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. 2023 Aug 4;14(8):1584.
doi: 10.3390/genes14081584.

Examination of Factors Affecting Site-Directed RNA Editing by the MS2-ADAR1 Deaminase System

Md Thoufic Anam Azad  1   2 , Umme Qulsum  1   3 , Toshifumi Tsukahara  1   4
Affiliations

Examination of Factors Affecting Site-Directed RNA Editing by the MS2-ADAR1 Deaminase System

Md Thoufic Anam Azad et al. Genes (Basel). .

Abstract

Adenosine deaminases acting on RNA (ADARs) have double-stranded RNA binding domains and a deaminase domain (DD). We used the MS2 system and specific guide RNAs to direct ADAR1-DD to target adenosines in the mRNA encoding-enhanced green fluorescence protein. Using this system in transfected HEK-293 cells, we evaluated the effects of changing the length and position of the guide RNA on the efficiency of conversion of amber (TAG) and ochre (TAA) stop codons to tryptophan (TGG) in the target. Guide RNAs of 19, 21 and 23 nt were positioned upstream and downstream of the MS2-RNA, providing a total of six guide RNAs. The upstream guide RNAs were more functionally effective than the downstream guide RNAs, with the following hierarchy of efficiency: 21 nt > 23 nt > 19 nt. The highest editing efficiency was 16.6%. Off-target editing was not detected in the guide RNA complementary region but was detected 50 nt downstream of the target. The editing efficiency was proportional to the amount of transfected deaminase but inversely proportional to the amount of the transfected guide RNA. Our results suggest that specific RNA editing requires precise optimization of the ratio of enzyme, guide RNA, and target RNA.

Keywords: RNA editing; efficiency; gene therapy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The editing efficiencies of the six guide RNAs, located upstream or downstream of MS2-RNA. * Indicates p value < 0.05 and statistically significant.
Figure 2
Figure 2
The effect of the amount of guide RNA on the efficiency of target and off-target RNA editing. HEK-293 cells were transfected with 500 ng of MS2-ADADR1-DD and 250, 500, or 1000 ng of the 21 nt upstream guide RNA. The solid bar indicates editing of the target site and dotted bars indicate off-target editing. * Indicates p value < 0.05 and statistically significant.
Figure 3
Figure 3
The effect of the amount of MS2-ADAR1-DD on the efficiency of target and off-target RNA editing. HEK-293 cells were transfected with 500 ng of the 21 nt upstream guide RNA and 250, 500, or 1000 ng of MS2-ADADR1-DD. The solid bar indicates editing of the target site and dotted bars indicate off-target editing. * Indicates p value < 0.05 and statistically significant and ** indicates p value < 0.01 is statistically more significant.
Figure 4
Figure 4
The effect of the amount of MS2-ADADR1-DD on the efficiency of target and off-target editing in cells expressing a double repeated 19 nt 2× upstream guide RNA. HEK-293 cells were transfected with 500 ng of the guide RNA and 250, 500, or 1000 ng of MS2-ADADR1-DD. The solid bar indicates editing of the target site and dotted bars indicate off-target editing. * Indicates p value < 0.05 and statistically significant.
Figure 5
Figure 5
The effect of the amount of double repeated 19 nt 2× upstream guide RNA on the efficiency of target and off-target editing. HEK-293 cells were transfected with 500 ng of MS2-ADADR1-DD and 250, 500, or 1000 ng of the guide RNA. The solid bar indicates editing of the target site and dotted bars indicate off-target editing. * Indicates p value < 0.05 and statistically significant.
Figure 6
Figure 6
The efficiency of conversion of stop codon (TAA) to the tryptophan codon (TGG). HEK-293 cells were transfected with 500 ng of MS2-ADADR1-DD and 250 ng of the 21 nt upstream guide RNA. The efficiencies of editing of the 5′ (TAA) and 3′ (TAA) adenosines are shown. Replicates of the experimental data are presented side by side.
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