Abstract
Site‐directed adenosine-to-inosine (A-to-I) RNA editing is a novel transcriptome engineering approach that allows to recode genetic information at RNA level by specific deamination of adenosines. Because the resulting inosine is read as guanosine by cellular machines and during translation, site-directed RNA editing (SDRE) does ultimately introduce A-to-G base substitutions into mRNAs. This opens up the opportunity to recode Start- and Stop-codons, splice signals, miRNA recognition sites, and 12 of the 20 canonical amino acids. Thus SDRE allows a wide range of interventions, including the manipulation of residues relevant for signaling or protein function, and the ability to correct G-to-A point mutations. Our R/G guideRNA SDRE system allows to steer wild-type human ADAR (adenosine deaminase acting on RNA) enzymes towards selected target adenosines within user-defined mRNAs. The genetically encodable R/G gRNA design, which was developed during this thesis based on successive rounds of rational design, allows for the first time to recruit endogenous human ADAR enzymes. In contrast to all other SDRE approaches, the R/G gRNA system does therefore not require the overexpression of an editase. Transcriptome engineering can now be achieved by the simple application of a plasmid or adenovirus encoded short guideRNA. During this thesis, several fundamental questions, which had to precede the further development of SDRE into a therapeutic application, could be solved. The R/G gRNA system became the first SDRE approach to prove editing of endogenous mRNAs. In addition, it was used to recode the recessive PINK1 W437X amber loss-of-function mutation in HeLa cells and could show that SDRE allows the functional rescue of PINK1-Parkin-mediated mitophagy, which is linked to the etiology of Parkinson's disease. Our R/G gRNAs have high potential to become a next generation drug for the precise correction of disease-causing point mutations and the tuneable manipulation of protein function. In doing so, they would complement existing genome and transcriptome manipulation strategies by enabling interventions out of reach for currently available molecular tools.
Author and committee
dc:creator, dc:contributor.*- Author
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- Reautschnig, Philipp
Identifiers
dc:identifier.*- Identifier
- hdl:10900/89731