A Humanized Yeast Model for Studying TRAPP Complex Mutations; Proof-of-Concept Using Variants from an Individual with a TRAPPC1-Associated Neurodevelopmental Syndrome.

Variants in membrane trafficking proteins are known to cause rare disorders with severe symptoms. The highly conserved transport protein particle (TRAPP) complexes are key membrane trafficking regulators that are also involved in autophagy. Pathogenic genetic variants in specific TRAPP subunits are linked to neurological disorders, muscular dystrophies, and skeletal dysplasias. Characterizing these variants and their phenotypes is important for understanding the general and specialized roles of TRAPP subunits as well as for patient diagnosis. Patient-derived cells are not always available, which poses a limitation for the study of these diseases. Therefore, other systems, like the yeast Saccharomyces cerevisiae, can be used to dissect the mechanisms at the intracellular level underlying these disorders. The development of CRISPR/Cas9 technology in yeast has enabled a scar-less editing method that creates an efficient humanized yeast model. In this study, core yeast subunits were humanized by replacing them with their human orthologs, and TRAPPC1, TRAPPC2, TRAPPC2L, TRAPPC6A, and TRAPPC6B were found to successfully replace their yeast counterparts. This system was used for studying the first reported individual with an autosomal recessive disorder caused by biallelic TRAPPC1 variants, a girl with a severe neurodevelopmental disorder and myopathy. We show that the maternal variant (TRAPPC1 p.(Val121Alafs*3)) is non-functional while the paternal variant (TRAPPC1 p.(His22_Lys24del)) is conditional-lethal and affects secretion and non-selective autophagy in yeast. This parallels defects seen in fibroblasts derived from this individual which also showed membrane trafficking defects and altered Golgi morphology, all of which were rescued in the human system by wild-type TRAPPC1. This study suggests that humanized yeast can be an efficient means to study TRAPP subunit variants in the absence of human cells and can assign significance to variants of unknown significance (VUS). This study lays the foundation for characterizing further TRAPP variants through this system, rapidly contributing to disease diagnosis.

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.

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.

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.