Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Chinese hamster ovary (CHO) cells are crucial for therapeutic protein production.
• Random integration of transgenes leads to unstable and variable expression in CHO cells.
• Targeted gene integration is needed for consistent protein manufacturing.

Purpose of the Study:

• To develop an efficient method for targeted gene integration in CHO cells.
• To utilize CRISPR/Cas9 genome editing for precise transgene insertion.
• To achieve stable and homogeneous transgene expression for biopharmaceutical production.

Main Methods:

• Employed CRISPR/Cas9 genome editing system for targeted gene insertion.
• Designed a donor plasmid with a gene of interest (GOI) and homology arms.
• Utilized drug selection for identification of successfully integrated cell lines.

Main Results:

• Achieved efficient and precise integration of a 3.7-kb gene expression cassette into specific CHO cell loci.
• Demonstrated homogeneous transgene expression in targeted CHO cell lines.
• Established a reliable method for generating isogenic cell lines with consistent protein production.

Conclusions:

• CRISPR/Cas9 mediated targeted integration offers a robust solution for unstable transgene expression in CHO cells.
• This approach facilitates the development of cell lines with predictable and consistent therapeutic protein yields.
• The strategy advances the generation of high-quality biopharmaceuticals through improved cell line engineering.