Accelerating stable recombinant cell line development by targeted integration

To provide a high-throughput screening method for human ether-a-go-go-gene–related gene (hERG) K+ channel inhibition, a new recombinant cell line, in which single action potential (AP)–induced cell death was produced by gene transfection. Mutated human cardiac Na+ channel Nav1.5 (IFM/Q3), which shows extremely slow inactivation, and wild-type inward rectifier K+ channel, Kir2.1, were stably co-expressed in HEK293 cells (IFM/Q3+Kir2.1). In IFM/Q3+Kir2.1, application of single electrical stimulation (ES) elicited a long AP lasting more than 30 s and led cells to die by more than 70%, whereas HEK293 co-transfected with wild-type Nav1.5 and Kir2.1 fully survived. The additional expression of hERG K+ channels in IFM/Q3+Kir2.1 shortened the duration of evoked AP and thereby markedly reduced the cell death. The treatment of the cells with hERG channel inhibitors such as nifekalant, E-4031, cisapride, terfenadine, and verapamil, recovered the prolonged AP and dose-dependently facilitated cell death upon ES. The EC50 values to induce the cell death were 3 µM, 19 nM, 17 nM, 74 nM, and 3 µM, respectively, whereas 10 µM nifedipine did not induce cell death. Results indicate the high utility of this cell system for hERG K+ channel safety assay.

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Controlling Ratios of Plasmid-Based Double Cut Donor and CRISPR/Cas9 Components to Enhance Targeted Integration of Transgenes in Chinese Hamster Ovary Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22052407 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2407

Author(s):

Sung Wook Shin ◽

Dongwoo Kim ◽

Jae Seong Lee

Keyword(s):

Cell Line ◽

Chinese Hamster Ovary ◽

Cho Cells ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Cho Cell ◽

Cell Line Development ◽

Cho Cell Line ◽

Vector Systems ◽

Targeted Integration

Chinese hamster ovary (CHO) cells are the most valuable expression host for the commercial production of biotherapeutics. Recent trends in recombinant CHO cell-line development have focused on the site-specific integration of transgenes encoding recombinant proteins over random integration. However, the low efficiency of homology-directed repair upon transfection of Cas9, single-guide RNA (sgRNA), and the donor template has limited its feasibility. Previously, we demonstrated that a double-cut donor (DCD) system enables highly efficient CRISPR/Cas9-mediated targeted integration (TI) in CHO cells. Here, we describe several CRISPR/Cas9 vector systems based on DCD templates using a promoter trap-based TI monitoring cell line. Among them, a multi-component (MC) system consisting of an sgRNA/DCD vector and Cas9 expression vector showed an approximate 1.5-fold increase in knock-in (KI) efficiency compared to the previous DCD system, when a systematically optimized relative ratio of sgRNA/DCD and Cas9 vector was applied. Our optimization efforts revealed that concurrently increasing sgRNA and DCD components relative to Cas9 correlated positively with KI efficiency at a single KI site. Furthermore, we explored component bottlenecks, such as effects of sgRNA components and applicability of the MC system on simultaneous double KI. Taken together, we improved the DCD vector design by tailoring plasmid constructs and relative component ratios, and this system can be widely used in the TI strategy of transgenes, particularly in CHO cell line development and engineering.

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