Site-directed RNA editing by adenosine deaminase acting on RNA for correction of the genetic code in gene therapy

Site-directed RNA editing is an important technique for correcting gene sequences and ultimately tuning protein function. In this study, we engineered the deaminase domain of adenosine deaminase acting on RNA (ADAR1) and the MS2 system to target-specific adenosines, with the goal of correcting G-to-...

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Bibliographic Details
Published in:Gene therapy 2017-12, Vol.24 (12), p.779-786
Main Authors: Azad, Md T A, Bhakta, S, Tsukahara, T
Format: Article
Language:English
Subjects:
Adenosine
Adenosine - genetics
Adenosine deaminase
Adenosine Deaminase - metabolism
Alleles
Amber
Blotting, Western
Codon, Terminator
Codons
Deoxyribonucleic acid
DNA
DNA sequencing
Editing
Fluorescence
Gene polymorphism
Gene therapy
Genetic Code
Genetic disorders
Genetic Therapy
Green fluorescent protein
Green Fluorescent Proteins - genetics
Health aspects
Humans
Inosine - genetics
Methods
Mutation
Point Mutation
Proteins
Restriction fragment length polymorphism
RNA Editing
RNA processing
RNA, Guide, CRISPR-Cas Systems
RNA-binding protein
RNA-Binding Proteins - metabolism
Stop codon
Structure
Western blotting
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Summary:Site-directed RNA editing is an important technique for correcting gene sequences and ultimately tuning protein function. In this study, we engineered the deaminase domain of adenosine deaminase acting on RNA (ADAR1) and the MS2 system to target-specific adenosines, with the goal of correcting G-to-A mutations at the RNA level. For this purpose, the ADAR1 deaminase domain was fused downstream of the RNA-binding protein MS2, which has affinity for the MS2 RNA. To direct editing to specific targets, we designed guide RNAs complementary to target RNAs. The guide RNAs directed the ADAR1 deaminase to the desired editing site, where it converted adenosine to inosine. To provide proof of principle, we used an allele of enhanced green fluorescent protein (EGFP) bearing a mutation at the 58th amino acid (TGG), encoding Trp, into an amber (TAG) or ochre (TAA) stop codon. In HEK-293 cells, our system could convert stop codons to read-through codons, thereby turning on fluorescence. We confirmed the specificity of editing at the DNA level by restriction fragment length polymorphism analysis and sequencing, and at the protein level by western blotting. The editing efficiency of this enzyme system was ~5%. We believe that this system could be used to treat genetic diseases resulting from G-to-A point mutations.
ISSN:0969-7128
1476-5462
DOI:10.1038/gt.2017.90