{"id":{"repo_id":"tuebingen","oai_identifier":"oai:publikationen.uni-tuebingen.de:10900/83827"},"canonical_url":"https://search.dev.ndltd.org/etd/tuebingen/oai:publikationen.uni-tuebingen.de:10900/83827","repository":{"repo_id":"tuebingen","name":"Universität Tübingen","base_url":"https://publikationen.uni-tuebingen.de/oai/request"},"display":{"title":"Establishing Site-Directed A-to-I RNA Editing in Cell Culture","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Vogel, Paul"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2019,"date_issued":"2019-08-21","date_published":"2019-08-21","updated_at":"2026-08-21T22:21:56Z","subjects":[],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["hdl:10900/83827"],"render_values":[{"text":"hdl:10900/83827","href":null,"code":true}]}]},"links":{"outbound_url":null,"outbound_label":null,"outbound_source":null},"source_record":{"url":"https://publikationen.uni-tuebingen.de/oai/request?verb=GetRecord&metadataPrefix=mets&identifier=oai%3Apublikationen.uni-tuebingen.de%3A10900%2F83827","prefix":"mets"},"metadata_groups":[{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2019-08-21"]},{"key":"dc:type","label":"Dc Type","values":["PhDThesis"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["hdl:10900/83827"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.other","label":"Dc Description Other","values":["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."]},{"key":"dc:title","label":"Title","values":["Establishing Site-Directed A-to-I RNA Editing in Cell Culture"]}]}],"canonical_facts":{"dc:date.issued":["2019-08-21"],"dc:description.other":["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."],"dc:identifier":["hdl:10900/83827"],"dc:title":["Establishing Site-Directed A-to-I RNA Editing in Cell Culture"],"dc:type":["PhDThesis"]},"updated_at":"2026-08-21T22:21:56Z"}