Almost immediately after its inception as a biotechnological tool, CRIPSR/Cas9 showed that it was more than just a genome editor. Deactivated Cas9 (dCas9) is a precision RNA-guided DNA-binding domain with a growing range of effector domains that can be swapped out like LEGO.
When it comes to epigenome editing with dCas9, histones have led the way. dCas9-LSD1 induces repressive histone methylation and dCas9-p300 induces activating histone acetylation. However, DNA methylation has been surprisingly absent from the dCas9 party…until now.
A talented team from Croatia has brought us the long awaited dCas9-DNMT3A, in which linkers fuse the catalytic region of DNMT3A to dCas9. When put in a cell, dCas9-DNMT3A recruits another DNMT3A (to form a functional dimer) as well as (catalytically inactive but stimulatory) DNMT3L.
The team provided some proof-of-principle applications by transfecting HEK293 cells. They targeted gene promoters (BACH2 and IL6ST) and followed up with pyrosequencing.
Here’s what they found:
- A single construct (~10 kb) delivers targeted CpG methylation in a ∼35 bp region that is centered 27 bp downstream from the protospacer adjustment motif (PAM).
- However, the study did note some secondary activity 25 bp upstream.
- Co-expressing multiple distinct sgRNAs for a target promoter allowed for synergistic methylation of a wider region and also results in gene repression.
- By tracking the temporal dynamics, they observed that methylation levels increased over the first few days, hit a peak after 6-7 days, and then began to decrease until reaching a plateau.
dCas9-DNMT3A ultimately offers up a new tool for both functional genomics and the development of precision medicine. One day the authors hope that it will allow for epigenetic therapy that doesn’t depend on global alterations to the epigenome. For now, we’re just left wondering when TET and DNA Demethylation will join the dCas9 party.
Learn on about precisely inducing DNA methylation in Nucleic Acids Research, March 2016.