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Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
Published on: June 16, 2017
Protective antigen-mediated delivery of an anti-CRISPR protein for precision genome editing
Axel O Vera1,2, Nicholas L Truex1, Vedagopuram Sreekanth2,3,4
1Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139.
Abstract:
Precise control over the dosage of Cas9-based technologies is essential because off-target effects, mosaicism, chromosomal aberrations, immunogenicity, and genotoxicity can arise with prolonged Cas9 activity. Type II anti-CRISPR proteins (Acrs) inhibit and control Cas9 but are generally impermeable to the cell membrane due to their size and anionic charge. Moreover, existing Acr delivery methods are long-lived and operate within hours (e.g., viral and nonviral vectors) or require external devices (e.g., electroporation), limiting therapeutic applications. To address these problems, we developed a protein-based anti-CRISPR delivery platform, LFN-Acr/PA, which delivers Acrs into cells within minutes. LFN-Acr/PA is a nontoxic, two-component protein system derived from anthrax toxin, where protective antigen (PA) proteins bind receptors widespread in human cells, forming a pH-triggered endosomal pore that an engineered Acr (LFN-Acr) binds and uses to enter the cell. In the presence of PA, LFN-Acr enters human cells (e.g., immortalized cell lines, embryonic stem cells, and 3D cell cultures) at concentrations as low as 2.5 pM to inhibit up to 95% of Cas9-mediated knockout, knock-in, transcriptional activation, and base editing. Timing LFN-Acr delivery reduces off-target base editing and increases Cas9 specificity by 41%. LFN-Acr/PA is the most potent known cell-permeable CRISPR-Cas inhibition system, significantly improving the utility of CRISPR for genome editing.
Insights
A novel protein delivery system, LFN-Acr/PA, enables rapid cellular entry of anti-CRISPR proteins (Acrs) to precisely control Cas9 activity. This enhances CRISPR genome editing safety and efficacy by minimizing unwanted genetic modifications.
Area of Science:
- Molecular Biology
- Biotechnology
- Gene Editing
Background:
- CRISPR-Cas9 technology offers powerful genome editing but requires precise dosage control to prevent off-target effects, mosaicism, and genotoxicity.
- Type II anti-CRISPR proteins (Acrs) can inhibit Cas9 activity, but their delivery into cells is challenging due to membrane impermeability and limitations of current methods like viral vectors or electroporation.
Purpose of the Study:
- To develop a rapid and efficient cell-permeable delivery platform for anti-CRISPR proteins (Acrs) to enhance the safety and precision of CRISPR-Cas9 genome editing.
- To create a protein-based system that overcomes the limitations of existing Acr delivery methods for therapeutic applications.
Main Methods:
- Developed LFN-Acr/PA, a two-component protein system derived from anthrax toxin, utilizing protective antigen (PA) for cell entry and an engineered Acr (LFN-Acr).
- Demonstrated LFN-Acr/PA's ability to deliver LFN-Acr into various human cell types, including immortalized cell lines, embryonic stem cells, and 3D cultures, within minutes.
- Quantified the inhibition of Cas9-mediated editing (knockout, knock-in, transcriptional activation, base editing) at low LFN-Acr concentrations.
Main Results:
- LFN-Acr/PA efficiently delivered LFN-Acr into human cells at concentrations as low as 2.5 pM.
- Achieved up to 95% inhibition of Cas9-mediated genome editing functions.
- Demonstrated that timed delivery of LFN-Acr reduced off-target base editing and increased Cas9 specificity by 41%.
Conclusions:
- LFN-Acr/PA represents the most potent known cell-permeable CRISPR-Cas inhibition system to date.
- This platform significantly improves the precision and safety of CRISPR-Cas9 genome editing, expanding its therapeutic potential.
- The rapid delivery mechanism offers a significant advantage over existing methods for controlling Cas9 activity in real-time.
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