Affiliation:
1. Department of Biology, Faculty of Science, Shinshu University, 3-1-1 Asahi, Matsumoto 390-8621, Nagano, Japan
Abstract
Dysregulation of mRNA processing results in diseases such as cancer. Although RNA editing technologies attract attention as gene therapy for repairing aberrant mRNA, substantial sequence defects arising from mis-splicing cannot be corrected by existing techniques using adenosine deaminase acting on RNA (ADAR) due to the limitation of adenosine-to-inosine point conversion. Here, we report an RNA editing technology called “RNA overwriting” that overwrites the sequence downstream of a designated site on the target RNA by utilizing the RNA-dependent RNA polymerase (RdRp) of the influenza A virus. To enable RNA overwriting within living cells, we developed a modified RdRp by introducing H357A and E361A mutations in the polymerase basic 2 of RdRp and fusing the C-terminus with catalytically inactive Cas13b (dCas13b). The modified RdRp knocked down 46% of the target mRNA and further overwrote 21% of the mRNA. RNA overwriting is a versatile editing technique that can perform various modifications, including addition, deletion, and mutation introduction, and thus allow for repair of the aberrant mRNA produced by dysregulation of mRNA processing, such as mis-splicing.
Subject
Inorganic Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Computer Science Applications,Spectroscopy,Molecular Biology,General Medicine,Catalysis
Reference44 articles.
1. RAN and Disease;Cooper;Cell,2009
2. Kataoka, N., Mayeda, A., and Ohno, K. (2019). RNA Diseases in Humans-From Fundamental Research to Therapeutic Application, Frontiers.
3. Hybrid restriction enzymes: Zinc finger fusions to Fok I cleavage domain;Kim;Proc. Natl. Acad. Sci. USA,1996
4. Breaking the code of DNA binding specificity of TAL-type III effectors;Boch;Science,2009
5. Targeting DNA double-strand breaks with TAL effector nucleases;Christian;Genetics,2010