A metabolic CRISPR-Cas9 screen in Chinese hamster ovary cells identifies glutamine-sensitive genes

Abstract Over the past decades, optimization of media formulation and feeding strategies have fueled a many-fold improvement in CHO-based biopharmaceutical production. While Design of Experiments (DOE) and media screens have led to many advances, genome editing offers another avenue for enhancing cell metabolism and bioproduction. However the complexity of metabolism, involving thousands of genes, makes it unclear which engineering strategies will result in desired traits. Here we developed a comprehensive pooled CRISPR screen for CHO cell metabolism, including ∼16,000 gRNAs against ∼2500 metabolic enzymes and regulators. We demonstrated the value of this screen by identifying a glutamine response network in CHO cells. Glutamine is particularly important since it is often substantially over-fed to drive increased TCA cycle flux but can lead to accumulation of toxic ammonia. Within the glutamine-response network, the deletion of a novel and poorly characterized lipase, Abhd11 , was found to substantially increase growth in glutamine-free media by altering the regulation of the TCA cycle. Thus, the screen provides an invaluable targeted platform to comprehensively study genes involved in any metabolic trait.