An improved CRISPR-Cas9 protein-based method for knocking out insect Sf9 cell genes

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Insect cells are valuable for biopharmaceutical production but lack advanced genetic engineering tools.
• CRISPR-Cas9 technology offers powerful gene editing capabilities with limited application in insect cells.

Purpose of the Study:

• To develop and implement an efficient CRISPR-Cas9 pipeline for gene editing in Sf9 insect cells.
• To engineer Sf9 cells by knocking out the caspase initiator Sf-Dronc to enhance apoptosis resistance.
• To evaluate the impact of Sf-Dronc knockout on cell phenotype and the production of various biopharmaceutical products.

Main Methods:

• A ribonucleoprotein (RNP) complex delivery strategy using guide RNA and Cas9 enzyme was established for Sf9 cells.
• The pipeline was validated by targeting the fdl gene, achieving a 68% knockout rate.
• Sf-Dronc was targeted for knockout, and resulting cell lines were analyzed for apoptosis resistance, viability, and product yields using next-generation sequencing.

Main Results:

• The developed CRISPR-Cas9 pipeline demonstrated high efficiency, with a 68% knockout rate for the fdl gene.
• Engineered Sf9 cells with Sf-Dronc knockout exhibited increased resistance to apoptosis and delayed viability loss upon baculovirus infection.
• Sf-Dronc deletion resulted in a twofold increase in the production of influenza virus-like particles (VLPs) while having minimal impact on recombinant adeno-associated virus (rAAV) and PfRipr5 production.

Conclusions:

• An efficient CRISPR-Cas9 gene editing pipeline was successfully implemented in Sf9 insect cells.
• The engineered Sf9 cell lines demonstrate enhanced apoptosis resistance and improved production of specific biopharmaceuticals, particularly VLPs.
• This advancement facilitates the development of superior insect cell hosts for the biopharmaceutical industry.