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

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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.