Goldengate Assembly of a Synthetic Reader Effector Library for Epigenetic Engineering in Triple Negative Breast Cancer

Epigenetic therapy is gaining traction as a way to activate non-mutated tumor suppressor genes in cancer cells, which is an alternative to traditional chemotherapies that inhibit DNA replication. Triple negative breast cancer (TNBC) is untreatable with hormone therapy and shows relapse after chemotherapy, but responds moderately to leading epigenetic drugs. Currently, the design of epigenetic treatments for TNBC is restricted to small molecules that bind specific chromatin-modifying enzymes: histone modifiers EZH2 and HDACs, and DNA methyltransferases. Inhibition of these enzymes passively affects gene regulation, can affect cellular processes outside of chromatin, and is therefore not ideal for specific epigenetic modulations. Our overall goal is to engineer chromatin proteins that directly bind to modified histones in human cell nuclei for cancer epigenetics research and treatment. Our immediate goal is to screen a large library of synthetic reader-effector (SRE) proteins for anti-tumor activity in TNBC cells. To enable efficient identification of the most potent SREs, we have designed a platform based on the Golden Gate cloning method to produce a library of hundreds of synthetic reader-effector constructs. We have developed a method to produce donor plasmids for Golden Gate cloning, where PCR-amplified modules are ligated into a Kanamycin-resistant vector. Each module carries a standard set of type IIS sites and 4 b.p. overhangs to enable faithful assembly of the following four modular peptide variants into diverse SREs without frameshifts: histone binding domain 1 (>100 variants), linker, histone binding domain 2 (>100 variants), transcriptional effector/ fluorescent tag. We have demonstrated successful assembly by verifying the correct orientations and orders of the modules in an Ampicillin-resistant destination vector. In conclusion, we have designed a Golden Gate assembly platform to build diverse SREs. Work to assemble the complete library of SREs and screen for high-affinity binders of H3K27me3 in vitro (whole nucleosomes) is underway.