Our group has developed several new molecular technologies to study and exploit RNA modifications in mammalian cells. We developed a general method to evolve reverse-transcriptases (RT) to encodes RNA modifications in mutations for use in high-throughput sequencing analysis of the sites and abundances of RNA modifications, thus far focusing on m1A as an exemplar. We developed programmable RNA reader proteins by engineering RNA-targeting Cas systems with effector domains from RNA regulatory proteins in order to begin to study the role(s) of individual regulatory sites in the transcriptome. Finally, we developed an entirely human protein-based programmable RNA delivery systems, inspired by CRISPR/Cas9, which is 5-fold smaller than the current RNA-targeting Cas systems, providing us with a powerful new approach to probe transcriptome regulation and also providing a path toward clinical applications of programmed epitranscriptome regulatory systems.
Programmable RNA regulatory proteins. dCas13b fusion proteins can be used to site-specifically deliver regulatory proteins to the transcriptome. dCas13b-YTHDF1 targets transcripts to trigger assembly of translation machinery dCas13b-YTHDF2 triggers deadenylation and degradation.
Epitranscritomics. RNA modifications provide a critical layer of control of information flow in mammalian cells. RNA “reader” proteins are both important controllers of this process and provide opportunities to interact and control this regulation.
CIRTS, a "humanized" RNA-targeting CRISPR-Cas system. Epitranscriptomic regulation controls information flow through the central dogma and provides unique opportunities for manipulating cells at the RNA level. However, both fundamental studies and potential translational applications are impeded by a lack of methods to target specific RNAs with effector proteins. Here, we present CRISPR-Cas-inspired RNA targeting system (CIRTS), a protein engineering strategy for constructing programmable RNA control elements. We show that CIRTS is a simple and generalizable approach to deliver a range of effector proteins, including nucleases, degradation machinery, translational activators, and base editors to target transcripts. We further demonstrate that CIRTS are not only smaller than naturally-occurring CRISPR-Cas programmable RNA binding systems, but can also be built entirely from human protein parts. CIRTS provides a platform to probe fundamental RNA regulatory processes, while the human-derived nature of CIRTS provides a potential strategy to avoid immune issues when applied to epitranscriptome-modulating therapies.