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

开发Chem-CRISPR/dCas9系统以在单基因水平实现小分子介导的表观遗传调控

• 批准号: 509482700
• 负责人: Dr. Xinlai Cheng
• 金额: --
• 依托单位: Buchmann Institute for Molecular Life Sciences
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/509482700?language=en
• 关键词: Development; Chem; CRISPR; dCas9; system

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.

表观遗传畸变是人类癌症发生、促进和进展的主要驱动因素之一，也常常与获得性治疗耐药相关。临床前实验已报道了许多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，严重的靶向和脱靶毒性是与药物开发失败相关的主要因素之一。 CRISPR/dCas9技术为单基因水平上的精确表观遗传调控提供了强大的工具。这种方法需要外源表观遗传调节蛋白的异位表达，这会引起副作用。我们打算开发一个Chem-CRISPR/dCas9平台，其中CRISPR/dCas9可以将化学表观遗传激活剂/抑制剂带到所需的DNA序列附近，从而实现单基因水平的表观遗传调控。在我们最近的工作中，我们测试了几种位点选择性化学修饰方法，并成功地将 TT-clamp 系统应用于 Chem-CRISPR/dCas9 平台。我们证明，标记短 Phe(F)-Cys(C)-Pro(P)-Phe(F) 氨基酸序列的 (d)Cas9 可以被细胞中的全氟芳香族化合物特异性识别。我们将 FITC 融合到 FCPF 结合剂 (FITC-FCPF) 中，并实现了在活细胞中标记 Cas9FCPF。我们开发了一种携带沙利度胺（CRBN E3 连接酶的配体）的 PROTAC-FCPF 异双功能分子，并证明 PROTAC-FCPF 降解 (d)Cas9FCPF 和其他 CasFCPF 蛋白。在我们针对该提案的初步工作中，我们将 JQ1（一种泛 BET 抑制剂）缀合到 FCPF 结合剂（JQ1-FCPF）中，并证明 JQ1-FCPF 在 FRET 测定中诱导 dCas9FCPF 和 BRD4WT 的接近，这通过免疫共沉淀进一步得到证实。全基因组转录组学表明，在 JQ1-FCPF 和 Chem-CRISPR/dCas9FCPF 系统（具有靶向 C-MYC 启动子和增强子的 sgRNA）存在的情况下，C-MYC 受到特异性抑制。在这个拟议的项目中，我们将合成携带多种化学表观遗传调节剂的新型FCPF缀合物，以展示Chem-CRISPR/dCas9FCPF系统在单基因水平表观基因组编辑调控中的潜力和应用。