We have recently found that a large number of S. cerevisiae genes can be "humanized", i.e. the yeast gene is deleted and replaced with its human ortholog to generate viable strains. We have also observed several gene modules (pathways or complexes) which are comprised mostly of genes that can be humanized (Kachroo et al. (2015) Science, 348:921-925). The sterol biosynthesis pathway, which is common between yeast and humans (producing the precursors of ergosterol and cholesterol in them respectively) is one such pathway; 17 of the genes in it (many of them essential) can be individually humanized.
We have sought to investigate the possibility of higher order humanizations of an entire gene module (simultaneous humanization of multiple genes), using the sterol biosynthesis pathway as a starting point. We have developed a rapid, high-throughput approach based on CRISPR/Cas9 technology which allows us to easily and efficiently perform genetic engineering at several yeast loci. We have also investigated the utility of methods for combining modifications at several different loci in one strain. We expect that module-scale humanization will yield valuable insights into the evolution of species. In addition to this, this system is a promising platform for systematically investigating interactions between human genes, both in their wild-type as well as mutant forms or allelic variants. Since experiments with humanized yeast are much less labor and cost intensive than alternatives such as cultured human cells, they offer the possibility of probing human allelic variation on a greater scale than has been possible before.