Elimination of the "essential" Warburg effect in mammalian cells through a multiplex genome engineering strategy

Abstract

Innovative genome editing technologies are accumulating and are rapidly elucidating the genetic basis of many cell functions. However, many disease phenotypes and physiological processes are governed by complex networks involving many gene products. Thus, numerous phenotypes need to be unraveled using multiplex genome editing techniques and in silico analysis. Furthermore, these approaches must further leverage legacy knowledge to identify the genes involved and understand the cell response. As an example, we unraveled the core cellular network controlling the Warburg effect, also known as aerobic glycolysis. This effect involves the tendency of rapidly‐proliferating cells to consume excess glucose and secrete copious amounts of lactic acid, despite having adequate oxygen for more efficient oxidative phosphorylation. This response is a hallmark of cancer, immune cell expansion, and other processes with rapidly proliferating cells. However, efforts to test the purpose of Warburg metabolism have been stymied by the difficulty to generate cell lines that are unable to produce lactic acid, since genes involved have proven to be essential or synthetic lethal in immortalized cells. We discovered a genetic circuit involving multiple genes that control lactic acid secretion, and using a multiplex genome editing with CRISPR, we successfully eliminated lactic acid secretion and enabled the deletion of multiple "essential" genes. Surprisingly, the cells show improved metabolic and growth phenotypes, despite the elimination of this fundamental metabolic activity. To understand how immortalized mammalian cells can cope without this seemingly essential metabolic process, we conducted a comprehensive analysis of these cell lines using time‐course RNA‐Seq, metabolomics, and analysis with a genome‐scale metabolic network model we have developed for Chinese hamster ovary cells1. Thus, through a multiplex metabolic engineering effort and comprehensive systems biology analysis, we have been able to engineer out a hallmark phenotype of proliferating cells and begin to understand now a cell can survive without a seemingly essential process.Support or Funding InformationThe Novo Nordisk Foundation Center for Biosustainability (NNF10CC1016517)