While it’s never a great idea to stare directly into the sun, a DNA methyltransferase (DNMT) eclipse brought to you by the SunTag system may just steal your eyes during its totality. This designer epigenome editing system comes to you from the labs of Margaret A. Goodell and Wei Li at Baylor College of Medicine (Texas) and shades out CpG sites in a manner that’s sure to leave a mark.
The dCas9-SunTag-DNMT3A system utilizes deactivated Cas9 (dCas9) fused to a repetitive protein scaffold known as SunTag, which recruits multiple copies of effector domains fused to antibodies that recognize SunTag. While previous studies have applied this system for some precision DNA demethylation, this new study represents the first application of SunTag to power up precision DNA methylation.
Here’s what the talented team discovered when targeting a CpG island in the promoter and first exon of HOXA5 in human embryonic kidney (HEK293T) cells:
- Amplicon bisulfite sequencing revealed that methylation levels increased drastically from around 2-5% to 80%
- RT-qPCR revealed a matching 80% decrease in gene expression
- The effects don’t end there, as the methylation spread over a 4.5-kb window of the HOXA5 gene and into the neighboring HOXA6 gene
- Interestingly, the methylation wasn’t limited to CpGs, as even some CpHs (H = A or C or T) became methylated
- To look for genome-wide off-target effects the group used reduced representation bisulfite sequencing (RRBS) and RNA-Seq to demonstrate minimal off-target effects
The team also set their sights on a different feature; a CpG canyon belonging to the gene KLF4, which becomes methylated in a number of tumour types. By targeting this feature with dCas9-SunTag-DNMT3A, they caused a more localized, but dramatic increase in methylation with only a slight change in expression.
Overall, the dCas9-SunTag-DNMT3A system offers precision methylation with minimal off-target effects and demonstrates that multimerization of DNA methyltransferases can be vital for large-scale repressive effects.
Go stare at the SunTag over at Genome Biology, September 2017!