"GigaAssay – An adaptable high-throughput saturation mutagenesis assay " by Ronald Benjamin, Christopher J. Giacoletto et al.
 

GigaAssay – An adaptable high-throughput saturation mutagenesis assay platform

Document Type

Article

Publication Date

7-1-2022

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.

Identifier

85135286773 (Scopus)

Publication Title

Genomics

External Full Text Location

https://doi.org/10.1016/j.ygeno.2022.110439

e-ISSN

10898646

ISSN

08887543

PubMed ID

35905834

Issue

4

Volume

114

Grant

1547526

Fund Ref

National Institute of Allergy and Infectious Diseases

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