NEW YORK – Researchers at the UNC Eshelman School of Pharmacy, the National Institutes of Health, and elsewhere have developed a CRISPR-Cas9-based system that uses chemical epigenetic modifiers (CEMs) to enable dose-dependent activation of gene expression without the use of exogenous transcription regulatory proteins.
As the researchers reported in a study published in Nature Biotechnology on Monday, the system is composed of a catalytically inactive Cas9 (dCas9) complexed with FK506-binding protein (FKBP) and a CEM consisting of FK506 linked to a molecule that interacts with cellular epigenetic machinery. The CEMs are designed to activate the expression of target genes by recruiting components of the endogenous chromatin-activating machinery, eliminating the need for exogenous transcriptional activators.
"We show that CEMs upregulate gene expression at target endogenous loci up to 20-fold or more depending on the gene," the authors wrote. "We also demonstrate dose-dependent control of transcriptional activation, function across multiple diverse genes, reversibility of CEM activity, and specificity of our best-in-class CEM across the genome."
Other studies have demonstrated that recruiting the exogenous chromatin-modifying machinery enables the control of expression levels in a gene-specific manner, the researchers said. Adding dCas9 allows for the ability to precisely induce changes in expression.
In a previous study, the scientists demonstrated the ability of CEMs to modify chromatin and subsequently repress gene expression at engineered reporter loci. For this new study, they developed CEM-activating (CEMa) molecules that recruit endogenous gene-activating machinery, including CEM87, CEM88, and CEM114, which each bind to different chromatin-modifying enzymes of bromodomain inhibitors of histone acetyltransferases (HATs) or acetylated lysine reader proteins. The CEMa family is also compatible with dCas9-FKBP-based systems, which allows them to be directed to any gene.
To test for changes in gene expression, the researchers transfected HEK293T cells with a green fluorescent protein (GFP) reporter gene downstream of the TRE3G promoter and performed flow cytometry on cells co-expressing the guide RNA (gRNA) and dCas9 machinery. They then tested activating the reporter gene with a plasmid expressing dCas9-FKBP×1 or dCas9-FKBP×2 and one of the three CEMa molecules. After 48 hours of exposure, normalized GFP reporter expression was significantly increased in all cases as compared to untreated cells, the researchers found.
To confirm that the CEMa system was activating GFP in a controlled, FKBP-dependent manner, they analyzed cells expressing dCas9 alone treated with CEMa for 48 hours and found that CEM treatment did not significantly change GFP expression. They also tested whether the activation was a result of the CEMa molecule as a whole by expressing dCas9-FKBP×2 in cells and treating the cells with the inhibitor from which CEM87 was synthesized, FK506, or CEM87, and found that treatment with CEM87 was the only condition that increased GFP expression.
After optimizing the dCas9 system, the researchers conducted a time course with CEM87 and CEM114, the two most effective CEMa molecules. They transfected cells with plasmids expressing dCas9, MS2-FKBP×2, and GFP with either targeting or non-targeting gRNA. In comparison to untreated cells, treatment of cells with both CEM87 and CEM114 yielded high GFP expression after 24 hours and 48 hours. Experiments to find the optimal dose curves from CEM87 and CEM114 revealed a hook effect near treatments of 400 to 800 nanomolars, where the CEMa treatment became less effective.
"This could be a result of active inhibition of the desired chromatin regulator machinery meant for recruitment," the authors speculated. "Alternatively, there could be toxicity at high concentrations."
Researchers could use the dCas9-CEMa platform to control gene activity in a dose-dependent manner by varying the compound dose, they added, which could be useful for target validation studies.
The researchers then sought to determine how stable the activation of CEM87 and CEM114 is without constant CEMa treatment. They switched the CEM-containing medium with untreated medium or with medium containing excess FK506 and found that cells treated with CEM87 or CEM114 for 48 hours had significantly increased GFP expression. After two days of continued treatment, no treatment, or exposure to excess FK506, the cells under each condition continued to exhibit significantly activated GFP expression. After four days, the cells under all conditions continued to have significant activation.
However, after six days of excess FK506 in cells treated with CEM87 or CEM114 and after six days of no treatment in cells treated with CEM87, the increase in expression was no longer significant, indicating that the effects of the dCas9-CEMa system are reversible.
The study the versatility of the dCas9-CEMa system, the researchers tested it with gRNAs targeting the interleukin-1 receptor antagonist (IL1RN) locus, a weakly expressed gene. IL1RN expression in cells treated with CEM87 increased by nearly 93-fold, compared to control cells. The team also targeted the MYC1 locus, an area of the genome that is more highly expressed than the other targets, and found that CEM87 did not significantly increase its expression.
"By adapting the CEMa technology to dCas9 targeting constructs, we can use this system to theoretically target any gene in the genome by strategic gRNA design," the authors concluded. "We have demonstrated the ability to control the chromatin landscape and induce changes in the expression of endogenous mammalian disease-related genes in a direct, biologically relevant manner. This dCas9-CEMa technology paves the way for targeting disease-relevant genes to ask specific, pointed questions about the mechanisms of action in disease."