Development of a Chem-CRISPR/dCas9 system to achieve small-molecule-mediated epigenetic regulation at the single-gene level

表观遗传异常是人类癌症发生、发展和发展的主要驱动因素之一，也常常与获得性治疗抵抗有关。从临床前实验中已经报道了一些化学表观遗传调节剂。然而，动物模型和临床试验的结果表明，严重的靶上和靶外毒性是导致疫情发展失败的主要因素之一。CRISPR/dCas9技术为单基因水平的精确表观遗传调控提供了强有力的工具。这种方法需要异位表达外源表观遗传调节蛋白，这会导致副作用。我们打算开发一个Chem-CRISPR/dCas9平台，在该平台中，CRISPR/dCas9可以使化学表观遗传激活剂/抑制物接近所需的DNA序列，从而在单基因水平上实现表观遗传调控。在我们最近的工作中，我们测试了几种位置选择性化学修饰的方法，并成功地将TT-钳制系统适应于Chem-CRISPR/dCas9平台。我们证明了(D)Cas9标记Phe(F)-Cys(C)-Pro(P)-Phe(F)氨基酸序列是全氟芳香族化合物在细胞内特异性识别的。我们将FITC融合到FCPF结合剂(FITC-FCPF)中，实现了在活细胞中标记Cas9FCPF。我们开发了携带CRBN E3连接酶配体沙利度胺的PROTAC-FCPF异双功能分子，并证明了PROTAC-FCPF降解(D)Cas9FCPF和其他CasFCPF蛋白。在我们的初步工作中，我们将PAN BET抑制剂JQ1连接到FCPF结合蛋白(JQ1-FCPF)中，并在FRET实验中证明了JQ1-FCPF诱导了dCas9FCPF和BRD4WT的结合，这一点通过免疫共沉淀进一步得到证实。全基因组转录组学表明，C-MYC在JQ1-FCPF和Chem-CRISPR/dCas9FCPF系统中被特异性抑制，并有针对C-MYC启动子和增强子的sgRNAs。在这个拟议的项目中，我们将合成携带不同化学表观遗传调节子的新型FCPF偶联物，以展示Chem-CRISPR/dCas9FCPF系统在单基因水平上调控表观基因组编辑的潜力和应用。

Epigenetic aberration is one of the major driving factors in initiation, promotion, and progression of human cancer and is also often associated with acquired therapeutic resistance. A number of chemical epigenetic modulators have been reported from preclinical experiments. However, results from animal models and clinical trials implicated that severe on- and off-target toxicity is one of the leading factors related to failures of epidrug development. CRISPR/dCas9 technology provides a powerful tool for precisely epigenetic regulation at the single-gene level. This approach requires ectopic expression of exogenous epigenetic modulate proteins, which causes side effects. We intend to develop a Chem-CRISPR/dCas9 platform, in which CRISPR/dCas9 can bring chemical epigenetic activators/inhibitors in proximity to desired DNA sequences and thereby achieve epigenetic modulation at the single-gene level. In our recent work, we tested several approaches of site-selective chemical modifications and successfully adapted the TT-clamp system to Chem-CRISPR/dCas9 platform. We demonstrated that (d)Cas9 tagging the short Phe(F)-Cys(C)-Pro(P)-Phe(F) amino acid sequence was specifically recognized by perfluoroaromatics in cells. We fused FITC into the FCPF binder (FITC-FCPF) and achieved to label Cas9FCPF in live cells. We developed a PROTAC-FCPF heterobifunctional molecule carrying thalidomide, a ligand of CRBN E3 ligase, and demonstrated that PROTAC-FCPF degraded (d)Cas9FCPF and other CasFCPF proteins. In our preliminary work for this proposal, we conjugated JQ1, a pan BET inhibitor, into FCPF binder (JQ1-FCPF) and demonstrate that JQ1-FCPF induced proximity of dCas9FCPF and BRD4WT in FRET assay, which was further confirmed by co-immunoprecipitation. Genome-wide transcriptomics shows that C-MYC is specifically repressed in the presence of JQ1-FCPF and Chem-CRISPR/dCas9FCPF system with sgRNAs targeting promoters and enhancers of C-MYC. In this proposed project, we will synthesize novel FCPF conjugates carrying diverse chemical epigenetic modulators to demonstrate potentials and applications of Chem-CRISPR/dCas9FCPF system in the regulation of epigenome editing at the single-gene level.