Pooled CRISPR screening enables genotype-phenotype association in industrial microorganisms.

Industrial microorganisms combine genomic robustness with process resilience to achieve high-level bioproduction, and these traits are usually governed by complex, multi-gene interactions. Pooled CRISPR screening-assisted genotype-phenotype association (GPA), an emerging approach attracting increasing attention, has recently evolved into a powerful platform for systematically interrogating gene function in industrially relevant strains and for rapidly identifying genotypes that drive desired phenotypes. In this review, we frame the GPA workflow as "Design-Build-Screen-Apply" and focus on the middle two steps. We compare state-of-the-art library-building technologies and catalogue positive hits obtained with diverse enrichment and screening strategies, evaluating their respective strengths, limitations, and applicability. Relevant applications from the past five years are then summarized to illustrate how GPA deciphers industrially relevant traits and accelerates the construction of high-performance microbial cell factories. Finally, we explore how artificial intelligence (AI) can streamline pooled CRISPR GPA workflows and outline remaining challenges.