CRISPR/Cas9-assisted heterothallic conversion enables rapid hybridization of industrial Saccharomyces strains.

Efficient yeast breeding remains a major bottleneck in industrial biotechnology, particularly in food and beverage applications, where strain improvement must balance phenotypic innovation with regulatory constraints. Traditional hybridization procedures for natural Saccharomyces strains are experimentally limited by their diploid and homothallic nature, often resulting in labor-intensive processes of unpredictable duration. Here, we developed and validated a CRISPR/Cas9-assisted breeding framework that enables rapid, systematic, and informative hybridization by transiently converting homothallic strains into stable heterothallic mating partners through targeted HO gene inactivation. Using this strategy, we generated and characterized multiple intraspecific and interspecific hybrids challenging some industrially relevant traits, as sulfur dioxide release, acetic acid production, and the ability to restart stuck fermentations. In all cases, we generated the hybrids and assessed their performance within three months. Hybrid phenotypes followed inheritance patterns including parental-like, intermediate, and combinatorial traits, enabling rapid evaluation of cross utility. Moreover, cisgenic hybrids were generated by the restoration of the native HO locus, maintaining identical fermentative performance. Application to Saccharomyces cerevisiae × Saccharomyces uvarum interspecific crosses further demonstrated the versatility of the approach while revealing intrinsic biological constraints on trait combination. Overall, CRISPR/Cas9-assisted heterothallic conversion emerges as a versatile platform for accelerated yeast breeding, providing decision-enabling insights that support informed strain development across fermentation-based industries.