Pooled CRISPR screening enables genotype-phenotype association in industrial microorganisms

Area of Science:

• Microbiology
• Synthetic Biology
• Biotechnology

Background:

• Industrial microorganisms require genomic robustness and process resilience for high-level bioproduction, often involving complex multi-gene interactions.
• Pooled CRISPR screening-assisted genotype-phenotype association (GPA) is an emerging, powerful platform for systematically interrogating gene function in industrial strains.

Purpose of the Study:

• To review the Design-Build-Screen-Apply workflow of pooled CRISPR GPA, focusing on library construction and screening strategies.
• To illustrate how GPA deciphers industrially relevant traits and accelerates the development of high-performance microbial cell factories.
• To explore the potential of artificial intelligence (AI) in streamlining GPA workflows.

Main Methods:

• Comparison of state-of-the-art library-building technologies for pooled CRISPR GPA.
• Cataloguing and evaluation of enrichment and screening strategies used in GPA.
• Summarization of relevant GPA applications from the past five years.

Main Results:

• GPA effectively identifies genotypes driving desired phenotypes in industrial strains.
• Diverse screening strategies have varying strengths, limitations, and applicability for GPA.
• GPA has been successfully applied to decipher complex traits in microbial cell factories.

Conclusions:

• Pooled CRISPR GPA is a key technology for understanding and engineering industrial microorganisms.
• AI integration holds promise for optimizing GPA workflows and accelerating bioproduction advancements.
• Further research is needed to address remaining challenges in GPA implementation.