Large scale microfluidic CRISPR screening for increased amylase secretion in yeast

Area of Science:

• Biotechnology
• Molecular Biology
• Yeast Genetics

Background:

• Efficient recombinant protein production via secretion is crucial for biotechnology.
• Understanding cellular pathways for protein translation, processing, and export is essential for optimizing production.

Purpose of the Study:

• To identify genes and non-coding RNAs that regulate α-amylase secretion in *Saccharomyces cerevisiae*.
• To leverage CRISPR technology for large-scale genetic screening to enhance protein secretion.

Main Methods:

• Utilized droplet microfluidic screening combined with genome-wide CRISPR libraries in *S. cerevisiae*.
• Perturbed expression of coding and non-coding genes to identify regulators of α-amylase secretion.
• Validated findings through gene overexpression and deletion experiments.

Main Results:

• Identified 345 genes influencing α-amylase secretion.
• Discovered that modulating genes in vesicle trafficking, endosome-Golgi transport, phagophore assembly, cell cycle, and energy metabolism enhances secretion.
• Found long non-coding RNAs near genes involved in endosomal, Golgi, and vacuolar processes.

Conclusions:

• CRISPR-based screening is a powerful tool for identifying genetic targets to improve protein secretion.
• Specific cellular pathways, including those related to intracellular transport and energy, are key targets for enhancing recombinant protein production.
• Non-coding RNAs may also play a role in regulating protein secretion pathways.