Mapping the growth effect of previously hidden ubiquitin alleles using an overexpression based mutational scan

Deep mutational scanning has emerged as a powerful, high throughput approach to determine the growth effect of thousands of alleles at once in bulk competition. However, to date only the growth effect of mutant alleles in isolation has been determined. Building off previous work, we have created a library of all possible single point mutations in ubiquitin and determined the growth effect of mutants overexpressed in the presence of a wild type allele. Using this scan, we explained over half of the previously missing mutants in the single allele scan by showing that they exhibit deleterious effects when co-expressed with wild type ubiquitin. Additionally, unlike the single allele growth effect, these overexpression growth effects were distributed across the entire protein. This overexpression scan methodology can identify likely dominant mutant effects in any essential gene and is highly complementary with traditional deep mutational scanning approaches.