Genome editing has become an important aspect of Chinese hamster ovary (CHO) cell line engineering for improving the production of recombinant protein therapeutics. Currently, the engineering focus is directed toward expanding product diversity while controlling and improving product quality and yields. In this chapter, we present our protocol for using the genome editing tool Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to knock out engineering target genes in CHO cells. As an example, we describe how to knock out the glutamine synthetase (GS) gene, which increases the selection efficiency of the GS-mediated gene amplification system.
We thank Karen Kathrine Br\\u00F8ndum and Johnny Arnsdorf for optimizing and setting up FACS sorting and Nachon Charanyanonda Petersen for help with the transfection and FACS sorting. This work was supported by the Novo Nordisk Foundation.
