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.

Fig. 1.

Design and implementation of the GigaAssay for Tat transcriptional activation. A. Design of GigaAssay system. Propagation of the recombined cells under poison selection. Cell sorting based on GFP reporter expression. gDNA is isolated, and a targeted Tat amplicon library is prepared and sequenced by NGS. Schematic representation of Tat dependent LTR transactivation inducing GFP expression. B.-D. Epifluorescence microscopic images of LentiX293T/LTR-GFP cells transfected with GigaAssay plasmids: Empty vector/LTR-GFP (B. - control); wtTat/LTR-GFP (C, + control); and an inhibitory mutant [12], C27S-Tat/LTR-GFP (D, − control). E. Flow cytometry of GigaAssay controls in LentiX293T/LTR-GFP cell to define gates. F. Flow cytometry sorting of GigaAssay LentiX293T/LTR-GFP cell library cells with gates defined by − and + controls.

Fig. 2.

Summary of Tat mutant transcriptional activities and GigaAssay verification. Tat transactivation activity for a saturating mutagenesis GigaAssay. The activity represents the level of Tat transactivation activity score measured by GFP⁺ / (GFP⁺ + GFP⁻) reads for each UMI-barcode averaged for each mutant. A. Pie graph showing percentage of mutants with activities similar to known WT and LOF activities. B. Bin plot showing range of activities for Tat mutants (n = 1,615). C. Assay reproducibility and verification summary. D. Scatter plots for technical replicates. Transcriptional activity [GFP⁺/(GFP⁻ + GFP⁺)] correlation among replicate GigaAssays (R² = 0.99).

Fig. 3.

Heatmaps of p values for Tat mutant transcriptional activities in LentiX293T/LTR-GFP cells. q values for comparison of Tat mutant activity to sets of mutants with WT (A) or LOF activity (B). Keys for q value colors are shown.

Fig. 4.

Heatmap showing Tat-induced transcriptional activity for a saturating mutagenesis GigaAssay. Heatmap for mutated amino acid for each position in Tat. The color gradient represents the level of Tat transactivation activity score measured by GFP⁺ / (GFP⁺ + GFP⁻) reads for each UMI-barcode averaged for each mutant. Black boxes are the WT amino acids and grey boxes are null values. A color key is shown. Abbreviations are LOF = loss-of-function, SS = secondary structure, Surface – solvent accessible surface, PTM – post-translational modification.

Fig. 5.

Tat mutant impact on structure/function. All surface maps are on the WT Tat 3D structure (PDB: 1TEV) with one member of each pair rotated 180^o about the Y axis: A. Amino acid positions on Tat backbone. B. Regions of Tat [20]. C. Secondary structures. D. Solvent assessable surfaces are with residues with <10% solvent exposure colored blue. E. Tat positions that do not tolerate any substitution (C25, C27, C30, C33, C34, C37, and K41; red). F. Ala scanning substitutions. E. Pro scanning substitutions. F. Cys scanning substitutions. G. Gly scanning substitutions. F.-I. Residues colored black are for reference amino acids that match the type of scanning. A gradient of yellow with no activity to green with full activity is shown. Minimum (J), average (K), and maximum (L) transactivation activity heatmap for all substitutions. A gradient of red with WT activity to yellow with no activity is shown. Abbreviations are: Single letter amino acid code. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6.

3D structure surface plots of different properties and function of Tat. All surface maps are on WT Tat 3D structure (PDB: 1TEV): A-F. Physiochemical tolerance surface plots for polar charged amino acids, those separated by positively and negatively charged amino acids, small aliphatic, polar uncharged, and large hydrophobic amino acids, respectively (see Methods). MCC = Mathews Correlation Coefficient. A gradient of blue to white to magenta ranging from lower to higher MCC scores for each position for the class of amino acids indicated is shown. Panel G is repeated from Fig. 4B here for visual comparison. H. Regions of Tat truncation and missense mutants that lose (cyan) or retain (light grey) activity. I. Tat PTMs. J.-L. Tat PPIs in 3 groups. The color key for regions, secondary structure, PTMs, PPIs, PPVs, and Tat activity are as in Fig. 4. Abbreviations are: PTM = post translational modification; PPI = protein-protein interaction. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)