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. [Display omitted] •Cas9-induced gene drive efficiently converts heterozygous to homozygous yeast locus•Yeast mating and Cas9 selection combines two or more separate edits into a single strain•The method enables marker-less enrichment, recombination of genetically engineered loci•MERGE reveals a fitness-driven path to humanize α-proteasome core subunits in yeast Engineering entire biological processes in yeast, controlled for expression and genomic context, is challenging due to inefficient homology-directed repair (HDR) using linear exogenous repair templates, often resulting in non-quantifiable readouts. Thus, an inability to obtain the edit can be attributed to inefficient HDR or inviable genotype. Therefore, accomplishing multiplexed and scalable genomic editing necessitates compatible technology. We developed a CRISPR-Cas9-based combinatorial genome editing method (MERGE) that facilitates a fitness-driven combination of all individual genetic edits, eliminating the need for selection markers while using a quantitative readout of colony-forming units. Abdullah et al. use yeast mating and CRISPR-Cas9 selection to combine many individual genetic edits into a single strain. The method facilitates marker-less enrichment and recombination of genetically engineered loci (MERGE). MERGE engineers the entire genetic systems in yeast by exploring the fitness landscape