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.

CRISPR相关的Cas9系统可以在体内和体外调节致病等位基因，从而提高了基因组编辑治疗的可能性。除了基因靶向外，通过催化失活的dCas9进行的表观遗传调控也可能是癌症治疗的一种潜在形式。颗粒蛋白（GRN）是一种有效的多能促分裂原和生长因子，可通过维持肝干癌细胞的自我更新来促进癌症进展，在肝癌组织中上调，并与肝癌患者的肿瘤存活率降低相关。我们合成了一组靶向GRN的dCas9 Epi抑制子通过将dCas9的C末端与三个表观遗传抑制基因相连：DNMT3a（DNA甲基转移酶），EZH2（组蛋白3赖氨酸27甲基转移酶）和KRAB（与Krüppel相关的盒转录抑制域）。结合指导RNA（gRNA），dCas9 Epi抑制因子导致Hep3B肝癌细胞中GRN mRNA丰度大大降低。这些dCas9 Epi抑制剂在GRN启动子中启动了从头CpG DNA甲基化，并产生了有利于基因抑制的组蛋白编码，包括降低的H3K4甲基化，增加的H3K9甲基化和增强的HP1a结合。GRN的表观遗传敲低导致抑制细胞增殖，减少肿瘤球形成和减少细胞侵袭。这些变化至少部分是通过MMP / TIMP途径实现的。因此，这项研究证明了在开发针对肿瘤的表观遗传靶标中使用dCas9 Epi抑制子的潜在实用性。