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.

高通量检测系统在解析细胞基本功能的作用机制方面，产生了不成比例的深远影响。然而，能够直接评估分子功能的高通量检测方法仍较为匮乏。为应对这一挑战，本研究构建、开发并验证了GigaAssay——一种模块化高通量分子功能检测系统。在本研究的实施过程中，我们针对工程化构建的报告细胞系，检测了Tat转录因子结合人类免疫缺陷病毒（HIV, Human Immunodeficiency Virus）长末端重复序列（LTR, long terminal repeat）、招募宿主因子以驱动绿色荧光蛋白（GFP, Green Fluorescent Protein）报告基因表达的过程。在单管检测体系中，每一个细胞均被来自编码数千种Tat蛋白的文库中的单一病毒感染；每个互补DNA（cDNA, complementary DNA）分子均携带1至2处独特的错义突变，以及一个独一无二的分子标识符。感染后的细胞基于其GFP荧光信号进行流式分选。利用各分选组分的信号比值，计算每个独特Tat分子相对于野生型Tat的转录活性。本研究将该GigaAssay单管检测体系应用于探究变异体如何影响HIV Tat介导的绿色荧光蛋白报告基因反式激活过程。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进行了细胞检测，未遗漏任何单突变体。每个突变体均通过100余个带有独立独特分子标识符的条形码标记cDNA分子进行检测。通过五项独立的检测性能验证实验（包括基准数据集比对、单克隆验证以及重复实验对比），证实本研究结果具备极高的准确性；各项评估均表明该体系具有优异的可重复性、准确性与鲁棒性。最终的分析结果为人类细胞中Tat介导的转录过程的单分子追踪、结构与功能解析、耐受性研究以及基因内上位性分析提供了全新的认知。