Empowering Science with the World's First High Accuracy and High Throughput Functional Assay

Understanding the functional consequences of genetic mutations remains a central challenge in modern biology, with far-reaching implications for human health and disease. While early systematic methods like alanine scanning and phage display provided foundational insights into protein structure and function, the emergence of high-throughput approaches—such as Multiplexed Assays of Variant Effect (MAVEs)—and predictive tools powered by artificial intelligence have vastly expanded our ability to profile mutational landscapes. However, these methods are often constrained by trade-offs between accuracy, scalability, and biological relevance.This dissertation presents the development and application of the GigaAssay, the world’s first high-throughput functional assay capable of delivering both high accuracy and scalability. Built upon a modular, one-pot experimental framework, the GigaAssay enables the quantitative assessment of thousands of mutations simultaneously, while maintaining single-molecule resolution through the use of hundreds of unique molecular identifier (UMI) barcodes per variant. The technology's generalizability and robustness are demonstrated by exploring the mutation space of two vastly different proteins, HIV-1 Tat and HER2. In addition to detailing the experimental and computational innovations that underpin the GigaAssay, this work highlights its transformative applications in virology and oncology, offering new avenues for functional genomics, drug development, and precision medicine. By enabling systematic and reproducible functional interrogation of genetic variation at unprecedented scale and accuracy, the GigaAssay empowers a new era of biological discovery.