Overcoming the Undesirable CRISPR-Cas9 Expression in Gene Correction

Area of Science:

• Biotechnology
• Gene Therapy
• Molecular Biology

Background:

• CRISPR-Cas9 offers permanent genetic correction but faces challenges in safe delivery and immune response.
• Cas9, a foreign protein, can trigger immune elimination of gene-edited cells, hindering clinical application.
• Efficient and safe delivery of gene editing tools to target cells remains a critical hurdle.

Purpose of the Study:

• To engineer a novel delivery system for CRISPR-Cas9 gene editing.
• To overcome challenges of safe delivery, immune response, and gene targeting efficiency.
• To demonstrate site-specific gene targeting with limited Cas9 expression and achieve functional gene correction.

Main Methods:

• Development of a helper-dependent adenoviral (HD-Ad) vector for in vivo gene delivery.
• Co-delivery of CRISPR-Cas9/single-guide RNA and donor DNA (LacZ reporter or CFTR gene) to cultured cells.
• Analysis of HD-Ad vector genome integrity and Cas9 expression following gene integration.

Main Results:

• Successful co-delivery of CRISPR-Cas9/single-guide RNA and donor DNA using the HD-Ad vector.
• Compromised HD-Ad vector genome integrity upon donor DNA integration, leading to concurrent elimination of Cas9 expression.
• Demonstrated feasibility of site-specific gene targeting with transient Cas9 activity.
• Achieved stable CFTR gene expression and functional correction in cultured cells.

Conclusions:

• The engineered HD-Ad vector system enables efficient and safe co-delivery of CRISPR-Cas9 and donor DNA.
• Transient Cas9 expression via this system mitigates potential immune responses.
• This approach holds promise for in vivo gene therapy applications, including CFTR correction.