CRISPR-Cas13d for Gene Knockdown and Engineering of CHO Cells.

Shen, Chih-Che; Lin, Mei-Wei; Nguyen, Bao Khanh Thi; Chang, Chin-Wei; Shih, Jie-Ru; Nguyen, Mai Thanh Thi; Chang, Yi-Hao; Hu, Yu-Chen

Chinese hamster ovary (CHO) cell is the predominant cell chassis for biopharmaceutical production. Engineering cellular pathways related to cell death, metabolism and glycosylation in CHO cell is desired but challenging. Here we present a novel approach that exploits CRISPR-Cas13d for gene silencing and CHO cell engineering. CRISPR-Cas13d is a burgeoning system that exploits Cas13d nuclease and guide RNA (gRNA) for RNA cleavage and gene knockdown. We first showed that CRISPR-Cas13d effectively knocked down exogenous genes in CHO cell lines (K1, DG44 and DUXB11) commonly used for recombinant protein production. We next demonstrated that CRISPR-Cas13d robustly suppressed the expression of exogenous genes and various endogenous genes involved in gene amplification, apoptosis, metabolism and glycosylation (e.g. GS, BAK, BAX, PDK1 and FUT8) in CHO cells with efficiencies ranging from 60-80%, simply by transient transfection. By integrating the entire CRISPR-Cas13d system with the Sleeping Beauty system and optimal gRNA design, we further improved the knockdown efficiency and rapidly generated stable cells with ï‚»80-90% knockdown. With this approach, we knocked down FUT8 expression for >90% and significantly attenuated the IgG fucosylation. These data altogether implicated the potentials of CRISPR-Cas13d for gene regulation, glycoengineering and cell engineering of CHO cells.