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Related Experiment Video

Updated: May 12, 2026

Genome Editing in Mammalian Cell Lines using CRISPR-Cas
07:56

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Published on: April 11, 2019

Engineering IscB to develop highly efficient miniature editing tools in mammalian cells and embryos.

Niannian Xue1, Dishan Hong1, Dan Zhang1

  • 1Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.

Molecular Cell
|August 3, 2024
PubMed
Summary

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Engineered IscB proteins offer enhanced genome editing in mammalian cells. This miniature tool, eIscB-D, shows high efficiency for base editing and disease model generation in mice.

Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetics

Background:

  • IscB proteins are ancestral to Cas9 endonucleases, offering potential for small, versatile genome editing tools.
  • Current IscB activity in mammalian cells is limited, hindering their application.
  • Enhancing IscB function is crucial for developing novel gene-editing technologies.

Purpose of the Study:

  • To improve the activity and efficiency of IscB proteins in mammalian genome editing.
  • To develop engineered IscB variants (eIscB-D) and optimized RNA components (eωRNA) for enhanced gene editing.
  • To explore the application of engineered IscB in base editing and disease model generation.

Main Methods:

  • Introduced three specific substitutions into the IscB protein sequence.
Keywords:
DNA-binding domainIS200/IS605IscBbase editingdisease modelsgenome editingguide RNAminiature CRISPR nucleaseprotein engineering

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Last Updated: May 12, 2026

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  • Fused a sequence-non-specific DNA-binding domain to create the eIscB-D variant.
  • Engineered a shorter, more efficient ωRNA (eωRNA).
  • Tested the eIscB-D/eωRNA system in mouse cell lines and embryos.

    • Main Results:

    • Achieved an average 7.5-fold increase in activity after introducing substitutions in IscB.
    • The eIscB-D variant demonstrated up to 91.3% editing efficiency.
    • The engineered eIscB-D/eωRNA system showed a 20.2-fold increase in activity compared to the original IscB.
    • Successfully adapted eIscB-D for efficient cytosine and adenine base editing.
    • Demonstrated high activity in mouse cell lines and embryos for disease model generation.

    Conclusions:

    • Engineered IscB variants, particularly eIscB-D, significantly enhance genome editing capabilities in mammalian systems.
    • The eIscB-D/eωRNA system represents a powerful and efficient miniature genome-editing tool.
    • This technology holds promise for diverse applications, including base editing and rapid generation of disease models.