Systematic Evaluation of Site-Specific Recombinant Gene Expression for Programmable Mammalian Cell Engineering

Area of Science:

• Molecular Biology
• Biotechnology
• Cell Engineering

Background:

• Stable recombinant gene expression is crucial for biological research and biotechnology.
• Targeted gene integration into specific sites improves predictability but expression is complex.
• Investigating the interplay between integration sites and expression cassettes requires better tools.

Purpose of the Study:

• To systematically investigate how expression cassette components influence recombinant gene expression in defined integration sites.
• To develop a versatile CRISPR/Cas9-based toolbox for engineering mammalian cell lines with targeted gene integration.
• To enable rapid screening of regulatory elements for precise control of gene expression.

Main Methods:

• Development of a CRISPR/Cas9 toolbox for targeted integration of a landing pad in mammalian cells.
• Construction of site-specific recombinant cell lines for recombinase-mediated cassette exchange.
• Systematic screening of various 5' proximal regulatory elements and their impact on gene expression.

Main Results:

• Demonstrated that multiple components within expression cassettes significantly impact net recombinant gene expression.
• Successfully generated mammalian cell lines with predictable and tunable gene expression patterns.
• Identified distinct and robust expression profiles driven by different regulatory elements in CHO cells.

Conclusions:

• The developed toolbox enables precise engineering of gene expression for user-defined outputs.
• This approach facilitates the creation of cell lines with tailored gene expression for biotechnological applications.
• Programmable mammalian cell engineering is advanced through systematic analysis of regulatory elements and integration sites.