2026-09-29T09:58:53Zhttps://publikationen.uni-tuebingen.de/oai/requestoai:publikationen.uni-tuebingen.de:10900/838272019-10-30T06:44:37Zcom_10900_42126col_10900_42133
Publikationssystem UB Tuebingen
advisor
Stafforst, Thorsten (Prof. Dr.)
author
Vogel, Paul
2018-08-27T12:21:32Z
2019-08-21
1671905326
http://hdl.handle.net/10900/83827
Tools to manipulate genetic information without interfering at the DNA level are highly desirable in medicine and the life sciences. Recently, our group introduced the first engineered, RNA-guided deaminase. The approach relies on the in situ covalent bond formation between a benzylguanine-modified guide RNA (BG-gRNA) and a SNAP-tagged deaminase (SNAP-ADAR). Once the gRNA-deaminase conjugate is formed, it enables specific adenosine-to-inosine (A-to-I) substitutions in target RNAs. Since inosine is biochemically interpreted as guanosine by the cellular machinery, site-directed A-to-I editing provides the possibility to manipulate RNA and protein function. In this PhD project, it was aimed at elucidating the potential of the SNAP-ADAR approach for future applications. Therefore, the performance of the editing system in mammalian cells was comprehensively characterized. It could be shown that efficient site-directed RNA editing with SNAP-ADAR enzymes in cell culture requires the chemical modification of the BG-gRNA. A strong performance in the editing of endogenous transcripts was demonstrated in engineered cell lines stably expressing SNAP-ADAR enzymes. Editing yields up to 90% were achieved and remained stable even when several transcripts or multiple sites on a single transcript were concurrently targeted. Maximum editing was reached after 3 hours of BG-gRNA transfection and stayed unchanged for several days. Additionally, low concentrations (≥ 1.25 pmol/96-well) of the BG-gRNA were sufficient to obtain highest editing levels. The SNAP-ADAR approach holds great promise for the recoding of many functionally important amino acid residues as 11 out of the 16 adenosine-containing 5’-NAN triplets were editable between 50% and 90%. First evidence was provided that the editing system might be a valuable tool for the correction of disease-causing mutations. Moreover, the possibility of manipulating entire signaling networks was highlighted by the efficient and concurrent editing of two disease-relevant transcripts, KRAS and STAT1. Photo-controlled A-to-I editing was applied to direct protein localization within the cell by introducing alternative start or stop codons which allowed the expression of signals for nuclear and membrane translocation. NGS-based analysis revealed that wild-type SNAP-ADAR enzymes are highly precise editing machines. Their more active versions (SNAP-ADARQ enzymes) produced some off-target edits into the transcriptome, but the observed off-target activity appeared to be reducible by lowering the SNAP-ADAR protein amounts without great inhibition of the on-target editing. Nevertheless, these enzymes were one order more precise than editing machines applied by competing approaches. The chemical modification of the BG-gRNA was shown to suppress the off-target editing within a duplex formed by the BG-gRNA and the target RNA. The SNAP-ADAR approach outcompetes all well-characterized approaches for site-directed RNA editing due the best balance between efficiency and specificity.
en
ubt-podok
Establishing Site-Directed A-to-I RNA Editing in Cell Culture
PhDThesis
URL
https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/83827/1/Promotionsarbeit_Paul_Vogel.pdf
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Promotionsarbeit_Paul_Vogel.pdf
URL
https://publikationen.uni-tuebingen.de/xmlui/bitstream/10900/83827/3/Promotionsarbeit_Paul_Vogel.pdf.txt
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Promotionsarbeit_Paul_Vogel.pdf.txt