title: An efficient system for selectively altering genetic information within mRNAs.
abstract: 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 U A: G premature termination codon (PTC) to tryptophan (U G: G) 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.
authors.affiliation: Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus, San Juan, PR 00901, USA.
authors.forename: Maria Fernanda
authors.initials: MF
authors.lastname: Montiel-González
authors.affiliation: Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus, San Juan, PR 00901, USA.|Department of Pharmacology, University of Puerto Rico Medical Sciences Campus, San Juan, PR 00936, USA.
authors.forename: Isabel C
authors.initials: IC
authors.lastname: Vallecillo-Viejo
authors.affiliation: Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus, San Juan, PR 00901, USA jrosenthal@mbl.edu.|The Marine Biological Laboratory, University of Chicago, Woods Hole, MA 02543, USA.
authors.forename: Joshua J C
authors.initials: JJ
authors.lastname: Rosenthal
mesh_terms: D000243:Adenosine Deaminase / Q000737:chemistry / Q000235:genetics; D002138:Calibration; D018389:Codon, Nonsense; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D017930:Genes, Reporter; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D010455:Peptides / Q000235:genetics; D010843:Pichia / Q000235:genetics; D000072417:Protein Domains; D017393:RNA Editing; D012333:RNA, Messenger / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D014364:Tryptophan / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D000243:Adenosine Deaminase; D018389:Codon, Nonsense; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins; D010455:Peptides; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014364:Tryptophan; D000094704:RNA, Guide, CRISPR-Cas Systems; C485184:enhanced green fluorescent protein; C567955:ADARB1 protein, human