Migliara, A. | Search bioRxiv

bioRxiv2025

Engineering Inducible Cell Fate Transitions by Harnessing Epigenetic Silencing

During development, cell-cell communication induces state transitions that are maintained by epigenetic regulation. Here, we harness endogenous epigenetic silencing machinery to engineer synthetic circuits that induce stable gene expression changes. Using synthetic Notch receptors that control the chromatin regulators KRAB and Dnmt3L, we developed input-sensing switches that induce self-sustained silencing of target loci. We used these modules to construct circuits in which combinatorial inputs direct a choice among multiple alternative states. These epigenetic switches can also be inverted to yield sustained activation of target genes. We demonstrate that these epigenetic switches can be used to drive morphological changes triggered by transient cell-cell interactions, but that remain stable over many cell divisions, as is observed in development. Additionally, these circuits can be used to drive the reprogramming of fibroblasts to a neuron-like state. These synthetic epigenetic circuits represent an important step towards engineering cell populations capable of coordinated multi-cell state decisions.

synthetic biology↗

bioRxiv2022

In vitro selection of Engineered Transcriptional Repressors for targeted epigenetic silencing and initial evaluation of their specificity profile

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and requirement for life-long treatments. On the other hand, artificial nucleases can impose stable gene inactivation through induction of DNA Double Strand Breaks, but recent studies are questioning the safety of this approach. Targeted epigenetic editing via Engineered Transcriptional Repressors (ETRs) may represent a solution, as a single administration of specific ETRs combinations can lead to durable silencing without DNA breaks induction. ETRs are proteins containing a programmable DNA Binding Domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3Lwas shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert on-demand the repressive state by DNA demethylation, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper position on the target gene where to tether the ETRs in order to maximize on target and minimizing off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome. Taking CRISPR-Cas9 system as a paradigmatic DBD for ETRs, here we describe a protocol consisting in the in vitro screen of gRNAs coupled to the triple ETRs combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows to reduce the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final evaluation in the therapeutically relevant setting of interest.

synthetic biology↗