TSA-ultrasound synergy enhances CRISPR-Cas9 gene editing efficiency in diploid yeast.

The CRISPR-Cas9 system is invaluable for microbial engineering. However, its efficiency remains limited in numerous microorganisms, especially in polyploid yeasts where the compact chromatin structures pose significant physical barriers, which are major constraints in microbial engineering. To address this limitation, we developed and validated a standardized synergistic protocol using the diploid industrial yeast Candida glycerinogenes as a model. This protocol combines TSA-induced chromatin decondensation with brief, low-intensity ultrasonication to enhance Cas9 accessibility. Key parameters were systematically optimized, and 200 nM Trichostatin A (TSA) (10 h) followed by 200 W sonication (3 min) were established as the optimal condition set. Validation results showed that the protocol more than doubled single-gene knockout efficiencies (GPD1, TRP1) compared with conventional methods. Importantly, it enabled complex edits that were previously unattainable in C. glycerinogenes, including the precise deletion of a 7.8-kb fragment and the editing of an 11.4-kb region for functional genomics. In summary, this study establishes a simple and effective workflow that overcomes chromatin-based barriers in the polyploid industrial yeast C. glycerinogenes, providing a practical tool for genetic engineering and functional genomics in this and potentially other recalcitrant yeasts.