Site-directed RNA editing (SDRE) technologies have great potential for treating genetic\ndiseases caused by point mutations. Our group and other researchers have developed SDRE methods\nutilizing adenosine deaminases acting on RNA (ADARs) and guide RNAs recruiting ADARs to target\nRNAs bearing point mutations. In general, effcient SDRE relies on introducing numerous guide\nRNAs relative to target genes. However, achieving a large ratio is not possible for gene therapy\napplications. In order to achieve a realistic ratio, we herein developed a system that can introduce\nan equal number of genes and guide RNAs into cultured cells using a fusion protein comprising\nan ADAR fragment and a plasmid vector containing one copy of each gene on a single construct.\nWe transfected the single construct into HEK293T cells and achieved relatively high effciency (up to\n42%). The results demonstrate that effcient SDRE is possible when the copy number is similar for all\nthree factors (target gene, guide RNA, and ADAR enzyme). This method is expected to be capable of\nhighly ecient gene repair in vivo, making it applicable for gene therapy.
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