Bioorthogonal Chemical Epigenetic Modifiers Enable Dose-Dependent CRISPR Targeted Gene Activation in Mammalian Cells

CRISPR-Cas9 systems have been developed to regulate gene expression by using either fusions to epigenetic regulators or, more recently, through the use of chemically mediated strategies. These approaches have armed researchers with new tools to examine the function of proteins by intricately controlling expression levels of specific genes. Here we present a CRISPR-based chemical approach that uses a new chemical epigenetic modifier (CEM) to hone to a gene targeted with a catalytically inactive Cas9 (dCas9) bridged to an FK506-binding protein (FKBP) in mammalian cells. One arm of the bifunctional CEM recruits BRD4 to the target site, and the other arm is composed of a bumped ligand that binds to a mutant FKBP with a compensatory hole at F36V. This bump-and-hole strategy allows for activation of target genes in a dose-dependent and reversible fashion with increased specificity and high efficacy, providing a new synthetic biology approach to answer important mechanistic questions in the future.