Abstract
Many genetic diseases are caused by T-to-C point mutations. Hence, editing of mutated genes represents a promising strategy for treating these disorders. We engineered an artificial RNA editase by combining the deaminase domain of APOBEC1 (apolipoprotein B mRNA editing catalytic polypeptide 1) with a guideRNA (gRNA) which is complementary to target mRNA. In this artificial enzyme system, gRNA is bound to MS2 stem-loop, and deaminase domain, which has the ability to convert mutated target nucleotide C-to-U, is fused to MS2 coat protein. As a target RNA, we used RNA encoding blue fluorescent protein (BFP) which was derived from the gene encoding GFP by 199 T > C mutation. Upon transient expression of both components (deaminase and gRNA), we observed GFP by confocal microscopy, indicating that mutated 199C in BFP had been converted to U, restoring original sequence of GFP. This result was confirmed by PCR–RFLP and Sanger’s sequencing using cDNA from transfected cells, revealing an editing efficiency of approximately 21%. Although deep RNA sequencing result showed some off-target editing events in this system, we successfully developed an artificial RNA editing system using artificial deaminase (APOBEC1) in combination with MS2 system could lead to therapies that treat genetic disease by restoring wild-type sequence at the mRNA level.
Highlights
Many genetic diseases are caused by T-to-C point mutations
We took advantage of this phenomenon, regarding tight junction between the coat protein and stem loop, by combining the MS2 coat protein with the APOBEC 1 and MS2 stem loop with the gRNA (Fig. 1a, Supplementary Data 8), which in turn guided the deaminase to reach the specific target nucleotide and the whole system showed the activity by editing C-to-U
We successfully established an artificial deaminase system based on APOBEC1 and showed that it functions as designed in C-to-U RNA editing
Summary
Many genetic diseases are caused by T-to-C point mutations. editing of mutated genes represents a promising strategy for treating these disorders. Several other genome editing techniques have made it possible to manipulate genomic information in a targeted m anner[1,2,3,4,5] These methods include zinc-finger nucleases (ZFNs), transcription activator–like effector (TALE) nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9)[6,7]. These existing base editors sometimes create unwanted C-to-T alterations when more than one C is present in the enzyme’s five-base-pair editing window which the Gehrke et al, group has tried to reduce bystander mutations using an engineered human APOBEC3A (eA3A) domain with C as[912] These techniques are expected to find applications in the treatment of diseases, it remains difficult to achieve accurate genome editing in all affected cells. For the treatment of patients, trillions of cells and various tissues must be delivered with the gene editing tools, so genome
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