Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 21, Issue 14, Pages -Publisher
MDPI
DOI: 10.3390/ijms21144943
Keywords
RNA editing; site-directed RNA editing; adenosine deaminases acting on RNA; MS2 system; single construct; MS2-ADAR
Funding
- Japan Society for the Promotion of Science [26670167, 17H02204, 18K19288]
- Grants-in-Aid for Scientific Research [26670167, 17H02204, 18K19288] Funding Source: KAKEN
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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 repairin vivo, making it applicable for gene therapy.
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