In the newest spy movie hitting theaters this month, undercover agents sneak inside the innermost sanctum of fortress walls to hijack nuclear weapons…err nuclear machinery to upregulate gene expression. Spoiler alert, we’ll even reveal the agents’ identities: it’s catalytically dead Cas9 (dCas9) with a small molecule associate, teaming up to complete the mission.
While other researchers may have called this a Mission Impossible, a team led by Nathanial Hathaway (University of North Carolina, Chapel Hill, USA) and Jian Jin (Icahn School of Medicine, New York, USA) coordinated a cellular heist. They took advantage of “chemical epigenetic modifiers” (CEMs) to recruit endogenous chromatin machinery via a bromodomain or chromodomain warhead. The CEM molecule serves as a linker by additionally interacting with FK506-binding protein (FKBP), which is complexed with dCas9. All of this is assembled at the target gene promoter thanks to a guide RNA (gRNA) bringing dCas9 to the appropriate sequence.
Using a GFP reporter, they tested several variations of the system to optimize the recruitment of chromatin machinery and maximize gene expression, ultimately settling on an untagged dCas9 in combination with a modified gRNA that binds FKBP (dCas9/MS2-FKBPx2). Then they sent it out on assignment to activate endogenous genes.
Here’s the debrief:
- Using a gRNA targeting endogenous MYOD1, CEM87 treatment increases mRNA expression in a dose-dependent manner, with an optimal range between 50 nM and 200 nM
- CEM87 activates gene expression by recruiting the bromodomain BET family proteins
- Endogenous genes can be activated to varying degrees; treatment with 200 nM CEM87 increases expression of low- and moderate-expressing genes (MYOD1, IL1RN, OCT4) but not the highly expressed MYC1 gene
- Increased gene expression at the mRNA level is paralleled with increased protein expression
- Targeting CXCR4 for activation results in a 12.2-fold increase in RNA expression and a 5.6-fold increase in protein levels
Although the dCas9-CEM system was not the strongest activator of gene expression they tested, this technology is unique in its ability to incrementally adjust expression via the CEM dosage. Ultimately, this will make it a valuable tool for understanding the relationship between gene expression level and phenotype in development and disease.
Read the full dosage dossier in Nature Biotechnology, November 2019.