@article{acdaee0e7a98448999b4d769dbff45b4,
title = "GigaAssay – An adaptable high-throughput saturation mutagenesis assay platform",
abstract = "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.",
keywords = "High-throughput assay, Intragenic epistasis, Protein structure, Saturation mutagenesis, Tat, Transcription",
author = "Ronald Benjamin and Giacoletto, {Christopher J.} and FitzHugh, {Zachary T.} and Danielle Eames and Lindsay Buczek and Xiaogang Wu and Jacklyn Newsome and Han, {Mira V.} and Tony Pearson and Zhi Wei and Atoshi Banerjee and Lancer Brown and Valente, {Liz J.} and Shirley Shen and Deng, {Hong Wen} and Schiller, {Martin R.}",
note = "Funding Information: We thank Drs. Edwin Oh, and Richard Tillet from the UNLV Nevada Institute of Personalized Medicine Genome Acquisition and Analysis Core for access to a flow cytometer sorter and help with some NGS sequencing and interpretation for GigaAssay development. We Thanks Drs. Jefferson Kinney (University of Nevada, Las Vegas) and Tom Metzger (Roseman University) for use of their flow cytometer. We wish to acknowledge the help of Dr. Nora Caberoy with electroporation. We appreciate the discussions we had with Drs. Qing Wu and Michael F. Lin about statistical assessment of the GigaAssay results. We thank Dr. James Raymond for help with editing the manuscript. NIH : R21AI116411 , R15GM107983 , R21AI078708 , R56AI109156 , P20GM121325 , COBRE and the Governor{\textquoteright}s Office of Economic Development (Grant Number: 1547526 ), and the Prabhu endowed professorship. We also acknowledge the UNLV College of Science for a grant to develop the GigaAssay. Funding Information: We thank Drs. Edwin Oh, and Richard Tillet from the UNLV Nevada Institute of Personalized Medicine Genome Acquisition and Analysis Core for access to a flow cytometer sorter and help with some NGS sequencing and interpretation for GigaAssay development. We Thanks Drs. Jefferson Kinney (University of Nevada, Las Vegas) and Tom Metzger (Roseman University) for use of their flow cytometer. We wish to acknowledge the help of Dr. Nora Caberoy with electroporation. We appreciate the discussions we had with Drs. Qing Wu and Michael F. Lin about statistical assessment of the GigaAssay results. We thank Dr. James Raymond for help with editing the manuscript. NIH: R21AI116411, R15GM107983, R21AI078708, R56AI109156, P20GM121325, COBRE and the Governor's Office of Economic Development (Grant Number: 1547526), and the Prabhu endowed professorship. We also acknowledge the UNLV College of Science for a grant to develop the GigaAssay. Publisher Copyright: {\textcopyright} 2022",
year = "2022",
month = jul,
doi = "10.1016/j.ygeno.2022.110439",
language = "English (US)",
volume = "114",
journal = "Genomics",
issn = "0888-7543",
publisher = "Academic Press Inc.",
number = "4",
}