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.