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Updated: Jan 10, 2026

CRISPR Epigenome Editing in Human Cells using Plasmid DNA Transfection and mRNA Nucleofection Delivery
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CRISPR Epigenome Editing in Human Cells using Plasmid DNA Transfection and mRNA Nucleofection Delivery

Published on: May 30, 2025

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Nuclear Localization Signals Enable the Cellular Delivery of an Anti-CRISPR Protein to Control Genome Editing

Axel O Vera, Franklin J Avilés-Vázquez, Taekjip Ha

    Biorxiv : the Preprint Server for Biology
    |November 24, 2025
    PubMed

    Insights

    We developed 6×NLS-Acr, a cell-permeable anti-CRISPR protein (Acr), to enhance CRISPR-Cas genome editing precision. This inhibitor rapidly enters cells, significantly improving editing specificity and therapeutic potential.

    Area of Science:

    • Molecular Biology
    • Biotechnology
    • Gene Editing

    Background:

    • Precise Cas9 regulation is crucial for minimizing off-target effects and genotoxicity in genome editing.
    • Current anti-CRISPR proteins (Acrs) face delivery challenges due to cell membrane impermeability.
    • Existing delivery methods like vectors or electroporation have limitations for therapeutic applications.

    Purpose of the Study:

    • To develop a cell-permeable anti-CRISPR protein (Acr) for enhanced CRISPR-Cas genome editing.
    • To investigate the cellular entry mechanism and inhibitory function of the novel Acr.
    • To assess the efficacy of the cell-permeable Acr in various human cell types.

    Main Methods:

    • Engineered a cell-permeable Acr (6×NLS-Acr) using nuclear localization signals (NLSs).
    • Utilized fluorescent tagging to visualize 6×NLS-Acr cellular entry and binding to Cas9·gRNA complexes.
    • Tested 6×NLS-Acr transduction efficiency and Cas9 inhibition in diverse human cell models, including stem cells and 3D cultures.

    Main Results:

    • 6×NLS-Acr demonstrated rapid cellular transduction (within 5 min) with an IC50 of 0.47 µM.
    • Achieved up to 99% inhibition of Cas9 activity and nearly 100% increase in genome-editing specificity.
    • Successfully delivered 6×NLS-Acr into various human cell types, including stem cells and 3D cultures.

    Conclusions:

    • 6×NLS-Acr is a highly effective cell-permeable inhibitor of CRISPR-Cas9 activity.
    • This novel Acr significantly enhances genome editing precision and specificity.
    • 6×NLS-Acr holds considerable potential for advancing CRISPR-based therapeutics.

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