Chinese hamster ovary (CHO) cells are the most widely used cells for producing biopharmaceuticals. Engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the Chinese hamster genome were computationally predicted and are frequently inaccurate. Here, we revised TSS annotations for 15,308 Chinese hamster genes and 4,478 non-coding RNAs based on experimental data from CHO-K1 cells and 10 hamster tissues. The experimental realignment and discovery of TSSs now expose previously hidden motifs, such as the TATA box. We further demonstrate, by targeting the glycosyltransferase gene Mgat3, how accurate annotations readily facilitate activating silent genes by CRISPRa to obtain more human-like glycosylation. Together, we envision our annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.

Learning Objectives:

1. The mapping of TSSs in Chinese Hamster Ovary cells is improved using nascent capped-RNA sequencing methods combined with samples across multiple hamster tissues.

2. The experimental TSSs is used for CRISPR activation of the silenced protein Mgat3 in the Chinese Hamster.