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Site-directed RNA Editing to Manipulate RNA and Protein Function

Periodic Reporting for period 3 - RNArepair (Site-directed RNA Editing to Manipulate RNA and Protein Function)

Reporting period: 2018-08-01 to 2020-01-31

In a process called gene expression, the genetic information, which is stored in the DNA, is read-out to produce the proteins of the cell. This process goes over an intermediate matrice that is called messenger RNA. For a long time, people considered this RNA intermediate to have only a messenger function. However, during the last two decades it became increasingly clear that the RNA intermediate enable an additional layer of control. Very recently, we and others have started to develop tools that allow to reprogram the genetic information at the RNA level by a process called site-directed RNA editing. Such tools allow to reprogramm specific sites in specific mRNA and, in the consequence, to change the composition of the resulting protein. We are particularly interested in the manipulation of protein (mis)function that is related to human disease. Many human diseases are linked to inherited miscomposition of proteins, often lacking any causal treatment in the clinics. Our tools may open new avenues for the treatment and/or better mechanistic understanding of human disease.
However, to make such tools efficient and precise is a major challenge that requires to combine expertise from chemistry and life sciences. Coming from a chemistry background, we have established an entirely novel access to assemble an editing tool inside the cell. Within this project we aim to better understand our RNa targeting strategy by comprehensively characterizing all its properties. Furthermore, we aim to develop several alternative methods to deliver the tool into relevant target tissues. Finally, we wish to implement the tool in an animal model. The action is required to make the basic research community and the applied biotech companies familiar with our novel RNA targeting approach and to foster the development of site-directed RNA editing as a platform for therapy and advanced RNA-targeting strategies.
The SNAP-ADAR tool has been comprehensively characterized in terms of potency, efficiency, codon scope, duration of the effect, concurrent editing, editing of endogenous transcripts, off-target editing in the mRNA/gRNA duplex, global off-target editing. We found the SNAP-ADAR system to be very powerful, to enable efficient, editing at various codons and transcripts with sufficient potency and duration. A single genomic copy of the editase allows efficient editing with sufficient specificity. We found ways to suppress off-target editing in the mRNA/gRNA duplex by optimizing the chemistry of the guideRNA.
We produced the first viral systems to become more flexible with the delivery of the tool. We started to implement further startegies to apply/deliver the tool into relevant cells.
Finally, we implemented a first photocontrol startegy and tested it successfully in cell culture and in vivo in an annelid (Platynereis dumerilii). We also implemented RNA editing for the inclusion N- and C-terminal protein localization signals and achieved photocontrol over protein localization.
With the current state of the project, we set a new benchmark for site-directed RNA editing. The SNAP-ADAR tool is the most comprehensively studied tool today and achieves an excellent balance of editing efficiency and off-target effects due to its unique assembly strategy. We hope, by the end of the project to give the proof that our startegy enables otherwise inaccessible means to study and manipulate disease-relevant processes.