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RNA Overwriting of Cellular mRNA by Cas13b-Directed RNA-Dependent RNA Polymerase of Influenza A Virus

Shinzi Ogasawara1, Sae Ebashi1

  • 1Department of Biology, Faculty of Science, Shinshu University, 3-1-1 Asahi, Matsumoto 390-8621, Nagano, Japan.

International Journal of Molecular Sciences
|June 28, 2023
PubMed

Insights

This study introduces RNA overwriting, a novel gene therapy. This technique repairs aberrant mRNA by overwriting sequences, offering a versatile solution for diseases caused by mRNA processing dysregulation.

Area of Science:

  • Molecular Biology
  • Gene Therapy
  • RNA Editing Technologies

Background:

  • Dysregulation of messenger RNA (mRNA) processing is implicated in various diseases, including cancer.
  • Current RNA editing technologies, such as adenosine deaminase acting on RNA (ADAR), are limited to adenosine-to-inosine point conversions and cannot correct substantial sequence defects like mis-splicing.
  • There is a need for advanced RNA editing tools capable of broader sequence repair.

Purpose of the Study:

  • To develop a novel RNA editing technology for repairing aberrant mRNA, specifically addressing limitations of existing methods.
  • To enable sequence overwriting downstream of a target site on mRNA within living cells.
  • To demonstrate the versatility of the new RNA editing technique for various modifications.

Main Methods:

  • Development of a novel RNA editing technology termed 'RNA overwriting'.
  • Utilized the RNA-dependent RNA polymerase (RdRp) from influenza A virus.
  • Engineered a modified RdRp by introducing specific mutations (H357A and E361A) and fusing it with catalytically inactive Cas13b (dCas13b) for targeted delivery and activity within cells.

Main Results:

  • The modified RdRp successfully knocked down 46% of the target mRNA.
  • The RNA overwriting technology achieved a 21% overwriting rate on the target mRNA.
  • Demonstrated the capability of RNA overwriting for diverse modifications, including insertions, deletions, and point mutations.

Conclusions:

  • RNA overwriting is a versatile and effective RNA editing technology.
  • This technique can repair aberrant mRNA resulting from mRNA processing dysregulation, such as mis-splicing.
  • RNA overwriting holds potential as a therapeutic strategy for diseases linked to mRNA processing defects.
Keywords:
Cas13RNA editingRNA-dependent RNA polymerase (RdRp)influenza A virusmis-splicing

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