GigaAssay – an adaptable high-throughput saturation mutagenesis assay for Tat-driven transcription
Description
High throughput assay systems have had a disproportionally large impact on understanding on uncovering the mechanisms of how basic cells functions. However, high throughput assays that directly assess molecular functions are limited. To address this challenge, Herein we engineered, developed, and tested the GigaAssay, a modular high throughput molecular function assay system. In this implementation, we measured how the Tat transcription factor binds HIV the long terminal repeat and recruits host factors to drives expression of a GFP reporter in an engineered reporter cell line. In a one pot assay system, each cell was infected with one virus from a library encoding 1,000s of Tat proteins, each cDNA molecule with one or two unique missense mutations and a unique molecular identifier. Infected cells are flow sorted based on their GFP fluorescence readout. The ratio of signal from each bin is used the calculate the transcriptional activity of each unique Tat molecule is compared to wild type Tat. This GigaAssay one pot assay system was adapted to study how variants impact HIV Tat-driven transactivation of a green fluorescent protein reporter. We assayed cells with individually randomly barcoded cDNAs for all 1,615 Tat single and 3,429 double amino acid substitutions with no single mutant dropout. Each mutant was assayed with more than 100 separately unique molecular identifier barcoded cDNA molecules for each mutant. The results were verified to have high accuracy with five independent assay performance assessments with benchmark data, individually tested clones, and replicate comparisons all indicate exceptional reproducibility, accuracy, and robustness. The resulting analyses yields new insights into the value of tracking single molecules, structure, function, tolerance, and intragenic epistasis of Tat driven transcription in human cells.
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