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Related Experiment Video

Updated: Jun 21, 2025

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Published on: November 1, 2024

Engineering miniature IscB nickase for robust base editing with broad targeting range.

Linxiao Han¹, Yueer Hu¹, Qiqin Mo¹

¹Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, Shanghai, China.

Nature Chemical Biology

|July 8, 2024

Summary

Researchers engineered miniature base editors, IminiBEs, from IscB for efficient genome editing. Fusing Sso7d protein created SIminiBEs, further enhancing editing efficiency and broadening target recognition for precise genetic modifications.

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Last Updated: Jun 21, 2025

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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Published on: March 25, 2020

Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

IscB, a Cas9 homolog, offers a smaller size advantageous for viral delivery systems.
The native OgeuIscB exhibits low editing efficiency in mammalian cells, limiting its therapeutic potential.

Purpose of the Study:

To engineer high-efficiency miniature base editors from OgeuIscB for improved genomic mutation capabilities.
To enhance the editing efficiency and target site recognition of IscB-based base editors.

Main Methods:

Engineering of OgeuIscB nickase and its cognate ωRNA to create IminiBEs (miniature base editors).
Fusion of the Sso7d DNA-binding protein to IminiBEs to create SIminiBEs.
Assessment of editing efficiency and target-adjacent motif recognition of IminiBEs and SIminiBEs in mammalian cells.

Main Results:

IMiniCBE achieved an average editing efficiency of 67%, and IminiABE achieved 52%.
SIminiBEs demonstrated a two- to threefold increase in editing efficiency at challenging sites.
IMiniCBE and SIminiCBE expanded target recognition beyond the canonical NWRRNA motif to include NNRR, NNRY, and NNYR.

Conclusions:

IMiniBEs and SIminiBEs represent efficient and versatile miniature base editing tools.
These engineered editors facilitate site-specific genomic mutations with enhanced efficiency and broader targeting scope.
The development holds promise for advanced gene editing applications requiring precise genomic alterations.