The CRISPR-associated Cas9 system can modulate disease-causing alleles both in vivo and ex vivo, raising the possibility of therapeutic genome editing. In addition to gene targeting, epigenetic modulation by the catalytically inactive dCas9 may also be a potential form of
cancer therapy.
Granulin (GRN), a potent pluripotent
mitogen and
growth factor that promotes
cancer progression by maintaining self-renewal of hepatic stem
cancer cells, is upregulated in
hepatoma tissues and is associated with decreased
tumor survival in patients with
hepatoma. We synthesized a group of dCas9 epi-suppressors to target GRN by tethering the C terminus of dCas9 with three epigenetic suppressor genes: DNMT3a (
DNA methyltransferase), EZH2 (
histone 3
lysine 27
methyltransferase), and KRAB (the Krüppel-associated box transcriptional repression domain). In conjunction with guide RNAs (gRNAs), the dCas9 epi-suppressors caused significant decreases in GRN
mRNA abundance in Hep3B
hepatoma cells. These dCas9 epi-suppressors initiated de novo CpG DNA methylation in the GRN promoter, and they produced
histone codes that favor gene suppression, including decreased H3K4 methylation, increased H3K9 methylation, and enhanced HP1a binding. Epigenetic knockdown of GRN led to the inhibition of cell proliferation, decreased
tumor sphere formation, and reduced cell invasion. These changes were achieved at least partially through the
MMP/TIMP pathway. This study thus demonstrates the potential utility of using dCas9 epi-suppressors in the development of epigenetic targeting against
tumors.