Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing.
Enviado por Guillermo Yudowski el
Título | Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing. |
Publication Type | Journal Article |
Year of Publication | 2013 |
Autores | Montiel-Gonzalez, MFernanda, Vallecillo-Viejo, I, Yudowski, GA, Rosenthal, JJC |
Journal | Proc Natl Acad Sci U S A |
Volume | 110 |
Issue | 45 |
Pagination | 18285-90 |
Date Published | 2013 Nov 5 |
ISSN | 1091-6490 |
Palabras clave | Adenosine Deaminase, Animals, Base Sequence, Blotting, Western, Codon, Nonsense, Cystic Fibrosis Transmembrane Conductance Regulator, Fluorescence, Genetic Engineering, Green Fluorescent Proteins, HEK293 Cells, Humans, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Oligonucleotides, RNA Editing, Xenopus |
Abstract | Adenosine deaminases that act on RNA are a conserved family of enzymes that catalyze a natural process of site-directed mutagenesis. Biochemically, they convert adenosine to inosine, a nucleotide that is read as guanosine during translation; thus when editing occurs in mRNAs, codons can be recoded and the changes can alter protein function. By removing the endogenous targeting domains from human adenosine deaminase that acts on RNA 2 and replacing them with an antisense RNA oligonucleotide, we have engineered a recombinant enzyme that can be directed to edit anywhere along the RNA registry. Here we demonstrate that this enzyme can efficiently and selectively edit a single adenosine. As proof of principle in vitro, we correct a premature termination codon in mRNAs encoding the cystic fibrosis transmembrane conductance regulator anion channel. In Xenopus oocytes, we show that a genetically encoded version of our editase can correct cystic fibrosis transmembrane conductance regulator mRNA, restore full-length protein, and reestablish functional chloride currents across the plasma membrane. Finally, in a human cell line, we show that a genetically encoded version of our editase and guide RNA can correct a nonfunctional version of enhanced green fluorescent protein, which contains a premature termination codon. This technology should spearhead powerful approaches to correcting a wide variety of genetic mutations and fine-tuning protein function through targeted nucleotide deamination. |
DOI | 10.1073/pnas.1306243110 |
Alternate Journal | Proc. Natl. Acad. Sci. U.S.A. |
PubMed ID | 24108353 |
PubMed Central ID | PMC3831439 |
Grant List | 1P30NS069258 / NS / NINDS NIH HHS / United States 2 U54 NS039405-06 / NS / NINDS NIH HHS / United States 2G12RR003051 / RR / NCRR NIH HHS / United States 8G12MD007600 / MD / NIMHD NIH HHS / United States DA023444 / DA / NIDA NIH HHS / United States G12 MD007600 / MD / NIMHD NIH HHS / United States G12 RR003051 / RR / NCRR NIH HHS / United States R01 NS064259 / NS / NINDS NIH HHS / United States R01 NS064259 / NS / NINDS NIH HHS / United States |