| 今後の研究の推進方策 |
ADAR1, ADAR2, and CRISPR-Cas 13X.1 plasmids will be ordered from Addgene. The
plasmid cloning will be done afterwards. An in vitro study using myotubes from DMD model mdx mice will be conducted to obtain a proof of concept. The restoration of full-length dystrophin protein will be confirmed at mRNA and protein levels. The Sanger sequencing for mRNA base editing, Immunofluorescence for dystrophin protein expression, and western blot for full-length protein expression will be considered as restoration validity tests. A suitable protocol for effective RNA editing will be optimized by exploring the following experiments: screening guide RNAs, titration of vector, screening editor enzyme, screening linker between the Editor and Cas13 protein, and off-target effect using RNA seq.
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