Construction of a TAT-Cas9-EGFP Site-Specific Integration Eukaryotic Cell Line Using Efficient PEG10 Modification

Area of Science:

• Molecular Biology
• Gene Editing Technologies

Background:

• CRISPR/Cas9 is vital for eukaryotic genome modification, with homology-directed repair (HDR) knock-in (KI) essential for disease modeling and gene therapy.
• Low HDR-mediated KI efficiency is a significant limitation in genetic engineering applications.
• HEK293 cells, particularly the 293T cell line, are valuable for recombinant protein production, especially when integrated site-specifically at the AAVS1 locus for stable expression.

Purpose of the Study:

• To investigate the impact of 5' end PEG10 modification on site-specific gene integration efficiency.
• To enhance the homology-directed repair (HDR) mediated knock-in (KI) efficiency at the AAVS1 locus in 293T cells.
• To establish a high-expression, site-specific integration 293T cell line for TAT-Cas9-EGFP production.

Main Methods:

• Utilized CRISPR/Cas9 system with a 5' end PEG10 modification.
• Performed site-specific gene integration at the AAVS1 locus in the 293T cell line.
• Assessed knock-in efficiency for a 1.8 kb target fragment.

Main Results:

• The 5' end PEG10 modification increased knock-in efficiency by 1.9-fold for a 1.8 kb target fragment.
• Knock-in efficiency improved from 26% to 49% with the modification.
• Successfully established a high-expression, site-specific integration 293T cell line for TAT-Cas9-EGFP.

Conclusions:

• 5' end PEG10 modification significantly enhances HDR-mediated KI efficiency.
• This optimized system provides a reliable method for creating stable, high-expression cell lines for protein production.
• The developed 293T cell line serves as a valuable resource for recombinant protein manufacturing.