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

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.