Gene expression regulation at the RNA level has emerged as a key point of control of information flow through the central dogma. RNA stability, trafficking, and translation efficiency are heavily regulated by canonical RNA processing mechanisms such as capping, splicing, and polyadenylation as well as chemical RNA modifications. With an ever-increasing number of reported regulatory layers, there is an increased need for new tools to disentangle these complex regulatory networks in a site- and transcript-specific manner within their endogenous environment. Additionally, RNA has become an attractive target for therapeutic intervention due to its rapid turnover and short lifetime allowing for multigenic targeting and higher safety profiles compared to DNA-targeting approaches. In this thesis, we report the development and optimization of programmable RNA effectors to both study and control gene expression regulation at the RNA level. We first developed programmable RNA “reader” proteins, which allow us to interrogate the effects of specific regulatory proteins on single transcripts. Our first-generation programmable epitranscriptomic readers rely on RNA-targeting CRISPR/Cas systems as the programmable delivery vehicle. While powerful, these microbially derived CRISPR/Cas systems are large and present immunogenicity issues when applied in non-native human contexts, limiting their potential future therapeutic applications. Therefore, we next developed a second-generation platform for engineering programmable RNA effector proteins that are significantly smaller than the CRISPR/Cas systems and are built entirely from human parts. Our CRISPR/Cas-inspired RNA targeting system (CIRTS) involves mining the human proteome for functional domains and combining them to create programmable RNA effector proteins. Next, we engineered a small molecule-inducible CIRTS biosensor system for temporal control of CIRTS activity in cells and in vivo. Finally, we set out to develop a second-generation CIRTS with improved efficacy, novel delivery methods, and the first steps towards preclinical applications. Taken together, CIRTS, which can induce transcript-specific RNA decay, protein production and RNA edits, will find applications in synthetic biology to control the genetic information flow at the RNA level.



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