Examination of Factors Affecting Site-Directed RNA Editing by the MS2-ADAR1 Deaminase System
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 c...
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| Format: | Article |
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MDPI AG
2023-08-01
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| Series: | Genes |
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| Online Access: | https://www.mdpi.com/2073-4425/14/8/1584 |
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| author | Md Thoufic Anam Azad Umme Qulsum Toshifumi Tsukahara |
| author_facet | Md Thoufic Anam Azad Umme Qulsum Toshifumi Tsukahara |
| author_sort | Md Thoufic Anam Azad |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T23:55:53Z |
| format | Article |
| id | doaj.art-ffa7a570fc804c2a82e812e8d7cd8d0f |
| institution | Directory Open Access Journal |
| issn | 2073-4425 |
| language | English |
| last_indexed | 2024-03-10T23:55:53Z |
| publishDate | 2023-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Genes |
| spelling | doaj.art-ffa7a570fc804c2a82e812e8d7cd8d0f2023-11-19T01:15:25ZengMDPI AGGenes2073-44252023-08-01148158410.3390/genes14081584Examination of Factors Affecting Site-Directed RNA Editing by the MS2-ADAR1 Deaminase SystemMd Thoufic Anam Azad0Umme Qulsum1Toshifumi Tsukahara2Area of Bioscience, Biotechnology and Biomedical Engineering Research Area, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City 923-1292, Ishikawa, JapanArea of Bioscience, Biotechnology and Biomedical Engineering Research Area, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City 923-1292, Ishikawa, JapanArea of Bioscience, Biotechnology and Biomedical Engineering Research Area, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi City 923-1292, Ishikawa, JapanAdenosine 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.https://www.mdpi.com/2073-4425/14/8/1584efficiencygene therapyRNA editing |
| spellingShingle | Md Thoufic Anam Azad Umme Qulsum Toshifumi Tsukahara Examination of Factors Affecting Site-Directed RNA Editing by the MS2-ADAR1 Deaminase System Genes efficiency gene therapy RNA editing |
| title | Examination of Factors Affecting Site-Directed RNA Editing by the MS2-ADAR1 Deaminase System |
| title_full | Examination of Factors Affecting Site-Directed RNA Editing by the MS2-ADAR1 Deaminase System |
| title_fullStr | Examination of Factors Affecting Site-Directed RNA Editing by the MS2-ADAR1 Deaminase System |
| title_full_unstemmed | Examination of Factors Affecting Site-Directed RNA Editing by the MS2-ADAR1 Deaminase System |
| title_short | Examination of Factors Affecting Site-Directed RNA Editing by the MS2-ADAR1 Deaminase System |
| title_sort | examination of factors affecting site directed rna editing by the ms2 adar1 deaminase system |
| topic | efficiency gene therapy RNA editing |
| url | https://www.mdpi.com/2073-4425/14/8/1584 |
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