CRISPR library screening to develop HEK293-derived cell lines with improved lentiviral vector titers

Area of Science:

• Biotechnology
• Molecular Biology
• Gene Therapy

Background:

• Lentiviral (LV) vectors are key tools for gene therapy and treating diseases.
• Increasing demand for clinical applications necessitates scalable production of purified LV vectors.

Purpose of the Study:

• To develop and apply CRISPR library screening methods for identifying genetic modifications that enhance LV vector production in human cells.
• To discover specific genes whose perturbation can significantly increase lentiviral vector titers.

Main Methods:

• Utilized LV vector-based Human CRISPR Activation and Knockout libraries (Calabrese and Brunello) to modify HEK293 and HEK293T cells.
• Employed iterative cycles of cell expansion, LV vector genome rescue via transfection, harvesting, and sequential transduction.
• Identified enriched guide RNAs (gRNAs) targeting genes influencing LV vector production using Next-Generation Sequencing (NGS).

Main Results:

• Successfully implemented a CRISPR screening workflow to identify genetic perturbations affecting LV vector production.
• Enriched specific gRNAs targeting genes that modulate lentiviral vector yields.
• Identified potential target genes, such as TTLL12, an inhibitor of antiviral innate immunity, for further investigation.

Conclusions:

• CRISPR screening is an effective strategy for discovering genetic targets to improve lentiviral vector production.
• Perturbations of identified genes, like TTLL12, hold promise for engineering cell lines with enhanced LV vector productivity.
• Further validation is required to confirm the impact of identified genes on lentiviral vector titers and therapeutic applications.