Every researcher loves a good tool, and lately, it seems that CRISPR might be an even more modular tool than PCR. Attesting to the modularity of CRISPR/Cas9 is catalytically deactivated Cas9 (dCas9). dCas9 has the precision of Cas9, when combined with sgRNA, and can be fused to a modular effector domain that allows for extra functions like epigenome editing. Now, thanks to a talented team from China, our dCas9 tool-kit has grown even larger with TET1 offering up precision DNA demethylation.
The dCas9 Epigenome Editing Effector Toolbox
So far, a number of approaches have paved the way to control the epigenome at desired regulatory elements and thus control resulting transcription. Here are a few examples of what effectors the dCas9 epigenome editing toolbox has in store:
- dCas9-LSD1 brings forth histone methylation for precision gene expression repression.
- dCas9-p300 results in histone acetylation that precisely activates gene expression.
- dCas9-DNMT3A utilizes DNA methylation to precisely repress gene expression, as shown by more than one group.
However, the concept of epigenome editing isn’t new to CRISPR/Cas9 and previous genome editing technologies, such as Zinc Fingers (ZFs) and Transcription Activator-Like Effectors (TALEs), have long been showing off the potential for precision modification of the epigenome.
Inducing DNA Demethylation with TET Enzymes
One epigenome editing application that really grabbed our attention was the use of the TALE system and the TET enzyme family. TET enzymes are responsible for active DNA demethylation, where a series of oxidative reactions gradually remove DNA methylation and produce by-products, such as hydroxymethylation, which can also serve as epigenetic marks in their own right.
Both TALE-TET1 and TALE-TET2 approaches have shown that the catalytic domains of the TET enzymes can be fused to a DNA-binding domain from a genome editing system, where they result in DNA demethylation and subsequently activate gene expression. However, ZFs and TALEs rely on protein engineering for their DNA binding, which is much more time consuming and limiting than designing a sgRNA.
Here are the features of the dCas9 system:
- dCas9 is fused to the catalytic domain (CD) of TET1 by a flexible linker.
- The sgRNA makes uses of a previously described upgrade, known as sgRNA 2.0.
- sgRNA 2.0 contains specific RNA sequences in choice locations, which can be recognized by an MS2 coat protein that is fused to the effector domain (TET1-CD).
- Thus, there is a double whammy of DNA demethylation caused by the TET1-CD coming from dCas9 and the multiple MS2 coat proteins.
In order to demonstrate the applicability of this system to humans, the team turned to human cells lines. Here’s the scoop:
- The genes chosen for sgRNA design were known to be repressed by DNA hypermethylation, which was also confirmed by 5-aza treatment.
- By targeting CpGs in the promoters of RANKL, MAGEB2, or MMP2, they induced DNA demethylation and activated transcription.
- In their experiments, they carried out a number of optimizations related to the region of sgRNA targeting, titers, and time course.
- When the optimal design was reached, the system behaved as expected and only “tenuous off-target effects” were observed.
- The dCas9-TET1 system was shown to work in HEK-293FT, SH-SY5Y, and HeLa cell lines.
- The team also confirmed the functionality of their system by deactivating the TET1-CD.
Ultimately, this designer system demonstrates the powerful potential that sgRNA 2.0 and aptamers offer up to any CRISPR-based system. dCas9-TET1’s ability to easily target genes of choice will enable new experimentation into the basic functional roles of DNA demethylation, such as screens of gene promoters. It could also enable the development of precision medicine.
Go learn how to demethylate your gene of choice over at Cell Discovery, May 2016