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

Karen Julie la Cour Karottki; Hooman Hefzi; Songyuan Li; Lasse Ebdrup Pedersen; Philipp N Spahn; Chintan Joshi; David Ruckerbauer; Juan A Hernandez Bort; Alex Thomas; Jae Seong Lee; Nicole Borth; Gyun Min Lee; Helene Faustrup Kildegaard; Nathan E Lewis

doi:10.1016/j.ymben.2021.03.017

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

Metab Eng. 2021 Jul:66:114-122. doi: 10.1016/j.ymben.2021.03.017. Epub 2021 Apr 1.

Affiliations

¹ The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark.
² The Novo Nordisk Foundation Center for Biosustainability at the University of California, San Diego, USA; Department of Pediatrics, University of California, San Diego, USA; Department of Bioengineering, University of California, San Diego, USA.
³ The Novo Nordisk Foundation Center for Biosustainability at the University of California, San Diego, USA; Department of Pediatrics, University of California, San Diego, USA.
⁴ Austrian Centre of Industrial Biotechnology, Vienna, Austria; University of Natural Resources and Life Sciences, Vienna, Austria.
⁵ Austrian Centre of Industrial Biotechnology, Vienna, Austria.
⁶ The Novo Nordisk Foundation Center for Biosustainability at the University of California, San Diego, USA.
⁷ Department of Molecular Science and Technology, Ajou University, Suwon, 16499, Republic of Korea.
⁸ Department of Biological Sciences, Kaist, 291 Daehak-Ro, Yuseong-Gu, Daejeon, 305-701, Republic of Korea.
⁹ The Novo Nordisk Foundation Center for Biosustainability at the University of California, San Diego, USA; Department of Pediatrics, University of California, San Diego, USA; Department of Bioengineering, University of California, San Diego, USA; National Biologics Facility, Technical University of Denmark, Denmark. Electronic address: nlewisres@ucsd.edu.

Abstract

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.

Keywords: CHO; CRISPR pooled Screen; Glutamine; Metabolism.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
CHO Cells
CRISPR-Cas Systems*
Cricetinae
Cricetulus
Gene Editing
Glutamine* / genetics
Glutamine* / metabolism

Substances

Glutamine

Grants and funding

R35 GM119850/GM/NIGMS NIH HHS/United States