Development of an intein-mediated split-Cas9 system for gene therapy

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• CRISPR/Cas9 is a powerful gene editing tool but its large size limits delivery via adeno-associated virus (AAV) vectors.
• Efficient delivery of gene editing components is crucial for therapeutic applications.

Purpose of the Study:

• To develop a split-Cas9 system to bypass AAV packaging limitations.
• To assess the nuclease activity and therapeutic potential of the reconstituted split-Cas9 system.

Main Methods:

• A split-Cas9 system was engineered by fusing Cas9 halves with split-intein moieties.
• Intein-mediated trans-splicing reconstituted functional Cas9 upon co-expression.
• Activity was validated using reporter systems, endogenous gene targeting, and assessing homologous directed recombination (HDR).

Main Results:

• The split-intein-mediated split-Cas9 system demonstrated nuclease activity comparable to wild-type Cas9.
• A split-Cas9D10A nickase version also showed comparable activity.
• Efficient HDR was achieved using a double nick strategy and co-delivered repair templates.
• Crucially, split-Cas9 was successfully packaged, delivered, and reconstituted in cells via rAAV.

Conclusions:

• The developed split-Cas9 system effectively overcomes AAV packaging constraints for CRISPR/Cas9 delivery.
• This technology holds significant promise for advancing in vivo gene therapy applications.
• Intein-mediated reconstitution offers a viable strategy for efficient gene editing using rAAV vectors.