Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing

Significance RNA editing by adenosine deamination is a natural process of site-directed mutagenesis used by organisms to modify genetic information as it passes through RNA. In this paper we present an engineered RNA editing enzyme that can be induced to edit any adenosine that is chosen. We show that our system can efficiently correct a premature stop codon in the cystic fibrosis transmembrane conductance regulator in frog oocytes. Furthermore, a fully genetically encoded version of the system functions in human cells. As a general method, site-directed RNA editing could be a useful technique for correcting genetic mutations and modifying protein function. 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.

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