On its quests to edit the epigenome like no biotechnology ever has, our deactivated Cas9 (dCas9) hero has come across many powerful effectors. When seeking out precision DNA demethylation, it has previously seen a multi TET1 approach. This makes use of both a dCas9 and TET1 catalytic domain fusion (dCas9-TET1) and also sgRNA 2.0 to recruit additional TET1 catalytic domains.
However, on this never ending quest of precision demethylation, a talented team from the lab of Izuho Hatada in Japan have decided to ramp up that demethylation power. In order to achieve such awesome effects, they used SunTag: a protein scaffold linked to dCas9. SunTag consists of a repeating peptide array that recruits many copies of an antibody-protein fusion, which allows for multiple copies of an effector domain to be tethered dCas9. Specifically, the repeating peptide is GCN4 and it recruits an anti-GCN4 single-chain variable fragment (scFv) fused to the effector domain, which in this case is TET1.
First, in their experiments, the team examined mouse embryonic stem cells. They targeted the STAT3 binding site of Gfap, where demethylation results in differentiation into astrocytes. The methylation levels were analyzed by combined bisulfite restriction analysis (COBRA).
- They used a more conventional dCas9-TET1 fusion to establish a comparison point and produced a 14% decrease in methylation.
- Then, they modified SunTag by changing the linker length between the peptide array from 5 to 22 basepairs to accommodate for the bulk of the TET1. This designer modification allows for up 5 TET1 catalytic domains to be recruited and it resulted in a 38% decrease in methylation.
Basking in the sunnyside of their new system, the team then targeted the methylation sensitive CTCF binding sites of the imprinted gene H19 and examined the effects using bisulfite sequencing for methylation and qPCR for gene expression. They observed extensive demethylation and a 1.7 fold increase in expression (with 2 fold being the theoretical maximum). They also found that when aiming for a large region, targeting multiple sites with multiple sgRNAs improves the demethylation outcome.
Further showing off the power of their system, they examined human cancer cell lines, where they found they could precisely target highly efficient demethylation and induce gene expression with large scales of fold change. Finally, they went in vivo and used electroporation to edit fetal mouse brains. Overall, this designer system has a lot to offer for functional genomics studies and provides a powerful epigenetic twist in the quest for dCas9 transcriptional activators.
Go learn how to erase methylation from your loci of interest over at Nature Biotechnology, August 2016