Gene insertion in Saccharomyces cerevisiae using the CRISPR/Cas9 system

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR/Cas9 technology enables precise genome editing.
• Efficient gene insertion into Saccharomyces cerevisiae is crucial for genetic studies and strain engineering.

Purpose of the Study:

• To develop a rapid and efficient method for inserting target DNA sequences into specific genomic locations in Saccharomyces cerevisiae.
• To compare the efficiency of two gene insertion routes using the CRISPR/Cas9 system.

Main Methods:

• Designed two gene insertion strategies in Saccharomyces cerevisiae using the CRISPR/Cas9 system.
• Inserted a CRISPR array, containing a donor sequence, target gene, and guide sequence, into the Amp and crRNA sites of a pCRCT plasmid.
• Utilized a 100 bp dsDNA mutagenizing homologous recombination donor with homology arms flanking the Cas9 cutting site.

Main Results:

• Successfully inserted a 2.9 kb gene fragment into the target site of the Saccharomyces cerevisiae genome using a single pCRCTG plasmid and a 100 bp dsDNA donor.
• Demonstrated higher recombination efficiency when the CRISPR array was inserted into the crRNA site compared to the Amp site.

Conclusions:

• The developed CRISPR/Cas9-based recombination strategy is a powerful tool for creating yeast strains with targeted gene inserts.
• Optimizing CRISPR array insertion site (crRNA site) enhances recombination efficiency for gene editing in Saccharomyces cerevisiae.