GigaAssay – An adaptable high-throughput saturation mutagenesis assay platform

High-throughput assay systems have had a large impact on understanding the mechanisms of basic cell functions. However, high-throughput assays that directly assess molecular functions are limited. Herein, we describe the “GigaAssay”, a modular high-throughput one-pot assay system for measuring molecular functions of thousands of genetic variants at once. In this system, each cell was infected with one virus from a library encoding thousands of Tat mutant proteins, with each viral particle encoding a random unique molecular identifier (UMI). We demonstrate proof of concept by measuring transcription of a GFP reporter in an engineered reporter cell line driven by binding of the HIV Tat transcription factor to the HIV long terminal repeat. Infected cells were flow-sorted into 3 bins based on their GFP fluorescence readout. The transcriptional activity of each Tat mutant was calculated from the ratio of signals from each bin. The use of UMIs in the GigaAssay produced a high average accuracy (95%) and positive predictive value (98%) determined by comparison to literature benchmark data, known C-terminal truncations, and blinded independent mutant tests. Including the substitution tolerance with structure/function analysis shows restricted substitution types spatially concentrated in the Cys-rich region. Tat has abundant intragenic epistasis (10%) when single and double mutants are compared. [Display omitted] •There is no accurate high-throughput assay to study how mutants impact a gene function•We established an accurate, high-throughput functional assay of transcriptional activity of Tat mutants in human cells•We produced a comprehensive saturating mutagenic landscape of the Tat transcription factor and 1000 s of doubles mutants for intragenic epistasis•Structure / Activity / Tolerance 3D plots expand upon traditional SAR studies•The new GigaAssay should be extensible to the analysis of other functions and genes.•Our characterization of Tat mutants highlights the importance of intergenic epistasis in viral transciption infection and provides a framework for interpretation of Tat mutants and their effect on viral latency.