GigaAssay – an adaptable high-throughput saturation mutagenesis assay for Tat-driven transcription

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.高通量检测系统（high throughput assay systems）在解析基础细胞功能的作用机制方面，发挥了与其体量不成比例的巨大影响力。然而，可直接评估分子功能的高通量检测方法仍较为稀缺。为应对这一挑战，本研究设计、开发并验证了GigaAssay——一种模块化的高通量分子功能检测系统。在本研究的实施过程中，我们检测了Tat转录因子（Tat transcription factor）如何结合人类免疫缺陷病毒（HIV）的长末端重复序列（long terminal repeat, LTR），并招募宿主因子以驱动工程化报告细胞系中绿色荧光蛋白（green fluorescent protein, GFP）报告基因的表达。在单管检测系统（one pot assay system）中，每个细胞均被携带编码千余种Tat蛋白的文库中的一种病毒感染；每个互补DNA（complementary DNA, cDNA）分子均带有1至2个独特的错义突变位点，以及一个唯一的分子标识符（molecular identifier）。研究人员基于细胞的GFP荧光信号对感染后的细胞进行流式分选（flow sorting）。研究人员利用每个分选组分的信号比值，计算每个独特Tat分子的转录活性，并将其与野生型Tat（wild-type Tat）的活性进行对比。该单管检测版GigaAssay系统可被改造用于研究变异体如何影响HIV Tat介导的绿色荧光蛋白报告基因反式激活作用。我们针对全部1615种Tat单氨基酸替换突变体与3429种双氨基酸替换突变体，使用带有独立随机条形码的cDNA对细胞进行了检测，未出现任何突变体缺失的情况。针对每个突变体，研究人员均使用超过100个带有唯一分子标识符条形码的cDNA分子进行检测。通过五项独立的检测性能评估——包括基准数据集验证、单克隆实验验证以及重复实验比对——结果证实本方法具有极高的准确性，且展现出优异的重复性、精准度与鲁棒性（robustness）。本研究的分析结果为追踪人类细胞中Tat介导的转录过程的单分子特征、结构特性、功能效应、耐受性以及基因内上位效应（intragenic epistasis）提供了全新的研究视角。