Automation of high-throughput arrayed lentivirus production and titration

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• Generating arrayed genome-wide CRISPR libraries in lentiviral format is complex and resource-intensive.
• Existing methods are often laborious, time-consuming, and costly, hindering large-scale applications.

Purpose of the Study:

• To develop a fully automated workflow for lentivirus production and titration.
• To address the limitations of manual methods for generating arrayed lentiviral libraries.

Main Methods:

• Utilized a Biomek i7 Hybrid automated workstation integrated with multiple instruments and SAMI EX software.
• Implemented reverse transfection and triplicate wells for lentivector production in 96-well plates.
• Developed a titration method using U937-mCherry cells and a linear regression model for HEK293T cell conversion, validated across different flow cytometers.

Main Results:

• Achieved an average of three viral particles in transduction unit (TU) per producing HEK293T cell.
• Demonstrated strong reproducibility in titration across different flow cytometers (R² = 0.9).
• Produced high titers (median/mean ~1.2 x 10^6 TU/mL) for over 97% of 1,760 samples, exceeding the 2x10^5 TU/mL threshold.

Conclusions:

• The automated workflow provides a robust, scalable, and cost-effective platform for high-throughput arrayed lentiviral library production.
• This automation significantly streamlines the generation of essential tools for functional genomics research.
• The developed method enhances efficiency and reproducibility in lentiviral vector generation and titration.