for transgene expression and dCas9-based epigenome editing. Nucleic Acids Research 47:e23–e23. https://doi.org/10.1093/nar/gky1286

Hilton IB, D’Ippolito AM, Vockley CM, Thakore PI, Crawford GE, Reddy TE, Gersbach CA (2015) Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nature Biotechnology 33:510–517 . https://doi.org/10.1038/nbt.3199

Klann TS, Black JB, Chellappan M, Safi A, Song L, Hilton IB, Crawford GE, Reddy TE, Gersbach CA (2017) CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nature Biotechnology 35:561–568 . https://doi.org/10.1038/nbt.3853

  • Find plasmids from this publication at Addgene. 

Lei Y, Zhang X, Su J, Jeong M, Gundry MC, Huang Y-H, Zhou Y, Li W, Goodell MA (2017) Targeted DNA methylation in vivo using an engineered dCas9-MQ1 fusion protein. Nature Communications 8: . https://doi.org/10.1038/ncomms16026

  • Find plasmids from this publication at Addgene.

Liu XS, Wu H, Ji X, Stelzer Y, Wu X, Czauderna S, Shu J, Dadon D, Young RA, Jaenisch R (2016) Editing DNA Methylation in the Mammalian Genome. Cell 167:233–247.e17 . https://doi.org/10.1016/j.cell.2016.08.056

Maeder ML, Angstman JF, Richardson ME, Linder SJ, Cascio VM, Tsai SQ, Ho QH, Sander JD, Reyon D, Bernstein BE, Costello JF, Wilkinson MF, Joung JK (2013) Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. Nature Biotechnology 31:1137–1142 . https://doi.org/10.1038/nbt.2726

McDonald JI, Celik H, Rois LE, Fishberger G, Fowler T, Rees R, Kramer A, Martens A, Edwards JR, Challen GA (2016) Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation. Biology Open 5:866–874 . https://doi.org/10.1242/bio.019067

Mendenhall EM, Williamson KE, Reyon D, Zou JY, Ram O, Joung JK, Bernstein BE (2013) Locus-specific editing of histone modifications at endogenous enhancers. Nature Biotechnology 31:1133–1136 . https://doi.org/10.1038/nbt.2701

Stolzenburg S, Beltran AS, Swift-Scanlan T, Rivenbark AG, Rashwan R, Blancafort P (2015) Stable oncogenic silencing in vivo by programmable and targeted de novo DNA methylation in breast cancer. Oncogene 34:5427–5435 . https://doi.org/10.1038/onc.2014.470

Taghbalout A, Du M, Jillette N, et al (2019) Enhanced CRISPR-based DNA demethylation by Casilio-ME-mediated RNA-guided coupling of methylcytosine oxidation and DNA repair pathways. Nature Communications 10:. https://doi.org/10.1038/s41467-019-12339-7

Vojta A, Dobrinić P, Tadić V, Bočkor L, Korać P, Julg B, Klasić M, Zoldoš V (2016) Repurposing the CRISPR-Cas9 system for targeted DNA methylation. Nucleic Acids Research 44:5615–5628 . https://doi.org/10.1093/nar/gkw159

Williams RM, Senanayake U, Artibani M, Taylor G, Wells D, Ahmed AA, Sauka-Spengler T (2017). Genome and epigenome engineering CRISPR toolkit for probing in vivo cis-regulatory interactions in the chicken embryo. BioRxiv: https://doi.org/10.1101/135525.

  • Find plasmids from this publication at Addgene.

Xu X, Tao Y, Gao X, Zhang L, Li X, Zou W, Ruan K, Wang F, Xu G, Hu R (2016) A CRISPR-based approach for targeted DNA demethylation. Cell Discovery 2: . https://doi.org/10.1038/celldisc.2016.9

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