biostudies-literatureBenjamin RNIAID NIH HHSNHGRI NIH HHSNIGMS NIH HHS110439https://www.ebi.ac.uk/biostudies/studies/S-EPMC9420302biostudies-literatureUnknown114(4)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 GenomicsGigaAssay - An adaptable high-throughput saturation mutagenesis assay platform.PMC9420302R15 GM107983P20 GM121325R56 AI109156R43 HG012537R21 AI078708R21 AI116411Eames DBuczek LSchiller MRPearson TDeng HWWei ZBanerjee AHan MVBrown LGiacoletto CJValente LJShen SNewsome JBenjamin RFitzHugh ZTWu XfalseGigaAssay - 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 2022-01-01T00:00:00Z2022 Jul2026-04-08T11:02:05.905Z2025-04-21T14:53:47.15ZS-EPMC94203023590583410.1016/j.ygeno.2022.110439