An efficient system for selectively altering genetic information within mRNAs
Montiel-González, Maria Fernanda
Vallecillo-Viejo, Isabel C.
Rosenthal, Joshua J. C.
MetadataShow full item record
Site-directed RNA editing (SDRE) is a strategy to precisely alter genetic information within mRNAs. By linking the catalytic domain of the RNA editing enzyme ADAR to an antisense guide RNA, specific adenosines can be converted to inosines, biological mimics for guanosine. Previously, we showed that a genetically encoded iteration of SDRE could target adenosines expressed in human cells, but not efficiently. Here we developed a reporter assay to quantify editing, and used it to improve our strategy. By enhancing the linkage between ADAR's catalytic domain and the guide RNA, and by introducing a mutation in the catalytic domain, the efficiency of converting a UAG premature termination codon (PTC) to tryptophan (UGG) was improved from ∼11% to ∼70%. Other PTCs were edited, but less efficiently. Numerous off-target edits were identified in the targeted mRNA, but not in randomly selected endogenous messages. Off-target edits could be eliminated by reducing the amount of guide RNA with a reduction in on-target editing. The catalytic rate of SDRE was compared with those for human ADARs on various substrates and found to be within an order of magnitude of most. These data underscore the promise of site-directed RNA editing as a therapeutic or experimental tool.
© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Nucleic Acids Research 44 (2016): e157, doi:10.1093/nar/gkw738.
Suggested CitationArticle: Montiel-González, Maria Fernanda, Vallecillo-Viejo, Isabel C., Rosenthal, Joshua J. C., "An efficient system for selectively altering genetic information within mRNAs", Nucleic Acids Research 44 (2016): e157, DOI:10.1093/nar/gkw738, https://hdl.handle.net/1912/8580
The following license files are associated with this item: