Optimizing the CRISPR/Cas9 system for gene editing in Yarrowia lipolytica

Area of Science:

• Biotechnology
• Synthetic Biology
• Molecular Biology

Background:

• Yarrowia lipolytica is a robust yeast host for producing valuable chemicals.
• Efficient genome editing is crucial for advancing Y. lipolytica's biotechnological applications.
• Current CRISPR/Cas9 systems in Y. lipolytica face challenges with low homologous recombination efficiency and component variability.

Purpose of the Study:

• To optimize CRISPR/Cas9-mediated genome editing in Y. lipolytica.
• To enhance gene disruption and integration efficiencies for improved yeast cell factory construction.

Main Methods:

• Utilized the SCR1-tRNA promoter for sgRNA expression.
• Implemented a tRNA-sgRNA architecture for dual gene disruption.
• Deleted KU70 and overexpressed Rad52 and Sae2 to boost homologous recombination.
• Introduced a modified Cas9 (iCas9) variant.

Main Results:

• Achieved 92.5% gene disruption efficiency using SCR1-tRNA.
• Reached 57.5% dual gene disruption efficiency.
• Increased integration efficiency to 92.5% with KU70 deletion.
• Demonstrated enhanced homologous recombination via Rad52 and Sae2 overexpression.
• Showcased improved gene disruption and genome integration with iCas9.

Conclusions:

• Developed an optimized CRISPR/Cas9 system for efficient genome editing in Y. lipolytica.
• The enhanced system accelerates the development of Y. lipolytica based yeast cell factories.
• This work provides a powerful tool for genetic engineering in Y. lipolytica.