GigaAssay - An adaptable high-throughput saturation mutagenesis assay platform

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• High-throughput assays are crucial for understanding cell functions but assessing molecular functions directly remains challenging.
• Existing methods limit the simultaneous analysis of numerous genetic variants and their molecular functions.

Purpose of the Study:

• To introduce the GigaAssay, a modular, high-throughput, one-pot system for measuring molecular functions of thousands of genetic variants concurrently.
• To demonstrate the GigaAssay's proof of concept using the HIV Tat transcription factor and a GFP reporter system.

Main Methods:

• Utilized a viral library encoding thousands of Tat mutant proteins, each with a unique molecular identifier (UMI).
• Infected engineered reporter cells and employed flow cytometry to sort cells based on GFP fluorescence.
• Calculated transcriptional activity from fluorescence ratios across sorted cell populations.

Main Results:

• The GigaAssay achieved high accuracy (95%) and positive predictive value (98%) using UMIs, validated against benchmark data.
• Substitution tolerance analysis revealed specific mutation patterns concentrated in the Cys-rich region of Tat.
• Identified significant intragenic epistasis (10%) in Tat when comparing single and double mutants.

Conclusions:

• The GigaAssay is a powerful tool for high-throughput molecular function analysis of genetic variants.
• The system provides accurate and reliable data for structure-function studies, exemplified by HIV Tat.
• Findings offer new insights into Tat protein function, epistasis, and mutation tolerance.