Restoration of cytidine to uridine genetic code using an MS2-APOBEC1 artificial enzymatic approach.

By employing site-directed RNA editing (SDRE) to restore point-mutated RNA molecules, it is possible to change gene-encoded information and synthesize proteins with different functionality from a single gene. Thymine (T) to cytosine (C) point mutations cause various genetic disorders, and when they occur in protein-coding regions, C-to-uridine (U) RNA changes can lead to non-synonymous alterations. By joining the deaminase domain of apolipoprotein B messenger RNA (mRNA) editing catalytic polypeptide 1 (APOBEC1) with a guide RNA (gRNA) complementary to a target mRNA, we created an artificial RNA editase. We used an mRNA encoding blue fluorescent protein (BFP), obtained from the green fluorescent protein (GFP) gene through the introduction of a T > C mutation, as our target RNA. In a proof of principle experiment, we reverted the T > C mutation at the RNA level using our APOBEC1 site-directed RNA editing system, recovering GFP signal. Sanger sequencing of cDNA from transfected cells and polymerase chain reaction-restriction length polymorphism analysis validated this result, indicating an editing of approximately 21 %. Our successful development of an artificial RNA editing system using the deaminase APOBEC1, in conjunction with the MS2 system, may lead to the development of treatments for genetic diseases based on the restoration of specific types of wild type sequences at the mRNA level.