Single-cell imaging of protein dynamics of paralogs reveals mechanisms of gene retention.

Gene duplication is common across the tree of life, including yeast and humans, and contributes to genomic robustness. In this study, we examined changes in the subcellular localization and abundance of proteins in response to the deletion of their paralogs originating from the whole-genome duplication event, which is a largely unexplored mechanism of functional divergence. We performed a systematic single-cell imaging analysis of protein dynamics and screened subcellular redistribution of proteins, capturing their localization and abundance changes, providing insight into forces determining paralog retention. Paralogs showed dependency, whereby proteins required their paralog to maintain their native abundance or localization, more often than compensation. Network feature analysis suggested the importance of functional redundancy and rewiring of protein and genetic interactions underlying redistribution response of paralogs. Translation of non-canonical protein isoform emerged as a novel compensatory mechanism. This study provides new insights into paralog retention and evolutionary forces that shape genomes.

Rapid, scalable, combinatorial genome engineering by marker-less enrichment and recombination of genetically engineered loci in yeast.

A major challenge to rationally building multi-gene processes in yeast arises due to the combinatorics of combining all of the individual edits into the same strain. Here, we present a precise and multi-site genome editing approach that combines all edits without selection markers using CRISPR-Cas9. We demonstrate a highly efficient gene drive that selectively eliminates specific loci by integrating CRISPR-Cas9-mediated double-strand break (DSB) generation and homology-directed recombination with yeast sexual assortment. The method enables marker-less enrichment and recombination of genetically engineered loci (MERGE). We show that MERGE converts single heterologous loci to homozygous loci at ∼100% efficiency, independent of chromosomal location. Furthermore, MERGE is equally efficient at converting and combining multiple loci, thus identifying compatible genotypes. Finally, we establish MERGE proficiency by engineering a fungal carotenoid biosynthesis pathway and most of the human α-proteasome core into yeast. Therefore, MERGE lays the foundation for scalable, combinatorial genome editing in yeast.

Humanized yeast to model human biology, disease and evolution.

For decades, budding yeast, a single-cellular eukaryote, has provided remarkable insights into human biology. Yeast and humans share several thousand genes despite morphological and cellular differences and over a billion years of separate evolution. These genes encode critical cellular processes, the failure of which in humans results in disease. Although recent developments in genome engineering of mammalian cells permit genetic assays in human cell lines, there is still a need to develop biological reagents to study human disease variants in a high-throughput manner. Many protein-coding human genes can successfully substitute for their yeast equivalents and sustain yeast growth, thus opening up doors for developing direct assays of human gene function in a tractable system referred to as 'humanized yeast'. Humanized yeast permits the discovery of new human biology by measuring human protein activity in a simplified organismal context. This Review summarizes recent developments showing how humanized yeast can directly assay human gene function and explore variant effects at scale. Thus, by extending the 'awesome power of yeast genetics' to study human biology, humanizing yeast reinforces the high relevance of evolutionarily distant model organisms to explore human gene evolution, function and disease.