CRISPR-Mediated Genome Editing and Gene Repression in Scheffersomyces stipitis

Area of Science:

• Microbial biotechnology
• Synthetic biology
• Yeast genetics

Background:

• Scheffersomyces stipitis is a yeast with native xylose utilization and potential for producing health-promoting compounds.
• Current limitations include low transformation efficiency and lack of precise genetic engineering tools.
• The yeast primarily uses non-homologous end joining (NHEJ) for DNA repair, hindering accurate genome editing.

Purpose of the Study:

• To develop novel CRISPR-based tools for efficient and precise genome manipulation in Scheffersomyces stipitis.
• To establish homologous recombination (HR) as the dominant DNA repair pathway for genome editing.
• To enhance the yeast's utility as a microbial chassis for biotechnological applications.

Main Methods:

• Construction of a ku70Δku80Δ deficient strain to promote homologous recombination (HR).
• Development of a CRISPR-dCas9 platform incorporating the transcriptional repressor Mxi1 for gene knockdown.
• Optimization of yeast transformation protocols to significantly improve efficiency.

Main Results:

• Successfully established a ku70Δku80Δ deficient strain, making HR the dominant genome editing mechanism.
• Developed an efficient gene knockdown tool using CRISPR-dCas9 and Mxi1.
• Achieved a 191-fold increase in transformation efficiency compared to traditional methods.
• Demonstrated enhanced genome editing capabilities in Scheffersomyces stipitis.

Conclusions:

• This work provides essential CRISPR tools for advanced genetic manipulation of Scheffersomyces stipitis.
• The developed methods significantly improve transformation efficiency and enable precise genome editing.
• These advancements position Scheffersomyces stipitis as a more versatile microbial chassis for biotechnology and offer a model for other nonconventional yeasts.