Epigenetic Targeting of Granulin in Hepatoma Cells by Synthetic CRISPR dCas9 Epi-suppressors

Abstract

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.

Targeting the IGF1R Pathway in Breast Cancer Using Antisense lncRNA-Mediated Promoter cis Competition

“… 30 , 31 , 32 By tethering the C terminus of the catalytically inactive dCas9 to three epigenetic suppressor domains, we found that the synthetic dCas9 epi-suppressor gRNAs induced significant decreases in the transcription activity of the GRN target gene promoter in Hep3B hepatoma cells. 33 In this study, we have successfully applied the Cas9 system to introduce a powerful promoter in front of the IRAIN lncRNA. The Cas9-mediated ALIC approach leads to an increase in IRAIN antisense lncRNA, which induces the downregulation of the sense IGF1R coding RNA through the cis transcription competition over the overlapping promoter.…”

A novel FLI1 exonic circular RNA promotes metastasis in breast cancer by coordinately regulating TET1 and DNMT1

“…ChIP assay was used to examine the binding of TET1 to the FLI1 promoter and was performed using the method as described previously [50]. In brief, FECR1-overexpressing and vector control cells were fixed with 1% formaldehyde, sonicated on ice for 180 s (10 s on and 10 s off), and immunoprecipitated with an anti-TET1 antibody (Invitrogen, CA).…”

CRISPR/Cas: From Tumor Gene Editing to T Cell-Based Immunotherapy of Cancer

“…The group then designed gRNAs specific to the GRN promoter. Epigenetic targeting of GRN decreased tumor cell growth compared with the random gRNA control and dCas9 control groups ( 36 – 38 ), thus introducing a powerful epigenetic tool for oncogenes’ inhibition.…”

“…Recently, the CRISPR/Cas9 system has shed light on the underlying epigenetic irregularities and rendered researchers able to target these irregularities using the CRISPR/Cas9 platform. Wang et al (36) targeted granulin (GRN), a liver cancer stem cell marker, epigenetically using the CRISPR/Cas9 system. The system consisted of C-terminus of the catalytically inactive dCas9 fused to three epigenetic suppressor domains: DNMT3a, histone 3 K27 methyltransferase EZH2, and heterochromatin binding suppressor KRAB.…”