Systematic targeted gene deletion using the gene-synthesis method in fission yeast

Area of Science:

• Molecular Biology
• Yeast Genetics

Background:

• Systematic gene deletion is crucial for understanding gene function.
• Existing methods like serial-PCR and block-PCR are labor-intensive and require specialized skills.

Purpose of the Study:

• To develop a novel, systematic, and efficient gene-synthesis method for creating deletion cassettes in fission yeast.
• To enable large-scale gene deletion studies on a 96-well plate basis.

Main Methods:

• Designed a modular 2129-bp deletion cassette with a central KanMX4 selection marker and flanking artificial linker modules.
• Synthesized oligonucleotides on a 96-well plate, ligated them using ligase chain reaction, and amplified via nested PCR.
• Transformed deletion cassettes into fission yeast (Schizosaccharomyces pombe) and validated via PCR and tetrad analysis.

Main Results:

• Successfully deleted 563 genes in Schizosaccharomyces pombe using the novel method.
• Achieved a >90% success rate for gene deletion.
• Observed a low point-mutation rate of approximately 0.4 mutations per kb.

Conclusions:

• The developed gene-synthesis method provides an efficient and systematic approach for large-scale gene deletion in yeast.
• This method is adaptable for various yeast species and can be integrated with bar-coding for parallel analyses.
• Facilitates high-throughput functional genomics research.