Abstract
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.
Highlights
Industrial manufacturing of recombinant therapeutic proteins requires mammalian cells as expression hosts, among which Chinese hamster ovary (CHO) cells are the primary mammalian cells of choice
We demonstrated that donor templates, termed double-cut donors (DCDs)–which contain the target genomic site single-guide RNA (sgRNA)/protospacer adjacent motif (PAM) recognition sequences outside of homology sequences–markedly increased KI efficiency in CHO cells compared to conventional circular homology-directed repair (HDR) donors [13]
Based on the previous DCD system, we modified the clustered regularly interspaced short palindromic repeats of (CRISPR)–HDR plasmid vector design to further optimize the relative ratio of CRISPR–HDR components
Summary
Industrial manufacturing of recombinant therapeutic proteins requires mammalian cells as expression hosts, among which Chinese hamster ovary (CHO) cells are the primary mammalian cells of choice. The cell-line development (CLD) process using CHO cells is based on untargeted random integration of transgenes followed by a selection process and large-scale clone screening [1,2]. While the current CLD platform has generated high producing recombinant CHO cell lines, the process is labor-intensive and causes inconsistent expression of proteins of interest. Cellline lineleads leads homology-mediated sgRNA, double donor (DCD)template templateinto into monitoring monitoring cell to to homology-mediated targeted integration of human. Flow cytometry analysis of of DCD, MC, and AIO systems days after transfection. The vector constructs were transfected at equal amounts The vector constructs were transfected at equal amounts (5:5, w/w) inw/w)
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