Media and feed optimization have fueled many-fold improvements in mammalian biopharmaceutical production, but genome editing offers an emerging avenue for further 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 present a comprehensive pooled CRISPR screen for CHO cell metabolism, including ~16,000 gRNAs against ~2500 metabolic enzymes and regulators. Using this screen, we identified a glutamine response network in CHO cells. Glutamine is particularly important since it is often over-fed to drive increased TCA cycle flux, but toxic ammonia may accumulate. With the screen we found one orphan glutamine-responsive gene with no clear connection to our network. Knockout of this novel and poorly characterized lipase, Abhd11, substantially increased 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, and elucidate novel regulators of metabolism.
The authors wish to thank Nachon Charanyanonda Petersen for assistance in cell line generation and batch culture and Anna Koza, Alexandra Hoffmeyer, Pannipa Pornpitapong for assistance with NGS, Dr. Prashant Mali for packaging the gRNA library into the lentivirus, Dr. James A. Nathan for discussions regarding Abhd11 and Daria Sergeeva for co-drawing Fig. 1 . This work was supported by the Novo Nordisk Foundation ( NNF20SA0066621 , NNF10CC1016517 , and NNF16OC0021638 ) and NIGMS ( R35 GM119850 , NEL).
