| RRC ID |
63797
|
| 著者 |
Tohama T, Sakari M, Tsukahara T.
|
| タイトル |
Development of a Single Construct System for Site-Directed RNA Editing Using MS2-ADAR.
|
| ジャーナル |
Int J Mol Sci
|
| Abstract |
Site-directed RNA editing (SDRE) technologies have great potential for treating genetic diseases caused by point mutations. Our group and other researchers have developed SDRE methods utilizing adenosine deaminases acting on RNA (ADARs) and guide RNAs recruiting ADARs to target RNAs bearing point mutations. In general, efficient SDRE relies on introducing numerous guide RNAs relative to target genes. However, achieving a large ratio is not possible for gene therapy applications. In order to achieve a realistic ratio, we herein developed a system that can introduce an equal number of genes and guide RNAs into cultured cells using a fusion protein comprising an ADAR fragment and a plasmid vector containing one copy of each gene on a single construct. We transfected the single construct into HEK293T cells and achieved relatively high efficiency (up to 42%). The results demonstrate that efficient SDRE is possible when the copy number is similar for all three factors (target gene, guide RNA, and ADAR enzyme). This method is expected to be capable of highly efficient gene repair in vivo, making it applicable for gene therapy.
|
| 巻・号 |
21(14)
|
| 公開日 |
2020-7-13
|
| DOI |
10.3390/ijms21144943
|
| PII |
ijms21144943
|
| PMID |
32668759
|
| PMC |
PMC7404196
|
| MeSH |
Adenosine Deaminase / metabolism
Cell Nucleus / metabolism
Cells, Cultured
Gene Dosage
Genes, Reporter
Genes, Synthetic
Genetic Vectors / genetics
Green Fluorescent Proteins / genetics
HEK293 Cells
Humans
Levivirus / enzymology*
Mutagenesis, Site-Directed
Point Mutation
Promoter Regions, Genetic
RNA Editing*
RNA, Guide, Kinetoplastida / metabolism
Recombinant Fusion Proteins / metabolism
Transfection / methods*
Viral Proteins / metabolism
|
| IF |
4.556
|
| リソース情報 |
| ヒト・動物細胞 |
293T(RCB2202) |
