Awakening dormant glycosyltransferases in CHO cells with CRISPRa

Chinese hamster ovary (CHO) cells are the preferred workhorse for the biopharmaceutical industry, and CRISPR/Cas9 has proven powerful for generating targeted gene perturbations in CHO cells. Here, we expand the CRISPR engineering toolbox with CRISPR activation (CRISPRa) to increase transcription of endogenous genes. We successfully increased transcription of Mgat3 and St6gal1, and verified their activity on a functional level by subsequently detecting that the appropriate glycan structures were produced. This study demonstrates that CRISPRa can make targeted alterations of CHO cells for desired phenotypes. Chinese hamster ovary (CHO) cells are the preferred workhorse for the biopharmaceutical industry. Glycosylation is a critical quality attribute of biopharmaceuticals, but CHO glycans differ from their human counterparts. Here we demonstrate the use of CRISPR activation (CRISPRa) to increase human‐like glycosylation in CHO cells.