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Development of miniature base editors using engineered IscB nickase.

Dingyi Han1,2, Qingquan Xiao1,3,4, Yifan Wang1,2

  • 1Institute of Neuroscience, State Key Laboratory of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Research Center for Brain Science and Brain-Inspired Intelligence, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

Nature Methods
|May 25, 2023
PubMed
Summary

Engineered IscB (enIscB) enhances genome editing in mammalian cells. This miniature system, enIscB-T5E and miniature IscB-derived base editors (miBEs), offers efficient DNA editing with reduced translocation effects.

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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies

Background:

  • IscB is a miniature RNA-guided endonuclease, a potential ancestor to Cas9.
  • Its small size is advantageous for in vivo delivery, but low eukaryotic editing efficiency is a limitation.

Purpose of the Study:

  • To engineer IscB for high efficiency in mammalian systems.
  • To develop novel genome editing tools based on the enhanced IscB.

Main Methods:

  • Engineering of OgeuIscB and its ωRNA to create an efficient mammalian system (enIscB).
  • Fusion of enIscB with T5 exonuclease (T5E) to create enIscB-T5E.
  • Fusion of enIscB nickase with cytosine or adenosine deaminase to create miniature IscB-derived base editors (miBEs).

Main Results:

  • The engineered enIscB system demonstrated high efficiency in mammalian cells.
  • enIscB-T5E showed comparable targeting efficiency to SpG Cas9 with fewer chromosome translocations.
  • miBEs achieved robust DNA base conversion efficiency up to 92%.

Conclusions:

  • Engineered IscB (enIscB) systems, including enIscB-T5E and miBEs, are effective tools for genome editing in mammalian systems.
  • These miniature tools offer advantages for in vivo applications and precise DNA modifications.