CRISPR-Cas13d for Gene Knockdown and Engineering of CHO Cells

Abstract

Chinese hamster ovary (CHO) cells are the predominant cell chassis for biopharmaceutical production. Engineering cellular pathways related to cell death, metabolism, and glycosylation in CHO cells 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% to 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 ≈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.

Coupling CRISPR interference with FACS enrichment: New approach in glycoengineering of CHO cell lines for therapeutic glycoprotein production

“…With stable integration of the CRISPRi system targeting two sites upstream of TSS of the Fut8 gene, we were able to downregulate its expression by an average of ∼ 50% in the pool of CHO cells (Figure 1B and E). This is in contrast with the previous report about S. pyogenes-derived dCas9-KRAB based CRISPRi system not being effective in Fut8 gene repression, [45] pointing to a difference in the effectiveness of different CRISPRi systems as well as the need for screening for the optimal sgRNA. The simultaneous targeting of multiple sgRNAs to the same gene (Figure 1) had a similar effect as individual transfections (Figure S1) and did not lead to more potent gene downregulation, as reported previously.…”

“…Even more pronounced effect was observed on the Fc domain, where fucosylation was decreased by up to 60% (Figure 3C). Although using IgG as a model protein, the authors of a recently published study reported that knockdown of Fut8 gene expression for ∼ 80% to 90% led to a decrease in fucosylation for only 30%, [ 45 ] reaffirming that type of therapeutic protein as well as other cell line characteristic can significantly contribute to fucosylation variability. [ 40 ] The model protein used in the study is Fc‐fusion protein consisting of two structurally and functionally distinct protein domains, with multiple N‐glycosylation sites on the target‐binding domain and one on the Fc‐domain.…”

“…On the other hand, the tested CRISPRi system did not undermine cell growth as it was reported for CRISPR‐Cas13d. [ 45 ] High distribution of titer and galactosylation observed in enriched clones can be attributed to the clonal variation typically observed in CHO cell lines. [ 51 ]…”

Cas13d: A New Molecular Scissor for Transcriptome Engineering

“…Further, knockdown of the FUT8 gene causes attenuation of IgG fucosylation with 90% efficiency. Thus, their study has shown the generation of stable engineered CHO cells using the CRISPR-Cas13d platform, which has the ability to attenuate lactate accumulation and glycosylation and also shown apoptosis resistance which resulted in an increase in cell titer, enhance recombinant protein and antibody production during fed-batch culture ( Shen et al, 2020 ). Similarly, He et al, used active RfxCas13d to target metabolic genes including Pten, Pcsk9, and lncLstr, in mouse hepatocytes.…”

Specific knockdown of Htra2 by CRISPR-CasRx prevents acquired sensorineural hearing loss in mice

“…Studies on CasRx-mediated knockdown have been reported in bacteria, 25 plants, 26 and various cell types. 27 , 28 For example, Zhou et al. 13 alleviated neurological disease in mice by knocking down Ptbp1 using CasRx, and Jiang et al.…”