br Conflicts of interest br Acknowledgments This

Conflicts of interest
Acknowledgments This work was supported by the Project of Huzhou Municipal Science and Technology Bureau of Zhejiang Province (No. 2016GY45 to YGC) and he Project of Zhejiang Basic Public Benefit research of Zhejiang Province (No. LGF18H160005 to YGC)
Introduction With the rapid development of genomics, scientists have identified a great number of epigenetic marks in the genomes [1,2], and it is closely tied with cellular processes and diseases [3,4]. Investigating the function of the epigenetic marks and exploring how to manipulate them by gene editing tools are indispensable and beneficial for human health. Epigenetic manipulation has enormous potential in gene-associated disease therapy, animal disease modelling, drug screening and regenerative medicine. One of the major challenges of functional genomics is to invent technologies which can precisely regulate the epigenetic modifications. Previously, researchers mainly focused on small BMS-626529 regulating epigenetics, such as DNA methyltransferases and histone demethylase inhibitors. However, the utilization of these molecules is limited by their general change of epigenome and transcriptome. Notably, the targeted epigenome editing technologies by specific DNA-binding domains, such as ZFN and TALEs fused with epigenome-modifying enzymes, partially address this limitation. CRISPR/Cas9 system is the third generation of gene-editing technology after ZFN and TALEs, which only requires a Cas9 protein to target the specific genomic loci under the guidance of single-guide RNAs. Compared with the ZFN, TALEs and small molecule drugs, CRISPR/Cas9 system is of great convenience [[5], [6], [7]]. Moreover, the nuclease-inactive Cas9 (dead Cas9, dCas9) is engineered to fuse with epigenetic writers for the purpose of epigenetic modification and transcriptional regulation. The burgeoning dCas9-based epigenetic modification system can be a potential and powerful tool for modifying epigenetic marks. For example, by fusion with Kruppel-associated box (KRAB) or oligomers of p65 [8], fused dCas9 acts as a regulator of genes expression on specific loci guided by sgRNAs. Currently, the fusion of dCas9 with epigenetic modification enzymes, such as acetyltransferase p300 and DNA methylase DNMT3A, have been carried out to exert great epigenetic modification. However, the CRISPR/dCas9 system for histone methylation is rarely reported. Adipocytes are critical not only in maintaining lipid homeostasis and energy balance but also in producing and secreting important signaling molecules. In the process of adipocyte differentiation, a series of genes, which express in different spatiotemporal sequences, promoted lipid droplet accumulation and morphological changes. The C/EBPs family participates in cell proliferation and differentiation, metabolism, and inflammatory reactions, especially in hepatocytes, adipose cells and hematopoietic cells. C/ebpα is the first transcription factor that has been proved to play a fundamental role during adipocyte differentiation [9,10]. Epigenetic silencing of C/ebpα would be a potent strategy to tuning the adipogenic fate of adipogenic stem cells. In the current study, we repurposed the CRISPR/dCas9 system for epigenetic modification by including EZH2, a well-known histone methylation enzyme catalyzing trimethylation of H3K27 [11]. The system is comprised of three plasmids: a master vector (nuclease-null Cas9 protein), a sgRNA vector (the sgRNA incorporated with PP7 RNA aptamers) and an executive vector EZH2 fused to PCP. In the proof-of-concept study, we confirmed that CRISPR/dCas9 and EZH2 based epigenetic modification system (CRISPR/dCas9-EZH2 for short) targeting C/ebpα promoter can significantly down-regulate the expression of C/ebpα gene, leading to adipogenic differentiation inhibition of 3T3-L1 preadipocytes. Our study suggests that the CRISPR/dCas9-EZH2 system is a robust and potential epigenome-editing tool for manipulating histone methylation and knocking down gene expression.