CRISPR 101: Editing the Epigenome

Posted by Mary Gearing on Feb 14, 2017 10:44:08 AM


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.

Maeder et al. 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. Mendenhall et al. additionally fused a TAL effector to LSD1 histone demethylase to demethylate enhancer regions. 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! 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 avaliable from Addgene. For most of these constructs, catalytically dead modifiers are also available as controls.

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 the recently published pLV-dCas9-p300-P2A-PuroR for lentiviral expression.

CRISPR-acetylation.png

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.

CRISPR-demethylation.png

Repression

DNA Methyltransferase 3 Alpha

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.

CRISPR-methylation.png

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. Stolzenburg et al. compared ZFN-KRAB and ZFN-DNMT3A, finding that 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 - Hilton et al. found that 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!

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References

1. Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. 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. Nat Biotechnol. 2013 Oct 9. PMID: 24108092

2. Locus-specific editing of histone modifications at endogenous enhancers. Mendenhall EM, Williamson KE, Reyon D, Zou JY, Ram O, Joung JK, Bernstein BE. Nat Biotechnol. 2013 Sep 8. PMID: 24013198

3. A CRISPR-based approach for targeted DNA demethylation. Xu X, Tao Y, Gao X, Zhang L, Li X, Zou W, Ruan K, Wang F, Xu GL, Hu R. Cell Discov. 2016 May 3;2:16009. PMID: 27462456

4. Editing DNA Methylation in the Mammalian Genome. Liu XS, Wu H, Ji X, Stelzer Y, Wu X, Czauderna S, Shu J, Dadon D, Young RA, Jaenisch R. Cell. 2016 Sep 22;167(1):233-247.e17. PMID: 27662091

5. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Hilton IB, D'Ippolito AM, Vockley CM, Thakore PI, Crawford GE, Reddy TE, Gersbach CA. Nat Biotechnol. 2015 May;33(5):510-7. doi: 10.1038/nbt.3199. PMID: 25849900

6. CRISPR-Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Klann TS, Black JB, Chellappan M, Safi A, Song L, Hilton IB, Crawford GE, Reddy TE, Gersbach CA. Nat Biotechnol. 2017 Apr 3. doi: 10.1038/nbt.3853. PMID: 28369033

  • Find plasmids from this publication at Addgene. 

7. Repurposing the CRISPR-Cas9 system for targeted DNA methylation. Vojta A, Dobrinic P, Tadic V, Bockor L, Korac P, Julg B, Klasic M, Zoldos V. Nucleic Acids Res. 2016 Mar 11. PMID: 26969735

8. Stable oncogenic silencing in vivo by programmable and targeted de novo DNA methylation in breast cancer. Stolzenburg S, Beltran AS, Swift-Scanlan T, Rivenbark AG, Rashwan R, Blancafort P. Oncogene. 2015 Oct;34(43):5427-35. PMID: 25684141

9. Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation. McDonald JI, Celik H, Rois LE, Fishberger G, Fowler T, Rees R, Kramer A, Martens A, Edwards JR, Challen GA. Biol Open. 2016 May 11. PMID: 27170255

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