A rapid genetic screening system for identifying gene-specific suppression constructs for use in human cells

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Developing efficient gene suppression constructs (GSCs) is crucial for targeted gene regulation.
• Large-scale screening methods are needed to identify optimal GSCs from diverse libraries.
• Fission yeast offers a tractable system for genetic screening.

Purpose of the Study:

• To develop and validate a rapid, cell-based genetic screen in fission yeast for identifying efficient GSCs.
• To demonstrate the utility of this screening system for various target sequences, including human genes.
• To enable the discovery of GSCs applicable for gene regulation in human cells.

Main Methods:

• A fission yeast system was engineered where target sequences are fused to the 5' end of the lacZ reporter gene.
• Random fragment expression libraries derived from target sequences were screened using a lacZ gene-encoded colony color phenotype.
• The screen was tested by identifying GSCs for fission yeast ura4, human c-myc, and human Chk1.

Main Results:

• The screen successfully identified a range of GSCs, including rare efficient suppressors, for the tested genes.
• Specific GSCs for human c-myc were identified.
• The identified c-myc GSCs demonstrated regulation of both a c-myc-lacZ fusion gene and the endogenous c-myc gene in human cells.

Conclusions:

• The described fission yeast genetic screen is a powerful and rapid tool for identifying efficient gene suppression constructs.
• This method is versatile and applicable for discovering GSCs targeting any sequence for use in human cells.
• The validated GSCs show potential for precise regulation of gene expression in human systems.